Planarian flatworms undergo continuous internal turnover, wherein old cells are replaced by the division progeny of adult pluripotent stem cells (neoblasts). How cell turnover is carried out at the organismal level remains an intriguing question in planarians and other systems. While previous studies have predominantly focused on neoblast proliferation, little is known about the processes that mediate cell loss during tissue homeostasis. Here, we use the planarian epidermis as a model to study the mechanisms of cell removal. We established a covalent dye-labeling assay and image analysis pipeline to quantify the cell turnover rate in the planarian epidermis. Our findings indicate that the ventral epidermis is highly dynamic and epidermal cells undergo internalization via basal extrusion, followed by a relocation toward the intestine and ultimately digestion by intestinal phagocytes. Overall, our study reveals a complex homeostatic process of cell clearance that may generally allow planarians to catabolize their own cells.

Germ cells are regulated by local microenvironments (niches), which secrete instructive cues. Conserved developmental signaling molecules act as niche-derived regulatory factors, yet other types of niche signals remain to be identified. Single-cell RNA-sequencing of sexual planarians revealed niche cells expressing a nonribosomal peptide synthetase (nrps). Inhibiting nrps led to loss of female reproductive organs and testis hyperplasia. Mass spectrometry detected the dipeptide β-alanyl-tryptamine (BATT), which is associated with reproductive system development and requires nrps and a monoamine-transmitter-synthetic enzyme Aromatic L-amino acid decarboxylase (AADC) for its production. Exogenous BATT rescued the reproductive defects after nrps or aadc inhibition, restoring fertility. Thus, a nonribosomal, monoamine-derived peptide provided by niche cells acts as a critical signal to trigger planarian reproductive development. These findings reveal an unexpected function for monoamines in niche-germ cell signaling. Furthermore, given the recently reported role for BATT as a male-derived factor required for reproductive maturation of female schistosomes, these results have important implications for the evolution of parasitic flatworms and suggest a potential role for nonribosomal peptides as signaling molecules in other organisms.

Germ cells are regulated by local microenvironments (niches), which secrete instructive cues. Conserved developmental signaling molecules act as niche-derived regulatory factors, yet other types of niche signals remain to be identified. Single-cell RNA-sequencing of sexual planarians revealed niche cells expressing a non-ribosomal peptide synthetase (nrps). Inhibiting nrps led to loss of female reproductive organs and testis hyperplasia. Mass spectrometry detected the dipeptide β-alanyl-tryptamine (BATT), which is associated with reproductive system development and requires nrps and a monoamine-transmitter-synthetic enzyme (AADC) for its production. Exogenous BATT rescued the reproductive defects after nrps or aadc inhibition, restoring fertility. Thus, a non-ribosomal, monoamine-derived peptide provided by niche cells acts as a critical signal to trigger planarian reproductive development. These findings reveal an unexpected function for monoamines in niche-germ cell signaling. Furthermore, given the recently reported role for BATT as a male-derived factor required for reproductive maturation of female schistosomes, these results have important implications for the evolution of parasitic flatworms and suggest a potential role for non-ribosomal peptides as signaling molecules in other organisms.

Whole-mount in situ hybridization is a critical technique for analyzing gene expression in planarians. While robust in situ protocols have been developed, these protocols are laborious, making them challenging to incorporate in an academic setting, reducing throughput and increasing time to results. Here, the authors systematically tested modifications to all phases of the protocol with the goal of eliminating steps and reducing time without impacting quality. This modified protocol allows for whole-mount colorimetric in situ hybridization and multicolor fluorescence in situ hybridization to be completed in two days with a significant reduction in steps and hands-on processing time.

The flatworm planarian, Schmidtea mediterranea (Smed) is a master at regenerating and rebuilding whole animals from fragments. A full understanding of Smed\'s regenerative capabilities requires a high-resolution characterization of organs, tissues, and the adult stem cells necessary for regeneration in their native environment. Here, we describe a serial block face scanning electron microscopy (SBF-SEM) protocol, optimized for Smed specifically, for visualizing the ultrastructure of membranes and condensed chromosomes in this model organism.

The use of flow cytometry and fluorescence-activated cell sorting to roughly separate subpopulations of cells in Schmidtea mediterranea is long established. In this chapter, we describe a method for the immunostaining-either single or double-of live planarian cells, using mouse monoclonal antibodies reactive against S. mediterranea plasma membrane antigens. This protocol allows to sort live cells according to their membrane signature, offering the possibility to further characterize the cell populations in S. mediterranea in a variety of downstream applications, like transcriptomics and cell transplantation, also at the single-cell level.

Whole-mount in situ hybridization (WISH), colorimetric or fluorescent (FISH), allows for the visualization of endogenous RNA. For planarians, robust WISH protocols exist for small-sized animals (>5 mm) of the model species Schmidtea mediterranea and Dugesia japonica. However, the sexual strain of Schmidtea mediterranea studied for germline development and function reaches much larger body sizes in excess of 2 cm. The existing whole-mount WISH protocols are not optimal for such large specimens, owing to insufficient tissue permeabilization. Here, we describe a robust WISH protocol for 12-16 mm long sexually mature Schmidtea mediterranea individuals that could serve as a starting point for adapting WISH to other large planarian species.

Whole-mount in situ hybridization (WISH) is an extremely useful technique for visualizing specific mRNA targets and solving many biological questions. In planarians, this method is really valuable, for example, for determining gene expression profiles during whole-body regeneration and analyzing the effects of silencing any gene to determine their functions. In this chapter, we present in detail the WISH protocol routinely used in our lab, using a digoxigenin-labelled RNA probe and developing with NBT-BCIP. This protocol is basically that already described in Currie et al. (EvoDevo 7:7, 2016), which put together several modifications developed from several laboratories in recent years that improved the original protocol developed in the laboratory of Kiyokazu Agata in 1997. Although this protocol, or slight modifications of it, is the most common protocol in the planarian field for NBT-BCIP WISH, our results show that key steps such as the use and time of NAC treatment to remove the mucus need to be taken into account depending on the nature of the gene analyzed, especially for the epidermal markers.

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system.

Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to predict enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify genomic regions with histone modifications consistent with enhancer activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high-confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for predicted transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Footprinting analyses provided further evidence that these potential TF binding sites are likely to be occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of TFs in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. We found that our predicted GRNs were independently supported by existing TF RNAi/RNA-seq datasets, providing further evidence that our work predicts active enhancers that regulate adult stem cells and regenerative mechanisms.