Neoplastic transformation in the planarian: II. Ultrastructure of malignant reticuloma

Cadmium and phorbol ester induced tumorigenesis in the planarian, Dugesia dorotocephala, develops as a cocarcinogenic process involving initiation and promotion in the progression of neoplastic disease. Treatment of intact planarians with sublethal concentrations of cadmium sulfate and 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a type of infiltrating tumor that proved to be potentially lethal. Surgical transplantation of such tumorous tissues into otherwise healthy planarians resulted in the same histopathological progression to lethality, which confirmed the metastatic nature of the neoplasia. Electron microscopic studies revealed that both the chemically-induced and the transplantation-based tumors involved, exclusively, the proliferation and differentiation of abnormal reticular cells, referred to as reticuloma cells. Reticular cells normally are ameboid, phagocytic, and are thought to provide the planarian with a phylogenetic predecessor of an immune surveillance system. A considerable incidence of mitosis was observed within the tumor areas; and the sequence of differentiation, from transformed stem cells to mature but nonfunctional reticuloma cells, was elucidated. This profile of differentiation supports the concept of cellular derivation via stem cell dynamics as opposed to dedifferentiation. A variety of ultrastructural abnormalities were characterized: several of which tend to substantiate the anaplastic quality of the reticuloma, while others are more specifically diagnostic for malignancy. These findings further extend the potential usefulness of the planarian malignant reticuloma as a model system for the study of neoplastic stem cell diseases.     

Identification of immunological reagents for use in the study of freshwater planarians by means of whole-mount immunofluorescence and confocal microscopy

In recent years, interest in planarians as a model system for the study of metazoan regeneration, adult stem cell biology, and the evolution of metazoan body plans has been growing steadily. The availability of RNA interference (RNAi), BrdU-labeling of planarian stem cells, and thousands of planarian cDNA sequences soon to be released into public databases has opened planarians to molecular dissection. However, the successful application of large-scale RNAi-based screens, for example, will depend in part on the availability of markers to characterize the resulting phenotypes. Given the paucity of antibodies available for the study of planarian biology, we have screened various public and commercial antibody resources to identify immunoreagents capable of cross-reacting with planarian tissues. Here we report the identification and characterization of 33 such antibodies recognizing a wide variety of tissues in freshwater planarians.     

Structure and function of the reticular cell in the planarian Dugesia dorotocephala

Structural and functional characteristics of the reticular cell in the planarian Dugesia dorotocephala were studied by light and electron microscopy. Since the reticular cells have numerous glycogen granules, lipid droplets and some lysosomes in their cytoplasm, they are easily distinguishable from other cell types. They migrate into the injured tissue, cover the injured mesenchyme, and also phagocytize debris of degenerating cells. The reticular cells also recognize foreign invaders such as bacteria. The larger aggregates of killed bacteria are encapsulated by reticular cells and eliminated into the intestine, whereas small aggregates are phagocytized by reticular cells. When cell wall extract of bacteria was inserted into the planarian body before insertion of killed bacteria, reticular cells were found to respond more quickly and vigorously to subsequent insertion of killed bacteria, indicating that the reticular cell has an immune response memory. When planarians were treated with 0.3 ppm cadmium sulfate and 0.01 ppm TPA, reticuloma tumors were induced in 76% of exposed planarians, indicating the similarity to blood cell diseases in mammals such as leukemia or lymphoma which are also induced by TPA. When these tumors were transplanted into normal hosts, the tumor cells were attacked by host reticular cells. These observations indicate that planarian reticular cells are primitive blood cells, playing important roles in nutrient transportation, homeostatic control of cells, and in defence and immune surveillance systems.     

Bromodeoxyuridine specifically labels the regenerative stem cells of planarians

The singular regenerative abilities of planarians require a population of stem cells known as neoblasts. In response to wounding, or during the course of cell turnover, neoblasts are signaled to divide and/or differentiate, thereby replacing lost cell types. The study of these pluripotent stem cells and their role in planarian regeneration has been severely hampered by the reported inability of planarians to incorporate exogenous DNA precursors; thus, very little is known about the mechanisms that control proliferation and differentiation of this stem cell population within the planarian. Here we show that planarians are, in fact, capable of incorporating the thymidine analogue bromodeoxyuridine (BrdU), allowing neoblasts to be labeled specifically during the S phase of the cell cycle. We have used BrdU labeling to study the distribution of neoblasts in the intact animal, as well as to directly demonstrate the migration and differentiation of neoblasts. We have examined the proposal that a subset of neoblasts is arrested in the G2 phase of the cell cycle by double-labeling with BrdU and a mitosis-specific marker; we find that the median length of G2 (approximately 6 h) is sufficient to account for the initial mitotic burst observed after feeding or amputation. Continuous BrdU-labeling experiments also suggest that there is not a large, slow-cycling population of neoblasts in the intact animal. The ability to label specifically the regenerative stem cells, combined with the recently described use of double-stranded RNA to inhibit gene expression in the planarian, should serve to reignite interest in the flatworm as an experimental model for studying the problems of metazoan regeneration and the control of stem cell proliferation.     

Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting

The remarkable capability of planarian regeneration is mediated by a group of adult stem cells referred to as neoblasts. Although these cells possess many unique cytological characteristics (e.g. they are X-ray sensitive and contain chromatoid bodies), it has been difficult to isolate them after cell dissociation. This is one of the major reasons why planarian regenerative mechanisms have remained elusive for a long time. Here, we describe a new method to isolate the planarian adult stem cells as X-ray-sensitive cell populations by fluorescence-activated cell sorting (FACS). Dissociated cells from whole planarians were labeled with fluorescent dyes prior to fractionation by FACS. We compared the FACS profiles from X-ray-irradiated and non-irradiated planarians, and thereby found two cell fractions which contained X-ray-sensitive cells. These fractions, designated X1 and X2, were subjected to electron microscopic morphological analysis. We concluded that X-ray-sensitive cells in both fractions possessed typical stem cell morphology: an ovoid shape with a large nucleus and scant cytoplasm, and chromatoid bodies in the cytoplasm. This method of isolating X-ray-sensitive cells using FACS may provide a key tool for advancing our understanding of the stem cell system in planarians.     

Autophagy meets planarians

This review aims to demonstrate the importance of freshwater planarians as model organisms, particularly emphasizing those characteristics of the animal that make them a good model to study autophagy. The aim of this review is to provide a better understanding of autophagy in this model for the non-planarian reader, and elucidate the relevance of autophagy research in this peculiar model organism. Furthermore, I will try to synthesize the evidence showing the importance of autophagy in planarian body remodelling, and I will discuss some ideas about the role of autophagy in stem cell biology. In light of these new developments, it is likely that the planarian field will make an important contribution to the study of the molecular mechanisms involved in autophagy in the future.     

Djeyes absent (Djeya) controls prototypic planarian eye regeneration by cooperating with the transcription factor Djsix-1

A conserved network of nuclear proteins is crucial to eye formation in both vertebrates and invertebrates. The finding that freshwater planarians can regenerate eyes without the contribution of Pax6 suggests that alternative combinations of regulatory elements may control the morphogenesis of the prototypic planarian eye. To further dissect the molecular events controlling eye regeneration in planarians, we investigated the role of eyes absent (Djeya) and six-1 (Djsix-1) genes in Dugesia japonica. These genes are expressed in both regenerating eyes and in differentiated photoreceptors of intact adults. Through RNAi studies, we show that Djsix-1 and Djeya are both critical for the regeneration of normal eyes in planarians and genetically cooperate in vivo to establish correct eye cell differentiation. We further demonstrate that the genetic interaction is mediated by physical interaction between the evolutionarily conserved domains of these two proteins. These data indicate that planarians use cooperatively Djsix-1 and Djeya for the proper specification of photoreceptors, implicating that the mechanism involving their evolutionarily conserved domains can be very ancient. Finally, both Djsix-1 and Djeya double-stranded RNA are substantially more effective at producing no-eye phenotypes in the second round of regeneration. This is probably due to the significant plasticity of the planarian model system, based on the presence of a stable population of totipotent stem cells, which ensure the rapid cell turnover of all differentiated cell types.     

The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration

Planarian flatworms are an exception among bilaterians in that they possess a large pool of adult stem cells that enables them to promptly regenerate any part of their body, including the brain. Although known for two centuries for their remarkable regenerative capabilities, planarians have only recently emerged as an attractive model for studying regeneration and stem cell biology. This revival is due in part to the availability of a sequenced genome and the development of new technologies, such as RNA interference and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular level. Here, we highlight why planarians are an exciting tool in the study of regeneration and its underlying stem cell biology in vivo, and discuss the potential promises and current limitations of this model organism for stem cell research and regenerative medicine.     

A Bruno-like gene is required for stem cell maintenance in planarians

The regenerative abilities of freshwater planarians are based on neoblasts, stem cells maintained throughout the animal's life. We show that a member of the Bruno-like family of RNA binding proteins is critical for regulating neoblasts in the planarian Schmidtea mediterranea. Smed-bruno-like (bruli) mRNA and protein are expressed in neoblasts and the central nervous system. Following bruli RNAi, which eliminates detectable Bruli protein, planarians initiate the proliferative response to amputation and form small blastemas but then undergo tissue regression and lysis. We characterize the neoblast population by using antibodies recognizing SMEDWI-1 and Histone H4 (monomethyl-K20) and cell-cycle markers to label subsets of neoblasts and their progeny. bruli knockdown results in a dramatic reduction/elimination of neoblasts. Our analyses indicate that neoblasts lacking Bruli can respond to wound stimuli and generate progeny that can form blastemas and differentiate; yet, they are unable to self-renew. These results suggest that Bruli is required for stem cell maintenance.     

Study of possible involvement of MEK mitogen-activated protein kinase and TGF-β receptor in planarian regeneration processes using pharmacological inhibition analysis

Possible involvement of MEK mitogen-activated protein kinase and TGF-β receptor in the processes of regeneration and morphogenesis in freshwater planarian flatworms Schmidtea mediterranea was studied using a pharmacological inhibitor analysis. It was found that pharmacological inhibitors of these kinases significantly inhibit the regeneration of the head end of the animals and that this effect is realized due to inhibition of proliferative activity of neoblasts, planarian stem cells. It is shown that that the inhibition of the studied protein kinases in regenerating planarians markedly disturbs stem cell differentiation and morphogenesis.     

Planarian fibroblast growth factor receptor homologs expressed in stem cells and cephalic ganglions

The strong regenerative capacity of planarians is considered to reside in the totipotent somatic stem cell called the 'neoblast'. However, the signal systems regulating the differentiation/growth/migration of stem cells remain unclear. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system is thought to mediate various developmental events in both vertebrates and invertebrates. We examined the molecular structures and expression of DjFGFR1 and DjFGFR2, two planarian genes closely related to other animal FGFR genes. DjFGFR1 and DjFGFR2 proteins contain three and two immunoglobulin-like domains, respectively, in the extracellular region and a split tyrosine kinase domain in the intracellular region. Expression of DjFGFR1 and DjFGFR2 was observed in the cephalic ganglion and mesenchymal space in intact planarians. In regenerating planarians, accumulation of DjFGFR1-expressing cells was observed in the blastema and in fragments regenerating either a pharynx or a brain. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjFGFR1-expressing cells in the mesenchymal space decreased markedly. These results suggest that the DjFGFR1 protein may be involved in the signal systems controlling such aspects of planarian regeneration as differentiation/growth/migration of stem cells.     

Djrho2 is involved in regeneration of visual nerves in Dugesia japonica

The freshwater planarian is a powerful animal model for studying regeneration and stem cell activity in vivo.During regeneration,stem ceils (neoblasts in planarian) migrated to the wounding edge to re-build missing parts of the body.However, proteins involved in regulating cell migration during planarian regeneration have not been studied extensively.Here we report two small GTPase genes (Djrho2 and Djrho3) of Dugesia japonica (strain Pek-1).In situ hybridization results indicated that Djrho2 was expressed throughout the body with the exception of the pharynx region while Djrho3 was specifically expressed along the gastro-vaseular system.Djrho2 was largely expressed in neoblasts since its expression was sensitive to X-ray irradiation.In Djrho2-RNAi planarians, smaller anterior blaste-mas were observed in tail fragments during regeneration.Consistently, defective regeneration of visual nerve was detected by immu-nostainning with VC-1 antibody.These results suggested that Djrho2 is required for proper anterior regeneration in planairan.In contrast,no abnormality was observed after RNAi of Djrho3.We compared protein compositions of control and Djrho2-RNAi planarians using an optimized proteomic approach.Twenty-two up-regulated and 26 de-regulated protein spots were observed in the two-dimensional elec-trophoresis gels, and 17 proteins were successfully identified by Mass Spectrometry (MS) analysis.Among them, 6 actin-binding or cy-toskeleton-related proteins were found de-expressed in Djrho2-RNAi animals, suggesting that abnormal cytoskeleton assembling and cell migration were likely reasons of defected regeneration.     

Photoresponsivity and motility in the planarian Schmidtea mediterranea vary diurnally

ABSTRACT

The planarian flatworm has become one of the leading animal model systems for studying stem cell behavior and tissue regeneration. Recent studies have shown that components of the circadian clockwork have important roles in tissue homeostasis and repair. However, it remains unknown whether planarians exhibit circadian or diurnal rhythms in physiology or behavior. Here, we developed a behavioral assay to evaluate diurnal activity in planarians based upon their well-established propensity to swim away from light (negative phototaxis). We show evidence that the planarian Schmidtea mediterranea has diurnal variability in negative phototaxis as a function of daily variation in motility. We also demonstrate that variation in planarian motility over 48 h occurs with 24-h periodicity. Our data suggest that S. mediterranea may be a useful model for studying the interplay between the circadian system and tissue regeneration.     

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.     

Two msh/msx-related genes, Djmsh1 and Djmsh2, contribute to the early blastema growth during planarian head regeneration

Regeneration in planarians is an intriguing phenomenon, based on the presence of pluripotent stem cells, known as neoblasts. Following amputation, these cells activate mitotic divisions, migrate distally and undergo differentiation, giving rise to the regeneration blastema. We have identified two msh/msx-related genes, Djmsh1 and Djmsh2, which are expressed in distinct cell populations of the planarian Dugesia japonica and activated, with different patterns, during head regeneration. We demonstrate that RNA interference of Djmsh1 or Djmsh2 generates a delay in the growth of cephalic blastema, interfering with the dynamics of mitoses during its initial formation. Our data also reveal that the activity of the two planarian msh genes is required to regulate Djbmp expression during head regeneration. This study identifies, for the first time, a functional association between muscle segment homeobox (MSH) homeoproteins and BMP signaling during stem cell-based regeneration of the planarian head and provides a functional analysis of how msh genes may regulate in vivo the regenerative response of planarian stem cells.     

The power of regeneration and the stem-cell kingdom: freshwater planarians (Platyhelminthes)

The great powers of regeneration shown by freshwater planarians, capable of regenerating a complete organism from any tiny body fragment, have attracted the interest of scientists throughout history. In 1814, Dalyell concluded that planarians could "almost be called immortal under the edge of the knife". Equally impressive is the developmental plasticity of these platyhelminthes, including continuous growth and fission (asexual reproduction) in well-fed organisms, and shrinkage (degrowth) during prolonged starvation. The source of their morphological plasticity and regenerative capability is a stable population of totipotent stem cells--"neoblasts"; this is the only cell type in the adult that has mitotic activity and differentiates into all cell types. This cellular feature is unique to planarians in the Bilateria clade. Over the last fifteen years, molecular studies have begun to reveal the role of developmental genes in regeneration, although it would be premature to propose a molecular model for planarian regeneration. Genomic and proteomic data are essential in answering some of the fundamental questions concerning this remarkable morphological plasticity. Such information should also pave the way to understanding the genetic pathways associated with metazoan somatic stem-cell regulation and pattern formation.