The DNA Methyltransferase DMAP1 is Required for Tissue Maintenance and Planarian Regeneration.

The precise regulation of transcription is required for embryonic development, adult tissue turnover, and regeneration. Epigenetic modifications play a crucial role in orchestrating and regulating the transcription of genes. These modifications are important in the transition of pluripotent stem cells and their progeny. Methylation, a key epigenetic modification, influences gene expression through changes in histone tails and direct DNA methylation. Work in different organisms has shown that the DNA methyltransferase-1-associated protein (DMAP1) may associate with other molecules to repress transcription through DNA methylation. Thus, DMAP1 is a versatile protein implicated in a myriad of events, including pluripotency maintenance, DNA damage repair, and tumor suppression. While DMAP1 has been extensively studied in vitro, its complex regulation in the context of the adult organism remains unclear. To gain insights into the possible roles of DMAP1 at the organismal level, we used planarian flatworms that possess remarkable regenerative capabilities driven by pluripotent stem cells called neoblast. Our findings demonstrate the evolutionary conservation of DMAP1 in the planarian Schmidtea mediterranea. Functional disruption of DMAP1 through RNA interference revealed its critical role in tissue maintenance, neoblast differentiation, and regeneration in S. mediterranea. Moreover, our analysis unveiled a novel function for DMAP1 in regulating cell death in response to DNA damage and influencing the expression of axial polarity markers. Our findings provide a simplified paradigm for studying DMAP1's epigenetic regulation in adult tissues.

A Planarian Model System to Study Host-Pathogen Interactions.

This protocol is focused on using the recently established planarian infection model system to study host-pathogen interactions during fungal infection. Here, we describe in detail the infection of the planarian Schmidtea mediterranea with the human fungal pathogen Candida albicans. This simple and reproducible model system allows for rapid visualization of tissue damage throughout different infection timepoints. We note that this model system has been optimized for use with C. albicans, but should also be applicable for use with other pathogens of interest.

Collaborative Teaching plus (CT+): A Timely, Flexible, and Dynamic Course Design Implemented during Emergency Remote Teaching in an Introductory Biology Course.

Student-centered pedagogies promote student learning in college science, technology, engineering, and mathematics (STEM) classrooms. However, transitioning to active learning from traditional lecturing may be challenging for both students and instructors. This case study presents the development, implementation, and assessment of a modified collaborative teaching (CT) and team-based learning (TBL) approach (CT plus TBL, or CT+) in an introductory biology course at a Minority-Serving Institution. A logic model was formulated depicting the various assessment practices with the culminating goal of improving the student learning experience. We analyzed qualitative and quantitative data based on students and instructors' behaviors and discourse, and student midsemester and end-of-semester surveys. Our findings revealed that the integration of multiple instructors allowed for knowledge exchange in blending complementary behaviors and discourse practices during class sessions. In addition, the frequent ongoing assessments and incorporation of student feedback informed the CT+ design during both in-person and emergency remote teaching. Furthermore, this course design could be easily adapted to a variety of STEM courses in higher education, including remote instruction.

Non-traditional roles of immune cells in regeneration: an evolutionary perspective.

Immune cells are known to engage in pathogen defense. However, emerging research has revealed additional roles for immune cells, which are independent of their function in the immune response. Here, we underscore the ability of cells outside of the adaptive immune system to respond to recurring infections through the lens of evolution and cellular memory. With this in mind, we then discuss the bidirectional crosstalk between the immune cells and stem cells and present examples where these interactions regulate tissue repair and regeneration. We conclude by suggesting that comprehensive analyses of the immune system may enable biomedical applications in stem cell biology and regenerative medicine.

Analysis of DNA Double-Stranded Breaks Using the Comet Assay in Planarians.

Comet assay provides the opportunity to detect and characterize DNA strand breaks. Cellular lysing followed by embedding in agarose slide is used to visualize under an electrical current migration patterns corresponding to DNA fragments of different sizes. Here we describe the process of detecting and characterizing DNA damage by Comet assay on planarians, which is a model organism commonly used to understand the process of whole-body regeneration, stem cell regulation, and adult tissue maintenance.

Restoration of DNA integrity and the cell cycle by electric stimulation in planarian tissues damaged by ionizing radiation.

Exposure to high levels of ionizing γ radiation leads to irreversible DNA damage and cell death. Here, we establish that exogenous application of electric stimulation enables cellular plasticity and the re-establishment of stem cell activity in tissues damaged by ionizing radiation. We show that subthreshold direct current stimulation (DCS) rapidly restores pluripotent stem cell populations previously eliminated by lethally γ-irradiated tissues of the planarian flatworm Schmidtea mediterranea. Our findings reveal that DCS enhances DNA repair, transcriptional activity, and cell cycle entry in post-mitotic cells. These responses involve rapid increases in cytosolic Ca2+ concentration through the activation of L-type Cav channels and intracellular Ca2+ stores, leading to the activation of immediate early genes and ectopic expression of stem cell markers in post-mitotic cells. Overall, we show the potential of electric current stimulation to reverse the damaging effects of high-dose γ radiation in adult tissues. Furthermore, our results provide mechanistic insights describing how electric stimulation effectively translates into molecular responses capable of regulating fundamental cellular functions without the need for genetic or pharmacological intervention.

Direct Current Electric Stimulation Alters the Frequency and the Distribution of Mitotic Cells in Planarians.

Background: The use of direct current electric stimulation (DCS) is an effective strategy to treat disease and enhance body functionality. Thus, treatment with DCS is an attractive biomedical alternative, but the molecular underpinnings remain mostly unknown. The lack of experimental models to dissect the effects of DCS from molecular to organismal levels is an important caveat. Here, we introduce the planarian flatworm Schmidtea mediterranea as a tractable organism for in vivo studies of DCS. We developed an experimental method that facilitates the application of direct current electrical stimulation to the whole planarian body (pDCS). Materials and Methods: Planarian immobilization was achieved by combining treatment with anesthesia, agar embedding, and low temperature via a dedicated thermoelectric cooling unit. Electric currents for pDCS were delivered using pulled glass microelectrodes. The electric potential was supplied through a constant voltage power supply. pDCS was administered up to six hours, and behavioral and molecular effects were measured by using video recordings, immunohistochemistry, and gene expression analysis. Results: The behavioral immobilization effects are reversible, and pDCS resulted in a redistribution of mitotic cells along the mediolateral axis of the planarian body. The pDCS effects were dependent on the polarity of the electric field, which led to either increase in reductions in mitotic densities associated with the time of pDCS. The changes in mitotic cells were consistent with apparent redistribution in gene expression of the stem cell marker smedwi-1. Conclusion: The immobilization technique presented in this work facilitates studies aimed at dissecting the effects of exogenous electric stimulation in the adult body. Treatment with DCS can be administered for varying times, and the consequences evaluated at different levels, including animal behavior, cellular and transcriptional changes. Indeed, treatment with pDCS can alter cellular and transcriptional parameters depending on the polarity of the electric field and duration of the exposure.

Can neural signals override cellular decisions in the presence of DNA damage?

Cells within an organism are in constant crosstalk with their surrounding environment. Short and long-range signals influence cellular behavior associated with division, differentiation, and death. This crosstalk among cells underlies tissue renewal to guarantee faithful replacement of old or damaged cells over many years. Renewing tissues also offer recurrent opportunities for DNA damage and cellular transformation that tend to occur with aging. Most cells with extensive DNA damage have limited options such as halting cell cycle to repair DNA, undergo senescence, or programmed cell death. However, in some cases cells carrying toxic forms of DNA damage survive and proliferate. The underlying factors driving survival and proliferation of cells with DNA damage remain unknown. Here we discuss potential roles the nervous system may play in influencing the fate of cells with DNA damage. We present a brief survey highlighting the implications the nervous system has in regeneration, regulation of stem cells, modulation of the immune system, and its contribution to cancer progression. Finally, we propose the use of planarian flatworms as a convenient model organism to molecularly dissect the influence of neural signals over cellular fate regulation in the presence of DNA damage.

Whole planarian chromosome squash.

Whole planarian chromosome squash allows researchers to qualitatively analyze chromosome integrity. Treatment with colchicine is used to halt dividing cells within metaphase and does not require amputation or tissue puncturing. In combination with acetic-orcein, a stain-fixative for chromosomes, this strategy is suitable for animals with friable tissues caused by drug treatment, radiation, and RNA interference phenotypes. The whole planarian squash method presented here is a minimally invasive procedure that facilitates simultaneous analysis of chromosomal integrity in control and experimental animals. For complete details on the use and execution of this protocol, please refer to Peiris et al. (2016).

Measuring protein levels in planarians using western blotting.

In the planarian field, two techniques are mostly used for protein detection: immunohistochemistry (IHC) and western blotting. While IHC is great for visualizing the spatial distribution of proteins in whole organisms, it has limitations in antibody availability and issues related to nonspecific expression. The use of western blotting can circumvent nonspecific expression, providing a dependable way to quantify proteins of interest. Here, we present a standardized, easily reproducible protocol with details on protein extractions of whole planarians and western blotting. For complete details on the use and execution of this protocol, please refer to Ziman et al. (2020a).

TRAF-like Proteins Regulate Cellular Survival in the Planarian Schmidtea mediterranea.

Tissue homeostasis relies on the timely renewal of cells that have been damaged or have surpassed their biological age. Nonetheless, the underlying molecular mechanism coordinating tissue renewal is unknown. The planarian Schmidtea mediterranea harbors a large population of stem cells that continuously divide to support the restoration of tissues throughout the body. Here, we identify that TNF Receptor Associated Factors (TRAFs) play critical roles in cellular survival during tissue repair in S. mediterranea. Disruption with RNA-interference of TRAF signaling results in rapid morphological defects and lethality within 2 weeks. The TRAF phenotype is accompanied by an increased number of mitoses and cell death. Our results also reveal TRAF signaling is required for proper regeneration of the nervous system. Taken together, we find functional conservation of TRAF-like proteins in S. mediterranea as they act as crucial regulators of cellular survival during tissue homeostasis and regeneration.

Sirtuin-1 regulates organismal growth by altering feeding behavior and intestinal morphology in planarians.

Nutrient availability upon feeding leads to an increase in body size in the planarian Schmidtea mediterranea However, it remains unclear how food consumption integrates with cell division at the organismal level. Here, we show that the NAD-dependent protein deacetylases sirtuins are evolutionarily conserved in planarians, and specifically demonstrate that the homolog of human sirtuin-1 (SIRT1) (encoded by Smed-Sirt-1), regulates organismal growth by impairing both feeding behavior and intestinal morphology. Disruption of Smed-Sirt-1 with RNAi or pharmacological inhibition of Sirtuin-1 leads to reduced animal growth. Conversely, enhancement of Sirtuin-1 activity with resveratrol accelerates growth. Differences in growth rates were associated with changes in the amount of time taken to locate food and overall food consumption. Furthermore, Smed-Sirt-1(RNAi) animals displayed reduced cell death and increased stem cell proliferation accompanied by impaired expression of intestinal lineage progenitors and reduced branching of the gut. Taken together, our findings indicate that Sirtuin-1 is a crucial metabolic hub capable of controlling animal behavior, tissue renewal and morphogenesis of the adult intestine.

Poly(ADP-Ribose) Polymerase-3 Regulates Regeneration in Planarians.

Protein ADP-ribosylation is a reversible post-translational modification (PTM) process that plays fundamental roles in cell signaling. The covalent attachment of ADP ribose polymers is executed by PAR polymerases (PARP) and it is essential for chromatin organization, DNA repair, cell cycle, transcription, and replication, among other critical cellular events. The process of PARylation or polyADP-ribosylation is dynamic and takes place across many tissues undergoing renewal and repair, but the molecular mechanisms regulating this PTM remain mostly unknown. Here, we introduce the use of the planarian Schmidtea mediterranea as a tractable model to study PARylation in the complexity of the adult body that is under constant renewal and is capable of regenerating damaged tissues. We identified the evolutionary conservation of PARP signaling that is expressed in planarian stem cells and differentiated tissues. We also demonstrate that Smed-PARP-3 homolog is required for proper regeneration of tissues in the anterior region of the animal. Furthermore, our results demonstrate, Smed-PARP-3(RNAi) disrupts the timely location of injury-induced cell death near the anterior facing wounds and also affects the regeneration of the central nervous system. Our work reveals novel roles for PARylation in large-scale regeneration and provides a simplified platform to investigate PARP signaling in the complexity of the adult body.

Epithelial Infection With Candida albicans Elicits a Multi-System Response in Planarians.

Candida albicans is one of the most common fungal pathogens of humans. Prior work introduced the planarian Schmidtea mediterranea as a new model system to study the host response to fungal infection at the organismal level. In the current study, we analyzed host-pathogen changes that occurred in situ during early infection with C. albicans. We found that the transcription factor Bcr1 and its downstream adhesin Als3 are required for C. albicans to adhere to and colonize the planarian epithelial surface, and that adherence of C. albicans triggers a multi-system host response that is mediated by the Dectin signaling pathway. This infection response is characterized by two peaks of stem cell divisions and transcriptional changes in differentiated tissues including the nervous and the excretory systems. This response bears some resemblance to a wound-like response to physical injury; however, it takes place without visible tissue damage and it engages a distinct set of progenitor cells. Overall, we identified two C. albicans proteins that mediate epithelial infection of planarians and a comprehensive host response facilitated by diverse tissues to effectively clear the infection.

DNA damage and tissue repair: What we can learn from planaria.

Faithful renewal of aging and damaged tissues is central to organismal lifespan. Stem cells (SCs) generate the cellular progeny that replenish adult tissues across the body but this task becomes increasingly compromised over time. The age related decline in SC-mediated tissue maintenance is a multifactorial event that commonly affects genome integrity. The presence of DNA damage in SCs that are under continuous demand to divide poses a great risk for age-related disorders such as cancer. However, performing analysis of SCs with genomic instability and the DNA damage response during tissue renewal present significant challenges. Here we introduce an alternative experimental system based on the planaria flatworm Schmidtea mediterranea to address at the organismal level studies intersecting SC-mediated tissue renewal in the presence of genomic instability. Planaria have abundant SCs (neoblasts) that maintain high rates of cellular turnover and a variety of molecular tools have been developed to induce DNA damage and dissect how neoblasts respond to this stressor. S. mediterranea displays high evolutionary conservation of DNA repair mechanisms and signaling pathways regulating adult SCs. We describe genetically induced-DNA damage models and highlight body-wide signals affecting cellular decisions such as survival, proliferation, and death in the presence of genomic instability. We also discuss transcriptomic changes in the DNA damage response during injury repair and propose DNA repair as key component of tissue regeneration. Additional studies using planaria will provide insights about mechanisms regulating survival and growth of cells with DNA damage during tissue renewal and regeneration.

The planarian Schmidtea mediterranea is a new model to study host-pathogen interactions during fungal infections.

Candida albicans is one of the most common fungal pathogens of humans. Currently, there are limitations in the evaluation of C. albicans infection in existing animal models, especially in terms of understanding the influence of specific infectious stages of the fungal pathogen on the host. We show that C. albicans infects, grows and invades tissues in the planarian flatworm Schmidtea mediterranea, and that the planarian responds to infection by activating components of the host innate immune system to clear and repair host tissues. We study different stages of C. albicans infection and demonstrate that planarian stem cells increase division in response to fungal infection, a process that is likely evolutionarily conserved in metazoans. Our results implicate MORN2 and TAK1/p38 signaling pathways as possible mediators of the host innate immune response to fungal infection. We propose the use of planarians as a model system to investigate host-pathogen interactions during fungal infections.

SUMOylation controls stem cell proliferation and regional cell death through Hedgehog signaling in planarians.

Mechanisms underlying anteroposterior body axis differences during adult tissue maintenance and regeneration are poorly understood. Here, we identify that post-translational modifications through the SUMO (Small Ubiquitin-like Modifier) machinery are evolutionarily conserved in the Lophotrocozoan Schmidtea mediterranea. Disruption of SUMOylation in adult animals by RNA-interference of the only SUMO E2 conjugating enzyme Ubc9 leads to a systemic increase in DNA damage and a remarkable regional defect characterized by increased cell death and loss of the posterior half of the body. We identified that Ubc9 is mainly expressed in planarian stem cells (neoblasts) but it is also transcribed in differentiated cells including neurons. Regeneration in Ubc9(RNAi) animals is impaired and associated with low neoblast proliferation. We present evidence indicating that Ubc9-induced regional cell death is preceded by alterations in transcription and spatial expression of repressors and activators of the Hedgehog signaling pathway. Our results demonstrate that SUMOylation acts as a regional-specific cue to regulate cell fate during tissue renewal and regeneration.

Essential role for the planarian intestinal GATA transcription factor in stem cells and regeneration.

The cellular turnover of adult tissues and injury-induced repair proceed through an exquisite integration of proliferation, differentiation, and survival signals that involve stem/progenitor cell populations, their progeny, and differentiated tissues. GATA factors are DNA binding proteins that control stem cells and the development of tissues by activating or repressing transcription. Here we examined the role of GATA transcription factors in Schmidtea mediterranea, a freshwater planarian that provides an excellent model to investigate gene function in adult stem cells, regeneration, and differentiation. Smed-gata4/5/6, the homolog of the three mammalian GATA-4,-5,-6 factors is expressed at high levels in differentiated gut cells but also at lower levels in neoblast populations, the planarian stem cells. Smed-gata4/5/6 knock-down results in broad differentiation defects, especially in response to injury. These defects are not restricted to the intestinal lineage. In particular, at late time points during the response to injury, loss of Smed-gata4/5/6 leads to decreased neoblast proliferation and to gene expression changes in several neoblast subpopulations. Thus, Smed-gata4/5/6 plays a key evolutionary conserved role in intestinal differentiation in planarians. These data further support a model in which defects in the intestinal lineage can indirectly affect other differentiation pathways in planarians.

Alternative flow cytometry strategies to analyze stem cells and cell death in planarians.

Planarians possess remarkable stem cell populations that continuously support cellular turnover and are instrumental in the regeneration of tissues upon injury. Cellular turnover and tissue regeneration in planarians rely on the proper integration of local and systemic signals that regulate cell proliferation and cell death. Thus, understanding the signals controlling cellular proliferation and cell death in planarians could provide valuable insights for maintenance of adult body homeostasis and the biology of regeneration. Flow cytometry techniques have been utilized widely to identify, isolate, and characterize planarian stem cell populations. We developed alternative flow cytometry strategies that reduce the number of reagents and the time of sample preparation to analyze stem cells and cell death in planarians. The sensitivity of these methods is validated with functional studies using RNA interference and treatment with  Î³ irradiation or stressful conditions that are known to trigger cell death. Altogether, we provide a community resource intended to minimize adverse effects during ex vivo studies of stem cells and cell death in planarians.

The Akt signaling pathway is required for tissue maintenance and regeneration in planarians.

BACKGROUND: Akt (PKB) is a serine threonine protein kinase downstream of the phosphoinositide 3-kinase (PI3K) pathway. In mammals, Akt is ubiquitously expressed and is associated with regulation of cellular proliferation, metabolism, cell growth and cell death. Akt has been widely studied for its central role in physiology and disease, in particular cancer where it has become an attractive pharmacological target. However, the mechanisms by which Akt signaling regulates stem cell behavior in the complexity of the whole body are poorly understood. Planarians are flatworms with large populations of stem cells capable of dividing to support adult tissue renewal and regeneration. The planarian ortholog Smed-Akt is molecularly conserved providing unique opportunities to analyze the function of Akt during cellular turnover and repair of adult tissues. RESULTS: Our findings abrogating Smed-Akt with RNA-interference in the planarian Schmidtea mediterranea led to a gradual decrease in stem cell (neoblasts) numbers. The reduced neoblast numbers largely affected the maintenance of adult tissues including the nervous and excretory systems and ciliated structures in the ventral epithelia, which impaired planarian locomotion. Downregulation of Smed-Akt function also resulted in an increase of cell death throughout the animal. However, in response to amputation, levels of cell death were decreased and failed to localize near the injury site. Interestingly, the neoblast mitotic response was increased around the amputation area but the regenerative blastema failed to form. CONCLUSIONS: We demonstrate Akt signaling is essential for organismal physiology and in late stages of the Akt phenotype the reduction in neoblast numbers may impair regeneration in planarians. Functional disruption of Smed-Akt alters the balance between cell proliferation and cell death leading to systemic impairment of adult tissue renewal. Our results also reveal novel roles for Akt signaling during regeneration, specifically for the timely localization of cell death near the injury site. Thus, Akt signaling regulates neoblast biology and mediates in the distribution of injury-mediated cell death during tissue repair in planarians.