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.     

Phagocytic response of planarian reticular cells to heat-killed bacteria

The defence mechanism of the planarian Dugesia dorotocephala (Woodworth) against invasion of foreign material was studied by inserting heat-killed bacteria into an incision and then examining the tissues around the incision by light and electron microscopy. The incision was made behind the right eye of each planarian, and a small aggregate of heat-killed Mycobacterium tuberculosis H37Ra was inserted into it using a fine needle. Samples of tissues were collected at 2, 4, 6, 8, 10, 12, 24, and 48 h after insertion of bacteria. As early as 10 h after insertion, bacteria were found in phagosomes of reticular cells, which are mesenchymal cells similar to fixed parenchymal cells and known previously to phagocytize degenerate tissues. By 12 h, aggregates of bacteria were found encapsulated in extensions of reticular cells, and by 24 h encapsulated bacteria were found between epithelial cells of the intestinal wall. These findings indicate that foreign material can be phagocytized or encapsulated by reticular cells and expelled into the intestinal cavity; it is thus conceivable that reticular cells could act as an immune surveillance system against foreign invaders.     

Neoplastic transformation in the planarian: I. Cocarcinogenesis and histopathology

Although several investigators have reported that exposure to mammalian carcinogens induces abnormal tumorlike growths and teratogenic remodeling in planarians, there is no general agreement that these, or comparable responses in any other invertebrates, model mammalian carcinogenesis. To investigate this question, freshwater planarians of the species Dugesia dorotocephala were exposed to culture water containing an initiator and a promoter, either alone or in combination. Cadmium, a potent carcinogen, was used as an initiator in the protocol. Treatment with sublethal concentrations of cadmium sulfate produced a benign, but persistent, tumor in a small percentage of the planarians. The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester and well-known promoter, to the cadmium-containing solutions resulted in the induction of a progressive, potentially lethal, transplantable tumor in a large proportion of the treated flatworms. Light and electron microscopy revealed this particular tumor to be composed both of immature cells and of a single mature cell type: newly differentiated, but transformed, reticular cells. Further examination of the infiltrating tissue formations elucidated the profile of differentiation, from a population of mitotically active transformed stem cells through the transitional stages in the associated reticuloma. These results suggest that 1) the freshwater planarian displays the major phenomenology of mammalian cocarcinogenesis and that 2) the planarian reticuloma models several important features of a neoplastic stem cell disease.     

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.     

Insights into the histology of planarian flatworm Phagocata gracilis based on location specific,intact lipid information provided by GCIB-ToF-SIMS imaging

Planarian flatworms are known as the masters of regeneration, re-growing an entire organism from as little as 1/279th part of their body. While the proteomics of these processes has been studied extensively, the planarian lipodome remains relatively unknown. In this study we investigate the lipid profile of planarian tissue sections with imaging Time-of-Flight – Secondary-Ion-Mass-Spectrometry (ToF-SIMS). ToF-SIMS is a label-free technique capable of gathering intact, location specific lipid information on a cellular scale. Lipid identities are confirmed using LC-MS/MS. Our data shows that different organ structures within planarians have unique lipid profiles. The 22-carbon atom poly unsaturated fatty acids (PUFAs) which occur in unusually high amounts in planarians are found to be mainly located in the testes. Additionally, we observe that planarians contain various odd numbered fatty acid species, that are usually found in bacteria, localized in the reproductive and ectodermal structures of the planarian. An abundance of poorly understood ether fatty acids and ether lipids were found in unique areas in planarians as well as a new, yet unidentified class of potential lipids in planarian intestines. Identifying the location of these lipids in the planarian body provides insights into their bodily functions and, in combination with knowledge about their diet and their genome, enables drawing conclusions about planarian fatty acid processing.     

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.     

Purification,characterization and induction of a C-type lectin in the freshwater planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

Lectins are important components of the immune defense system of invertebrates. Given their important functions, numerous investigations have been carried out on the characterization and function of lectins in invertebrates. However, lectin studies with the freshwater planarian, an evolutionarily important animal, are rare. In this paper, we demonstrate agglutination of glutaraldehyde treated erythrocytes by a lectin with preference for rabbit erythrocytes. The result of hemagglutinating activity inhibition assays with several carbohydrates showed the most potent inhibitor was maltose. A natural lectin from the crude homogenates of freshwater planarian Dugesia japonica was purified by single step affinity chromatography using amylose-coupled agarose. The purified protein appeared as one band with a molecular mass of 350 kDa in PAGE, and as one band, approximately 56 kDa, in SDS-PAGE. The purified lectin showed dependence on calcium. The activity of the purified lectin was inhibited at temperatures greater than 50°C and showed a pH optimum between 5–8. The purified lectin also has binding activity to the Gram-negative bacteria E. coli, and the Gram-positive bacteria B. subtilis. Furthermore, the purified lectin obtained from injured and bacteria-induced planarians showed increased agglutinating activity against rabbit erythrocytes. These results suggest that the purified lectin may play an important role in the innate immunity of the freshwater planarian.     

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.     

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.     

Reconstruction of dopaminergic neural network and locomotion function in planarian regenerates

Planarian, an invertebrate flatworm, has a high capacity for regeneration when compared with other worms and animals. We show here for the first time that the reconstructed dopamine (DA) neural network regulates locomotion and behavior in planarian regenerates. The gene encoding tyrosine hydroxylase in the planarian Dugesia japonica (DjTH) was identified. DjTH protein was coexpressed with aromatic amino acid decarboxylase-like A (DjAADCA) in the planarian central nervous system (CNS). In addition, DjTH-knockdown planarians lost the ability to synthesize DA, but showed no change in 5-hydroxytryptamine synthesis. When the planarian body was amputated, DjTH-positive neurons were regenerated in the brain newly rebuilt from the tail piece at Day 3, and the DjTH-positive axonal and dendritic neural network in the CNS (dopaminergic tiara) was reconstructed at Days 5-7. At that time, autonomic locomotion and methamphetamine-induced hyperkinesia were also suppressed in DjTH-knockdown planarians. Planarian locomotion and behavior seem to be regulated in both cilia- and muscle-dependent manners. In DjTH-knockdown planarians, muscle-mediated locomotion and behavior were significantly attenuated. These results suggest that DA neurons play a key role in the muscle-mediated movement in planarians.     

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.     

Expression pattern of the expanded noggin gene family in the planarian Schmidtea mediterranea

Noggin genes are mainly known as inhibitors of the Bone Morphogenetic Protein (BMP) signalling pathway. Noggin genes play an important role in various developmental processes such as axis formation and neural differentiation. In vertebrates, inhibition of the BMP pathway is usually carried out together with other inhibitory molecules: chordin and follistatin. Recently, it has been shown in planarians that the BMP pathway has a conserved function in the maintenance and re-establishment of the dorsoventral axis during homeostasis and regeneration. In an attempt to further characterize the BMP pathway in this model we have undertaken an in silico search of noggin genes in the genome of Schmidtea mediterranea. In contrast to other systems in which between one and four noggin genes have been reported, ten genes containing a noggin domain are present in S. mediterranea. These genes have been classified into two groups: noggin genes (two genes) and noggin-like genes (eight genes). Noggin-like genes are characterized by the presence of an insertion of 50–60 amino acids in the middle of the noggin domain. Here, we report the characterization of this expanded family of noggin genes in planarians as well as their expression patterns in both intact and regenerating animals. In situ hybridizations show that planarian noggin genes are expressed in a variety of cell types located in different regions of the planarian body.     

Wnt signaling is required for antero-posterior patterning of the planarian brain

Wnt signaling functions in axis formation and morphogenesis in various animals and organs. Here we report that Wnt signaling is required for proper brain patterning during planarian brain regeneration. We showed here that one of the Wnt homologues in the planarian Dugesia japonica, DjwntA, was expressed in the posterior region of the brain. When DjwntA-knockdown planarians were produced by RNAi, they could regenerate their heads at the anterior ends of the fragments, but formed ectopic eyes with irregular posterior lateral branches and brain expansion. This suggests that the Wnt signal may be involved in antero-posterior (A-P) patterning of the planarian brain, as in vertebrates. We also investigated the relationship between the DjwntA and nou-darake/FGFR signal systems, as knockdown planarians of these genes showed similar phenotypes. Double-knockdown planarians of these genes did not show any synergistic effects, suggesting that the two signal systems function independently in the process of brain regeneration, which accords with the fact that nou-darake was expressed earlier than DjwntA during brain regeneration. These observations suggest that the nou-darake/FGFR signal may be involved in brain rudiment formation during the early stage of head regeneration, and subsequently the DjwntA signal may function in A-P patterning of the brain rudiment.     

A LIM-homeobox gene is required for differentiation of Wnt-expressing cells at the posterior end of the planarian body

Planarians have high regenerative ability, which is dependent on pluripotent adult somatic stem cells called neoblasts. Recently, canonical Wnt/β-catenin signaling was shown to be required for posterior specification, and Hedgehog signaling was shown to control anterior-posterior polarity via activation of the Djwnt1/P-1 gene at the posterior end of planarians. Thus, various signaling molecules play an important role in planarian stem cell regulation. However, the molecular mechanisms directly involved in stem cell differentiation have remained unclear. Here, we demonstrate that one of the planarian LIM-homeobox genes, Djislet, is required for the differentiation of Djwnt1/P-1-expressing cells from stem cells at the posterior end. RNA interference (RNAi)-treated planarians of Djislet [Djislet(RNAi)] show a tail-less phenotype. Thus, we speculated that Djislet might be involved in activation of the Wnt signaling pathway in the posterior blastema. When we carefully examined the expression pattern of Djwnt1/P-1 by quantitative real-time PCR during posterior regeneration, we found two phases of Djwnt1/P-1 expression: the first phase was detected in the differentiated cells in the old tissue in the early stage of regeneration and then a second phase was observed in the cells derived from stem cells in the posterior blastema. Interestingly, Djislet is expressed in stem cell-derived DjPiwiA- and Djwnt1/P-1-expressing cells, and Djislet(RNAi) only perturbed the second phase. Thus, we propose that Djislet might act to trigger the differentiation of cells expressing Djwnt1/P-1 from stem cells.     

Distribution of the stem cells (neoblasts) in the planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

It has been postulated that the high regeneration ability of planarians is supported by totipotent stem cells, called neoblasts. There have been a few reports showing the distribution of neoblasts in planarians. However, the findings were not completely consistent. To determine the distribution of neoblasts, we focused on proliferating cell nuclear antigen (PCNA), which is present in proliferative cells. We cloned and sequenced the cDNA of PCNA from the planarian Dugesia japonica and produced an antiserum recognizing the gene product. X-ray irradiation caused rapid loss of all PCNA-positive cells and loss of the neoblasts (which were morphologically defined by the presence of the chromatoid body), strongly suggesting that all PCNA-positive cells were true neoblasts. Using the antiserum, we were successful in identifying the neoblasts more clearly than any previous work. In addition to their dispersed distribution in the dorsal and ventral mesenchyme, the neoblasts were distributed as clusters along the midline and bilateral lines in the dorsal mesenchyme. We also examined the behavior of the neoblasts after decapitation. Decapitation did not seem to affect the migration of neoblasts far from the wound. We demonstrated here that DjPCNA is a powerful tool for identifying planarian neoblasts.Edited by D.A. Weisblat     

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.     

The planarian <Emphasis Type="Italic">nanos</Emphasis>-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration

Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A low percent ethanol method for immobilizing planarians

Planarians have recently become a popular model system for the study of adult stem cells, regeneration and polarity. The system is attractive for both undergraduate and graduate research labs, since planarian colonies are low cost and easy to maintain. Also in situ hybridization, immunofluorescence and RNA-interference (RNAi) gene knockdown techniques have been developed for planarian studies. However, imaging of live worms (particularly at high magnifications) is difficult because animals are strongly photophobic; they quickly move away from light sources and out of frame. The current methods available to inhibit movement in planarians include RNAi injection and exposure to cold temperatures. The former is labor and time intensive, while the latter precludes the use of many fluorescent reporter dyes. Here, we report a simple, inexpensive and reversible method to immobilize planarians for live imaging. Our data show that a short 1 hour treatment with 3% ethanol (EtOH) is sufficient to inhibit both the fine and gross movements of Schmidtea mediterranea planarians, of the typical size used (4-6 mm), with full recovery of movement within 3-4 hours. Importantly, EtOH treatment did not interfere with regeneration, even after repeated exposure, nor lyse epithelial cells (as assayed by H&E staining). We demonstrate that a short exposure to a low concentration of EtOH is a quick and effective method of immobilizing planarians, one that is easily adaptable to planarians of all sizes and will increase the accessibility of live imaging assays to planarian researchers.     

Bone morphogenetic protein is required for dorso-ventral patterning in the planarian Dugesia japonica

In order to clarify the function of the Djbmp (Dugesia japonica bone morphogenetic protein) gene in planarian body patterning, we carried out knockdown of this gene by RNA interference. When the planarians were treated with double-stranded RNA of Djbmp, a bulge formed on the dorsal side, with a dent in the middle of the bulge, and the body surface inside the dent was smoothened and less pigmented. In situ hybridization of the DjIFb gene, which is expressed in the body margin, revealed that the additional body margin was formed ectopically at the region surrounding the dent. The Djbmp-knockdown planarians often had a pair of incomplete nerve cords in the dorsal side, in addition to the original pair of ventral nerve cords. Taken together, we concluded that the Djbmp-knockdown induced formation of an ectopic ventral side, suggesting that Djbmp is required for the dorso-ventral body patterning in the planarian.