Quantitative analysis of cell types during growth,degrowth and regeneration in the planarians Dugesia mediterranea and Dugesia tigrina

A method of tissue maceration (dissociation) of planarian tissues into single cells was used to characterize the basic cell types in the planarians Dugesia mediterranea and Dugesia tigrina, and to determine the total cell number and distribution of cell types during growth, degrowth and regeneration.Using this method, 13 basic cell types have been determined for both species. The total number of cells increases with body length and volume whereas the distribution of cell types is only slightly affected. Growth and degrowth occur mainly through changes in total cell number leaving cell distribution only moderately affected. During regeneration, an increase in neoblast density in the blastema followed later on by increases in nerve cells are the more significant changes detected.These results are discussed in relation to mechanisms of cell renewal, blastema formation and maintenance of tissue polarity.Abbreviations nb neoblasts - nv nerve cells - ep epidermal cells - fp fixed parenchyma cells - g gastrodermal cells     

Planarian regeneration involves distinct stem cell responses to wounds and tissue absence

Regeneration requires signaling from a wound site for detection of the wound and a mechanism that determines the nature of the injury to specify the appropriate regenerative response. Wound signals and tissue responses to wounds that elicit regeneration remain poorly understood. Planarians are able to regenerate from essentially any type of injury and present a novel system for the study of wound responses in regeneration initiation. Newly developed molecular and cellular tools now enable study of regeneration initiation using the planarian Schmidtea mediterranea. Planarian regeneration requires adult stem cells called neoblasts and amputation triggers two peaks in neoblast mitoses early in regeneration. We demonstrate that the first mitotic peak is a body-wide response to any injury and that a second, local, neoblast response is induced only when injury results in missing tissue. This second response was characterized by recruitment of neoblasts to wounds, even in areas that lack neoblasts in the intact animal. Subsequently, these neoblasts were induced to divide and differentiate near the wound, leading to formation of new tissue. We conclude that there exist two functionally distinct signaling phases of the stem cell wound response that distinguish between simple injury and situations that require the regeneration of missing tissue.     

Recent identification of an ERK signal gradient governing planarian regeneration

Planarians have strong regenerative abilities derived from their adult pluripotent stem cell (neoblast) system. However, the molecular mechanisms involved in planarian regeneration have long remained a mystery. In particular, no anterior-specifying factor(s) could be found, although Wnt family proteins had been successfully identified as posterior-specifying factors during planarian regeneration (Gurley et al., 2008, Petersen and Reddien, 2008). A recent textbook of developmental biology therefore proposes a Wnt antagonist as a putative anterior factor (Gilbert, 2013). That is, planarian regeneration was supposed to be explained by a single decreasing gradient of the β-catenin signal from tail to head. However, recently we succeeded in demonstrating that in fact the extracellular-signal regulated kinases (ERK) form a decreasing gradient from head to tail to direct the reorganization of planarian body regionality after amputation (Umesono et al., 2013).     

Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria

Planarians have been a classic model system for the study of regeneration, tissue homeostasis, and stem cell biology for over a century, but they have not historically been accessible to extensive genetic manipulation. Here we utilize RNA-mediated genetic interference (RNAi) to introduce large-scale gene inhibition studies to the classic planarian system. 1065 genes were screened. Phenotypes associated with the RNAi of 240 genes identify many specific defects in the process of regeneration and define the major categories of defects planarians display following gene perturbations. We assessed the effects of inhibiting genes with RNAi on tissue homeostasis in intact animals and stem cell (neoblast) proliferation in amputated animals identifying candidate stem cell, regeneration, and homeostasis regulators. Our study demonstrates the great potential of RNAi for the systematic exploration of gene function in understudied organisms and establishes planarians as a powerful model for the molecular genetic study of stem cells, regeneration, and tissue homeostasis.     

Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea

In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post-amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians.     

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities¹. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a ''trunk'' fragment is cut from an intact worm, a new ''head'' will regenerate anteriorly and a ''tail'' will regenerate posteriorly restoring the original ''head-to-tail'' polarity of body structures prior to amputation (Figure 1A).Regeneration is driven by planarian stem cells, known as ''neoblasts'' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity^2-9.The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters¹⁰, represent an important system for studying evolution of the central nervous system^{11, 12} and have behavioral screening potential^{13, 14.} Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs^{8, 9, 15}. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals^16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.     

Role of c-Jun N-terminal kinase activation in blastema formation during planarian regeneration

The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts, which are the only mitotic somatic cells in adult planarians and are responsible for blastema formation after amputation. Little is known about the molecular mechanisms that drive blastema formation during planarian regeneration. Here we found that treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 blocked the entry of neoblasts into the M-phase of the cell cycle, while allowing neoblasts to successfully enter S-phase in the planarian Dugesia japonica. The rapid and efficient blockage of neoblast mitosis by treatment with the JNK inhibitor provided a method to assess whether temporally regulated cell cycle activation drives blastema formation during planarian regeneration. In the early phase of blastema formation, activated JNK was detected prominently in a mitotic region (the "postblastema") proximal to the blastema region. Furthermore, we demonstrated that undifferentiated mitotic neoblasts in the postblastema showed highly activated JNK at the single cell level. JNK inhibition by treatment with SP600125 during this period caused a severe defect of blastema formation, which accorded with a drastic decrease of mitotic neoblasts in regenerating animals. By contrast, these animals still retained many undifferentiated neoblasts near the amputation stump. These findings suggest that JNK signaling plays a crucial role in feeding into the blastema neoblasts for differentiation by regulating the G2/M transition in the cell cycle during planarian regeneration.     

Deep sequencing identifies new and regulated microRNAs in Schmidtea mediterranea

MicroRNAs (miRNAs) play important roles in directing the differentiation of cells down a variety of cell lineage pathways. The planarian Schmidtea mediterranea can regenerate all lost body tissue after amputation due to a population of pluripotent somatic stem cells called neoblasts, and is therefore an excellent model organism to study the roles of miRNAs in stem cell function. Here, we use a combination of deep sequencing and bioinformatics to discover 66 new miRNAs in S. mediterranea. We also identify 21 miRNAs that are specifically expressed in either sexual or asexual animals. Finally, we identified five miRNAs whose expression is sensitive to γ-irradiation, suggesting they are expressed in neoblasts or early neoblast progeny. Together, these results increase the known repertoire of S. mediterranea miRNAs and identify numerous regulated miRNAs that may play important roles in regeneration, homeostasis, neoblast function, and reproduction.     

A mortalin-like gene is crucial for planarian stem cell viability

In adult organisms, stem cells are crucial to homeostasis and regeneration of damaged tissues. In planarians, adult stem cells (neoblasts) are endowed with an extraordinary replicative potential that guarantees unlimited replacement of all differentiated cell types and extraordinary regenerative ability. The molecular mechanisms by which neoblasts combine long-term stability and constant proliferative activity, overcoming the impact of time, remain by far unknown. Here we investigate the role of Djmot, a planarian orthologue that encodes a peculiar member of the HSP70 family, named Mortalin, on the dynamics of stem cells of Dugesia japonica. Planarian stem cells and progenitors constitutively express Djmot. Transient Djmot expression in differentiated tissues is only observed after X-ray irradiation. DjmotRNA interference causes inability to regenerate and death of the animals, as a result of permanent growth arrest of stem cells. These results provide the first evidence that an hsp-related gene is essential for neoblast viability and suggest the possibility that high levels of Djmot serve to keep a p53-like protein signaling under control, thus allowing neoblasts to escape cell death programs. Further studies are needed to unravel the molecular pathways involved in these processes.     

JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH₂–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal.     

Stem cell-based growth, regeneration, and remodeling of the planarian intestine

Although some animals are capable of regenerating organs, the mechanisms by which this is achieved are poorly understood. In planarians, pluripotent somatic stem cells called neoblasts supply new cells for growth, replenish tissues in response to cellular turnover, and regenerate tissues after injury. For most tissues and organs, however, the spatiotemporal dynamics of stem cell differentiation and the fate of tissue that existed prior to injury have not been characterized systematically. Utilizing in vivo imaging and bromodeoxyuridine pulse-chase experiments, we have analyzed growth and regeneration of the planarian intestine, the organ responsible for digestion and nutrient distribution. During growth, we observe that new gut branches are added along the entire anteroposterior axis. We find that new enterocytes differentiate throughout the intestine rather than in specific growth zones, suggesting that branching morphogenesis is achieved primarily by remodeling of differentiated intestinal tissues. During regeneration, we also demonstrate a previously unappreciated degree of intestinal remodeling, in which pre-existing posterior gut tissue contributes extensively to the newly formed anterior gut, and vice versa. By contrast to growing animals, differentiation of new intestinal cells occurs at preferential locations, including within newly generated tissue (the blastema), and along pre-existing intestinal branches undergoing remodeling. Our results indicate that growth and regeneration of the planarian intestine are achieved by co-ordinated differentiation of stem cells and the remodeling of pre-existing tissues. Elucidation of the mechanisms by which these processes are integrated will be critical for understanding organogenesis in a post-embryonic context.     

Neoblast-enriched fraction rescues eye formation in eye-defective planarian 'menashi' Dugesia ryukyuensis

Planarians are well known for their remarkable regenerative capacity. This capacity to regenerate is thought to be due to the presence of totipotent somatic stem cells known as ‘neoblasts’, which have particular morphological characteristics. The totipotency of neoblasts was supported by Baguñà's experiment, which involved the introduction of donor cells into irradiated hosts. However, since Baguñà's experiment did not include the use of a phenotypic marker, the donor cells could not be traced. In the current study, a genetic mutant planarian, menashi, an eye‐defective mutant that lacks the pigmented area in the eyes, was established. This planarian is excellent for tracing the fate of cells after their introduction into irradiated hosts. To investigate the differentiation potency more directly, a neoblast‐rich fraction obtained from normal worms was transplanted into an X‐ray‐irradiated menashi strain. Planarians that survive X‐ray irradiation were developed, and we observed the pigment of the area in the eyes of the regenerating planarians. This result suggests that the neoblast‐rich fraction contains cells that can proliferate and differentiate. These cells can replace the cells and structures lost by X‐ray irradiation and ablation, and they can also differentiate into eye pigment cells.     

Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea

Tissue regeneration is a complex process that involves a mosaic of molecules that vary spatially and temporally. Insights into the chemical signaling underlying this process can be achieved with a multiplex and untargeted chemical imaging method such as mass spectrometry imaging (MSI), which can enable de novo studies of nervous system regeneration. A combination of MSI and multivariate statistics was used to differentiate peptide dynamics in the freshwater planarian flatworm Schmidtea mediterranea at different time points during cephalic ganglia regeneration. A protocol was developed to make S. mediterranea tissues amenable for MSI. MS ion images of planarian tissue sections allow changes in peptides and unknown compounds to be followed as a function of cephalic ganglia regeneration. In conjunction with fluorescence imaging, our results suggest that even though the cephalic ganglia structure is visible after 6 days of regeneration, the original chemical composition of these regenerated structures is regained only after 12 days. Differences were observed in many peptides, such as those derived from secreted peptide 4 and EYE53-1. Peptidomic analysis further identified multiple peptides from various known prohormones, histone proteins, and DNA- and RNA-binding proteins as being associated with the regeneration process. Mass spectrometry data also facilitated the identification of a new prohormone, which we have named secreted peptide prohormone 20 (SPP-20), and is up-regulated during regeneration in planarians.     

Identification and characterization of a fibroblast growth factor gene in the planarian Dugesia japonica

Planarians belong to the phylum Platyhelminthes and can regenerate their missing body parts after injury via activation of somatic pluripotent stem cells called neoblasts. Previous studies suggested that fibroblast growth factor (FGF) signaling plays a crucial role in the regulation of head tissue differentiation during planarian regeneration. To date, however, no FGF homologues in the Platyhelminthes have been reported. Here, we used a planarian Dugesia japonica model and identified an fgf gene termed Djfgf, which encodes a putative secreted protein with a core FGF domain characteristic of the FGF8/17/18 subfamily in bilaterians. Using Xenopus embryos, we found that DjFGF has FGF activity as assayed by Xbra induction. We next examined Djfgf expression in non-regenerating intact and regenerating planarians. In intact planarians, Djfgf was expressed in the auricles in the head and the pharynx. In the early process of regeneration, Djfgf was transiently expressed in a subset of differentiated cells around wounds. Notably, Djfgf expression was highly induced in the process of head regeneration when compared to that in the tail regeneration. Furthermore, assays of head regeneration from tail fragments revealed that combinatorial actions of the anterior extracellular signal-regulated kinase (ERK) and posterior Wnt/ß-catenin signaling restricted Djfgf expression to a certain anterior body part. This is the region where neoblasts undergo active proliferation to give rise to their differentiating progeny in response to wounding. The data suggest the possibility that DjFGF may act as an anterior counterpart of posteriorly localized Wnt molecules and trigger neoblast responses involved in planarian head regeneration.     

Expression and functional analysis of flotillins in Dugesia japonica

FLOTILLIN-1 and FLOTILLIN-2 are membrane rafts associated proteins that have been implicated in insulin and growth factor signaling, endocytosis, cell migration, proliferation, differentiation, cytoskeleton remodeling and membrane trafficking. Furthermore, FLOTILLINs also play important roles in the progression of cancer and neurodegenerative diseases. In this study, the roles of flotillins are investigated in planarian Dugesia japonica. The results show that Djflotillin-1 and Djflotillin-2 play a key role in homeostasis maintenance and regeneration process by regulating the proliferation of the neoblast cells, they are not involved in the maintenance and regeneration of the central nervous system in planarians.