Ets-1 transcription factor regulates glial cell regeneration and function in planarians.

Glia play multifaceted roles in nervous systems in response to injury. Depending on the species, extent of injury and glial cell type in question, glia can help or hinder the regeneration of neurons. Studying glia in the context of successful regeneration could reveal features of pro-regenerative glia that could be exploited for new human therapies. Planarian flatworms completely regenerate their nervous systems after injury - including glia - and thus provide a strong model system for exploring glia in the context of regeneration. Here, we report that planarian glia regenerate after neurons, and that neurons are required for correct glial numbers and localization during regeneration. We also identify the planarian transcription factor-encoding gene ets-1 as a key regulator of glial cell maintenance and regeneration. Using ets-1 (RNAi) to perturb glia, we show that glial loss is associated with altered neuronal gene expression, impeded animal movement and impaired nervous system architecture - particularly within the neuropil. Importantly, our work reveals the inter-relationships of glia and neurons in the context of robust neural regeneration.

Colorimetric Whole-Mount In Situ Hybridization in Planarians.

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.

WTAP regulates stem cells via TRAF6 to maintain planarian homeostasis and regeneration.

WTAP, a highly conserved Wilms' tumor 1 interacting protein, is involved in a variety of biological processes. However, functional studies of WTAP in planarians have not been reported. In this study, we examined the spatiotemporal expression pattern of planarian DjWTAP and investigated its functions in planarians regeneration and homeostasis. Knocking-down DjWTAP resulted in severe morphological defects leading to lethality within 20 days. Silencing DjWTAP promoted the proliferation of PiwiA+ cells but impaired the lineage differentiation of epidermal, neural, digestive, and excretory cell types, suggesting a critical role for DjWTAP in stem cell self-renewal and differentiation in planarian. To further investigate the mechanisms underlying the defective differentiation, RNA-seq was employed to determine the transcriptomic alterations upon DjWTAP RNA interference. Histone 4 (H4), Histone-lysine N-methyltransferase-SETMAR like, and TNF receptor-associated factor 6 (TRAF6), were significantly upregulated in response to DjWTAP RNAi. Knocking-down TRAF6 largely rescued the defective tissue homeostasis and regeneration resulted from DjWTAP knockdown in planarians, suggesting that DjWTAP maintains planarian regeneration and homeostasis via TRAF6.

Inhibition of ATM kinase rescues planarian regeneration after lethal radiation.

As stem cells divide, they acquire mutations that can be passed on to daughter cells. To mitigate potentially deleterious outcomes, cells activate the DNA damage response (DDR) network, which governs several cellular outcomes following DNA damage, including repairing DNA or undergoing apoptosis. At the helm of the DDR are three PI3-like kinases including Ataxia-Telangiectasia Mutated (ATM). We report here that knockdown of ATM in planarian flatworms enables stem cells to withstand lethal doses of radiation which would otherwise induce cell death. In this context, stem cells circumvent apoptosis, replicate their DNA, and recover function using homologous recombination-mediated DNA repair. Despite radiation exposure, atm knockdown animals survive long-term and regenerate new tissues. These effects occur independently of ATM's canonical downstream effector p53. Together, our results demonstrate that in planarians, ATM promotes radiation-induced apoptosis. This acute, ATM-dependent apoptosis is a key determinant of long-term animal survival. Our results suggest that inhibition of ATM in these organisms could, therefore, potentially favor cell survival after radiation without obvious effects on stem cell behavior.

Mitogen-like Cerium-Based Nanoparticles Protect Schmidtea mediterranea against Severe Doses of X-rays.

Novel radioprotectors are strongly demanded due to their numerous applications in radiobiology and biomedicine, e.g., for facilitating the remedy after cancer radiotherapy. Currently, cerium-containing nanomaterials are regarded as promising inorganic radioprotectors due to their unrivaled antioxidant activity based on their ability to mimic the action of natural redox enzymes like catalase and superoxide dismutase and to neutralize reactive oxygen species (ROS), which are by far the main damaging factors of ionizing radiation. The freshwater planarian flatworms are considered a promising system for testing new radioprotectors, due to the high regenerative potential of these species and an excessive amount of proliferating stem cells (neoblasts) in their bodies. Using planarian Schmidtea mediterranea, we tested CeO2 nanoparticles, well known for their antioxidant activity, along with much less studied CeF3 nanoparticles, for their radioprotective potential. In addition, both CeO2 and CeF3 nanoparticles improve planarian head blastema regeneration after ionizing irradiation by enhancing blastema growth, increasing the number of mitoses and neoblasts' survival, and modulating the expression of genes responsible for the proliferation and differentiation of neoblasts. The CeO2 nanoparticles' action stems directly from their redox activity as ROS scavengers, while the CeF3 nanoparticles' action is mediated by overexpression of "wound-induced genes" and neoblast- and stem cell-regulating genes.

Convergent evolution of a genotoxic stress response in a parasite-specific p53 homolog.

P53 is a widely studied tumor suppressor that plays important roles in cell-cycle regulation, cell death, and DNA damage repair. P53 is found throughout metazoans, even in invertebrates that do not develop malignancies. The prevailing theory for why these invertebrates possess a tumor suppressor is that P53 originally evolved to protect the germline of early metazoans from genotoxic stress such as ultraviolet radiation. This theory is largely based upon functional data from only three invertebrates, omitting important groups of animals including flatworms. Previous studies in the freshwater planarian flatworm Schmidtea mediterranea suggested that flatworm P53 plays an important role in stem cell maintenance and skin production, but these studies did not directly test for any tumor suppressor functions. To better understand the function of P53 homologs across diverse flatworms, we examined the function of two different P53 homologs in the parasitic flatworm Schistosoma mansoni. The first P53 homolog (p53-1) is orthologous to S. mediterranea P53(Smed-p53) and human TP53 and regulates flatworm stem cell maintenance and skin production. The second P53 homolog (p53-2) is a parasite-specific paralog that is conserved across parasitic flatworms and is required for the normal response to genotoxic stress in S. mansoni. We then found that Smed-p53 does not seem to play any role in the planarian response to genotoxic stress. The existence of this parasite-specific paralog that bears a tumor suppressor-like function in parasitic flatworms implies that the ability to respond to genotoxic stress in parasitic flatworms may have arisen from convergent evolution.

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.

Interactive toxicity of copper and cadmium in regenerating and adult planarians.

In a polluted environment, metals are present as complex mixtures. As a result, organisms are exposed to different metals at the same time, which affects both metal-specific as well as overall toxicity. Detailed information about the molecular mechanisms underlying the adverse effects of combined exposures remains limited in terms of different life stages. In this study, the freshwater planarian Schmidtea mediterranea was used to investigate developmental and physiological responses associated with a combined exposure to Cu and Cd. In addition, the cellular and molecular mechanisms underlying the provoked adverse effects were studied in different exposure scenarios. Mixed exposure resulted in a decline in survival, diverse non-lethal morphological changes, neuroregenerative impairments, altered behaviour and a limited repair capacity. Underlying to these effects, the cellular redox state was altered in all exposure conditions. In adult animals, this led to DNA damage and corresponding transcriptional changes in cell cycle and DNA repair genes. In regenerating animals, changes in hydrogen peroxide and glutathione contents led to regenerative defects. Overall, our results demonstrate that (1) developing organisms are more susceptible to metal exposures, and (2) the toxicity of an individual metal increases significantly in a mixed exposure scenario. These aspects have to be included in current risk assessment strategies.

Set1 Targets Genes with Essential Identity and Tumor-Suppressing Functions in Planarian Stem Cells.

Tumor suppressor genes (TSGs) are essential for normal cellular function in multicellular organisms, but many TSGs and tumor-suppressing mechanisms remain unknown. Planarian flatworms exhibit particularly robust tumor suppression, yet the specific mechanisms underlying this trait remain unclear. Here, we analyze histone H3 lysine 4 trimethylation (H3K4me3) signal across the planarian genome to determine if the broad H3K4me3 chromatin signature that marks essential cell identity genes and TSGs in mammalian cells is conserved in this valuable model of in vivo stem cell function. We find that this signature is indeed conserved on the planarian genome and that the lysine methyltransferase Set1 is largely responsible for creating it at both cell identity and putative TSG loci. In addition, we show that depletion of set1 in planarians induces stem cell phenotypes that suggest loss of TSG function, including hyperproliferation and an abnormal DNA damage response (DDR). Importantly, this work establishes that Set1 targets specific gene loci in planarian stem cells and marks them with a conserved chromatin signature. Moreover, our data strongly suggest that Set1 activity at these genes has important functional consequences both during normal homeostasis and in response to genotoxic stress.

Identification of novel yolk ferritins unique to planarians: planarians supply aluminum rather than iron to vitellaria in egg capsules.

All animals, other than Platyhelminthes, produce eggs containing yolk, referred to as "entolecithal" eggs. However, only Neoophora, in the phylum Platyhelminthes, produce "ectolecithal" eggs (egg capsules), in which yolk is stored in the vitelline cells surrounding oocytes. Vitelline cells are derived from vitellaria (yolk glands). Vitellaria are important reproductive organs that may be studied to elucidate unique mechanisms that have been evolutionarily conserved within Platyhelminthes. Currently, only limited molecular level information is available on vitellaria. The current study identified major vitellaria-specific proteins in a freshwater planarian, Dugesia ryukyuensis, using peptide mass fingerprinting (PMF) and expression analyses. Amino acid sequence analysis and orthology analysis via OrthoFinder ver.2.3.8 indicated that the identified major vitellaria-specific novel yolk ferritins were conserved in planarians (Tricladida). Because ferritins play an important role in Fe (iron) storage, we examined the metal elements contained in vitellaria and ectolecithal eggs, using non-heme iron histochemistry, elemental analysis based on inductively coupled plasma mass spectrometry and transmission electron microscopy- energy-dispersive X-ray spectroscopy analysis. Interestingly, vitellaria and egg capsules contained large amounts of aluminum (Al), but not Fe. The knockdown of the yolk ferritin genes caused a decrease in the volume of egg capsules, abnormality in juveniles, and increase in Al content in vitellaria. Yolk ferritins of D. ryukyuensis may regulate Al concentration in vitellaria via their pooling function of Al and protect the egg capsule production and normal embryogenesis from Al toxicity.

Regeneration in starved planarians depends on TRiC/CCT subunits modulating the unfolded protein response.

Planarians are able to stand long periods of starvation by maintaining adult stem cell pools and regenerative capacity. The molecular pathways that are needed for the maintenance of regeneration during starvation are not known. Here, we show that down-regulation of chaperonin TRiC/CCT subunits abrogates the regeneration capacity of planarians during starvation, but TRiC/CCT subunits are dispensable for regeneration in fed planarians. Under starvation, they are required to maintain mitotic fidelity and for blastema formation. We show that TRiC subunits modulate the unfolded protein response (UPR) and are required to maintain ATP levels in starved planarians. Regenerative defects in starved CCT-depleted planarians can be rescued by either chemical induction of mild endoplasmic reticulum stress, which leads to induction of the UPR, or by the supplementation of fatty acids. Together, these results indicate that CCT-dependent UPR induction promotes regeneration of planarians under food restriction.

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.

Mitochondrial state determines functionally divergent stem cell population in planaria.

Mitochondrial state changes were shown to be critical for stem cell function. However, variation in the mitochondrial content in stem cells and the implication, if any, on differentiation is poorly understood. Here, using cellular and molecular studies, we show that the planarian pluripotent stem cells (PSCs) have low mitochondrial mass compared with their progenitors. Transplantation experiments provided functional validation that neoblasts with low mitochondrial mass are the true PSCs. Further, the mitochondrial mass correlated with OxPhos and inhibiting the transition to OxPhos dependent metabolism in cultured cells resulted in higher PSCs. In summary, we show that low mitochondrial mass is a hallmark of PSCs in planaria and provide a mechanism to isolate live, functionally active, PSCs from different cell cycle stages (G0/G1 and S, G2/M). Our study demonstrates that the change in mitochondrial metabolism, a feature of PSCs is conserved in planaria and highlights its role in organismal regeneration.

Cysteinyl-specialized proresolving mediators link resolution of infectious inflammation and tissue regeneration via TRAF3 activation.

The recently elucidated proresolving conjugates in tissue regeneration (CTR) maresin-CTR (MCTR), protectin-CTR (PCTR), and resolvin-CTR (RCTR), termed cysteinyl-specialized proresolving mediators (cys-SPMs) each promotes regeneration, controls infection, and accelerates resolution of inflammation. Here, we sought evidence for cys-SPM activation of primordial pathways in planaria (Dugesia japonica) regeneration that might link resolution of inflammation and regeneration. On surgical resection, planaria regeneration was enhanced with MCTR3, PCTR3, or RCTR3 (10 nM), each used for RNA sequencing. The three cys-SPMs shared up-regulation of 175 known transcripts with fold-change > 1.25 and combined false discovery rate (FDR) < 0.002, and 199 canonical pathways (FDR < 0.25), including NF-κB pathways and an ortholog of human TRAF3 (TNFR-associated factor 3). Three separate pathway analyses converged on TRAF3 up-regulation by cys-SPMs. With human macrophages, three cys-SPMs each dose-dependently increased TRAF3 expression in a cAMP-PKA-dependent manner. TRAF3 overexpression in macrophages enhanced Interleukin-10 (IL-10) and phagocytosis of Escherichia coli IL-10 also increased phagocytosis in a dose-dependent manner. Silencing of mouse TRAF3 in vivo significantly reduced IL-10 and macrophage phagocytosis. TRAF3 silencing in vivo also relieved cys-SPMs' actions in limiting polymorphonuclear neutrophil in E. coli exudates. These results identify cys-SPM-regulated pathways in planaria regeneration, uncovering a role for TRAF3/IL-10 in regulating mammalian phagocyte functions in resolution. Cys-SPM activation of TRAF3 signaling is a molecular component of both regeneration and resolution of infectious inflammation.

CREB-binding protein (CBP) gene family regulates planarian survival and stem cell differentiation.

In developmental biology, the regulation of stem cell plasticity and differentiation remains an open question. CBP(CREB-binding protein)/p300 is a conserved gene family that functions as a transcriptional co-activator and plays important roles in a wide range of cellular processes, including cell death, the DNA damage response, and tumorigenesis. The acetyl transferase activity of CBPs is particularly important, as histone and non-histone acetylation results in changes in chromatin architecture and protein activity that affect gene expression. Many studies have described the conserved functions of CBP/p300 in stem cell proliferation and differentiation. The planarian Schmidtea mediterranea is an excellent model for the in vivo study of the molecular mechanisms underlying stem cell differentiation during regeneration. However, how this process is regulated genetically and epigenetically is not well-understood yet. We identified 5 distinct Smed-cbp genes in S. mediterranea that show different expression patterns. Functional analyses revealed that Smed-cbp-2 appears to be essential for stem cell maintenance. On the other hand, the silencing of Smed-cbp-3 resulted in the growth of blastemas that were apparently normal, but remained largely unpigmented and undifferentiated. Smed-cbp-3 silencing also affected the differentiation of several cell lineages including neural, epidermal, digestive, and excretory cell types. Finally, we analysed the predicted interactomes of CBP-2 and CBP-3 as an initial step to better understand their functions in planarian stem cell biology. Our results indicate that planarian cbp genes play key roles in stem cell maintenance and differentiation.

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.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Dynein assembly factor with WD repeat domains 1 (DAW1) is required for the function of motile cilia in the planarian Schmidtea mediterranea.

Motile cilia propel directed cell movements and sweep fluids across the surface of tissues. Orthologs of Dynein Assembly Factor with WD Repeat Domains 1 (DAW1) support normal ciliary beating by enhancing delivery of dynein complexes to axonemal microtubules. DAW1 mutations in vertebrates result in multiple developmental abnormalities and early or prenatal lethality, complicating functional assessment of DAW1 in adult structures. Planarian flatworms maintain cellular homeostasis and regenerate through differentiation of adult pluripotent stem cells, and systemic RNA-interference (RNAi) can be induced to analyze gene function at any point after birth. A single ortholog of DAW1 was identified in the genome of the planarian Schmidtea mediterranea (Smed-daw1). Smed-DAW1 is composed of eight WD repeats, which are 55% identical to the founding member of this protein family (Chlamydomonas reinhardtii ODA16) and 58% identical to human DAW1. Smed-daw1 is expressed in the planarian epidermis, protonephridial excretory system, and testes, all of which contain cells functionally dependent on motile cilia. Smed-daw1 RNAi resulted in locomotion defects and edema, which are phenotypes characteristic of multiciliated epidermis and protonephridial dysfunction, respectively. Changes in abundance or length of motile cilia were not observed at the onset of phenotypic manifestations upon Smed-daw1 RNAi, corroborating with studies showing that DAW-1 loss of function leads to aberrant movement of motile cilia in other organisms, rather than loss of cilia per se. However, extended RNAi treatments did result in shorter epidermal cilia and decreased abundance of ciliated protonephridia, suggesting that Smed-daw1 is required for homeostatic maintenance of these structures in flatworms.

Planarian cell number depends on blitzschnell, a novel gene family that balances cell proliferation and cell death.

Control of cell number is crucial to define body size during animal development and to restrict tumoral transformation. The cell number is determined by the balance between cell proliferation and cell death. Although many genes are known to regulate those processes, the molecular mechanisms underlying the relationship between cell number and body size remain poorly understood. This relationship can be better understood by studying planarians, flatworms that continuously change their body size according to nutrient availability. We identified a novel gene family, blitzschnell (bls), that consists of de novo and taxonomically restricted genes that control cell proliferation:cell death ratio. Their silencing promotes faster regeneration and increases cell number during homeostasis. Importantly, this increase in cell number leads to an increase in body size only in a nutrient-rich environment; in starved planarians, silencing results in a decrease in cell size and cell accumulation that ultimately produces overgrowths. bls expression is downregulated after feeding and is related to activity of the insulin/Akt/mTOR network, suggesting that the bls family evolved in planarians as an additional mechanism for restricting cell number in nutrient-fluctuating environments.

The regulation of rapamycin in planarian Dugesia japonica Ichikawa & Kawakatsu, 1964 regeneration according to TOR signaling pathway.

The freshwater planarian mostly lives in the upper reaches of springs and rivers. Generally, it is realized as a suitable warning indicator of environmental toxicants. The freshwater planarian Dugesia japonica has a powerful regenerative capability and can regenerate a new individual including a complete central nervous system in one week. Rapamycin is an inhibitor of mammalian TORC1 (target of rapamycin complex-1) and used in the treatment of some diseases like cancer, cardiovascular and neurological diseases. However, the roles of rapamycin in the regulation of planarian regeneration remain to be elucidated. In present study, freshwater planarians D. japonica were firstly treated with 1 µM rapamycin for 18 h exposures and the expression patterns of Djtor was analyzed by the whole-mount in situ hybridization (WISH). Our results indicated rapamycin could strongly inhibit Djtor expression in planarian D. japonica and cause asymmetric blastemas and neuronal defects in planarians. Furthermore, knockdown of Djtor gene in planarians using RNA interference resulted in the suppression of downstream autophagy genes. These findings suggested that rapamycin might regulate freshwater planarian regeneration via Djtor signaling pathway.