Localization of mitochondrial ribosomal RNA on the chromatoid bodies of marine planarian polyclad embryos

Electron-dense cytoplasmic structures, referred to as chromatoid bodies, are observed in the somatic stem cells, called neoblasts, and germline cells in adult planarians. Although it has been revealed that the chromatoid bodies morphologically resemble germline granules in Drosophila and Xenopus embryos, what essential role it plays in the planarian has remained unclear. In the present study, to examine whether chromatoid bodies in planarian embryos are responsible for germline formation, the presence and behavior of chromatoid bodies during embryogenesis were examined. Mitochondrial large ribosomal RNA and mitochondrial small ribosomal RNA were used as candidate markers for components of the chromatoid body. Starting from the fertilized egg, extramitochondrial signals of both RNA (mtrRNA) were observed. At the ultrastructural level, mtrRNA were localized on the surface of the chromatoid bodies. At subsequent stages, the signals of mtrRNA were observed in certain restricted blastomeres that contribute to the formation of larval structures. The signals gradually decreased from the gastrula stage. These results suggest that the chromatoid bodies associated with mtrRNA in embryogenesis are not germline granules. The chromatoid bodies of blastomeres may be concerned with the toti- or pluripotency and cell differentiation as proposed in adult planarian neoblasts.     

Drpiwi-1 is essential for germline cell formation during sexualization of the planarian Dugesia ryukyuensis

A piwi homolog is required for the regulation of stem cells, formation and maintenance of germline stem cells, and gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, both asexually and sexually, and develop and maintain germ cells and sexual organs. They have many pluripotent stem cells (neoblasts) that can differentiate into both somatic and germline stem cells. Thus, we searched for a piwi subfamily in the planarian Dugesia ryukyuensis. Four piwi homologs, identified as Drpiwi-1, -2, -3, and -4, were expressed in sexually reproductive worms. We then selectively destroyed the neoblasts by irradiating the worms with X-rays. In such worms, Drpiwi-1, -2, and -3 were not expressed at all, whereas Drpiwi-4 was expressed to the same degree as that in non-irradiated controls, indicating that Drpiwi-1, -2, and -3, but not Drpiwi-4, are expressed in neoblasts. During the regeneration process, Drpiwi-2(RNAi) and -3(RNAi) worms failed to regenerate after ablation, but Drpiwi-1 and -4(RNAi) worms regenerated. During the sexualizing process, Drpiwi-1(RNAi) worms failed to develop ovaries and testes, but somatic sexual organs were unaffected. Germ cell development was normal in Drpiwi-4(RNAi) worms. Therefore, Drpiwi-2 and -3 may be related to the regulation of neoblasts important for maintaining homeostasis, and Drpiwi-1 is essential for the development of germ cells but not somatic sexual organs. DrPiwi-1 is localized in the cytoplasm of stem cells and germline cells and may be involved in regulating some gene expression. We suggest that planarian Piwi controls germline formation via RNA silencing mechanisms.     

Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal

Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and differentiate. These results suggest that Spoltud-1 is required for long term stem cell self renewal.     

On the origin of neoblasts in freshwater planarians (Turbellaria)

Experiments on 1) regeneration of the cave-adapted planarian, Sphalloplana zeschi, 2) induction of sexuality in an asexual strain of Dugesia japonica japonica by feeding, and 3) culture of dissociated planarian cells, show that neoblasts originate from intestinal cells, i.e. phagocytic cells and granular clubs.     

ERK signaling controls blastema cell differentiation during planarian regeneration

The robust regenerative ability of planarians depends on a population of somatic stem cells called neoblasts, which are the only mitotic cells in adults and are responsible for blastema formation after amputation. The molecular mechanism underlying neoblast differentiation associated with blastema formation remains unknown. Here, using the planarian Dugesia japonica we found that DjmkpA, a planarian mitogen-activated protein kinase (MAPK) phosphatase-related gene, was specifically expressed in blastema cells in response to increased extracellular signal-related kinase (ERK) activity. Pharmacological and genetic [RNA interference (RNAi)] approaches provided evidence that ERK activity was required for blastema cells to exit the proliferative state and undergo differentiation. By contrast, DjmkpA RNAi induced an increased level of ERK activity and rescued the differentiation defect of blastema cells caused by pharmacological reduction of ERK activity. These observations suggest that ERK signaling plays an instructive role in the cell fate decisions of blastema cells regarding whether to differentiate or not, by inducing DjmkpA as a negative regulator of ERK signaling during planarian regeneration.     

Adenylate cyclase in regenerating tissues of the planarian Dugesia lugubris (O. Schmidt)

Adenylate cyclase (AC) was localized ultracytochemically in certain tissues of the regenerating planarian Dugesia lugubris. Studies were carried out from one hour after injury up to the 5th day of regeneration. It was found that the greatest amount of active AC appears during the initial hours of regeneration in the membranes of the muscle cells near the wound, in the epithelial cells surrounding the wound, and in rhabdite-forming cells and neoblasts.     

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.     

Localization of adenylate cyclase activity in the tissues of an intact planarian Dugesia lugubris (O. Schmidt)

Summary Adenylate cyclase was localized in tissues of an intact planarian Dugesia lugubris (O. Schmidt) by ultracytochemical methods. The enzyme was found in epithelium, muscles, secretory cells (mucous), and rhabdites forming cells and neoblasts. Adenylate cyclase occurred on the external side of cell membranes in cisterns of the endoplasmic reticulum, nuclear envelope and mitochondria. The problems of ultracytochemical localization of AC are discussed.     

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.     

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     

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.     

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.     

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.     

Cytological studies on the planarian neoblast

Summary The paper is a study of the cytology of the regeneration cells (neoblasts) in Planaria vitta.The morphology of the living cells has first been examined to provide a reference for an investigation of the fixed neoblasts as studied by ordinary cytological, cytochemical and electron microscopical technics.A rather selective staining method has been devised based on the strong basophilic properties of the scanty cytoplasm. The morphology of the fixed neoblasts and their distribution in the intact animal have been described, using this method.The marked cytoplasmic basophilia was found to be exclusively due to ribonucleic acid, and not to desoxyribonucleic acid or acid mucopolysaccharides.The cytoplasm contains moderate to considerable amounts of basic proteins. Tyrosine, cysteine/cystin, arginine, lysine and perhaps histidine were present, while tryptophan could not be demonstrated.No enzymes could be demonstrated apart perhaps from cytochrome oxidase.The mitochondria are small and inconspicuous and more or less evenly distributed throughout the cytoplasm. A Golgi apparatus could not be demonstrated.The electron microscopic picture is very characteristic, because of the high electron density of the cytoplasm. This density is the result of the presence of a great number of ribonucleoprotein granules. Most of the granules are free and only a minor part bound to the membranes of the endoplasmatic reticulum. The interesting features of the cell membrane are discussed in relation to the structure of the parenchyma.The cytochemical properties of the neoblast (RNA and sulfhydryl-groupcontaining protein) and the fine structure as revealed in the electron microscope characterize the neoblast as a morphogenetically active cell.     

A RING‐type E3 ligase controls anther dehiscence by activating the jasmonate biosynthetic pathway gene DEFECTIVE IN ANTHER DEHISCENCE1 in Arabidopsis

Suppression of expression of DAF [DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)‐Activating Factor], a gene that encodes a putative RING‐finger E3 ligase protein, causes non‐dehiscence of the anthers, alters pollen development and causes sterility in 35S:DAF RNAi/antisense Arabidopsis plants. This mutant phenotype correlates with the suppression of DAF but not with expression of the two most closely related genes, DAFL1/2. The expression of DAD1 was significantly reduced in 35S:DAF RNAi/antisense plants, and complementation with 35S:DAF did not rescue the dad1 mutant, indicating that DAF acts upstream of DAD1 in jasmonic acid biosynthesis. This assumption is supported by the finding that 35S:DAF RNAi/antisense plants showed a similar cellular basis for anther dehiscence to that found in dad1 mutants, and that external application of jasmonic acid rescued the anther non‐dehiscence and pollen defects in 35S:DAF antisense flowers. We further demonstrate that DAF is an E3 ubiquitin ligase and that its activity is abolished by C132S and H137Y mutations in its RING motif. Furthermore, ectopic expression of the dominant‐negative C132S or H137Y mutations causes similar indehiscence of anthers and reduction in DAD1 expression in transgenic Arabidopsis. This result not only confirms that DAF controls anther dehiscence by positively regulating the expression of DAD1 in the jasmonic acid biosynthesis pathway, but also supports the notion that DAF functions as an E3 ubiquitin ligase, and that the conserved RING‐finger region is required for its activity.