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Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the "germ-line multipotency program" and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells.

Limb regeneration involves re-establishing a limb development program from cells within adult tissues. Identifying molecular handles that provide insight into the relationship between cell differentiation status and cell lineage is an important step to study limb blastema cell formation. Here, using single cell PCR, focusing on newly isolated Twist1 sequences, we molecularly profile axolotl limb blastema cells using several progenitor cell markers. We link their molecular expression profile to their embryonic lineage via cell tracking experiments. We use in situ hybridization to determine the spatial localization and extent of overlap of different markers and cell types. Finally, we show by single cell PCR that the mature axolotl limb harbors a small but significant population of Twist1(+) cells., (Copyright © 2012. Published by Elsevier Inc.)

A hallmark of stem cells is the ability to sustainably generate stem cells themselves (self-renew) as well as differentiated cells. Although a full understanding of this ability will require clarifying underlying the primordial molecular and cellular mechanisms, how stem cells maintain their stem state and their population in the evolutionarily oldest extant multicellular organisms, sponges, is poorly understood. Here, we report the identification of the first stem cell-specific gene in demosponges, a homolog of Musashi (an evolutionarily conserved RNA binding protein that regulates the stem cell state in various organisms). EflMsiA, a Musashi paralog, is specifically expressed in stem cells (archeocytes) in the freshwater sponge Ephydatia fluviatilis. EflMsiA protein is localized predominantly in the nucleus, with a small fraction in the cytoplasm, in archeocytes. When archeocytes enter M-phase, EflMsiA protein diffuses into the cytoplasm, probably because of the breakdown of the nuclear membrane. In the present study, the existence of two types of M-phase archeocytes [(M)-archeocytes] was revealed by a precise analysis of the expression levels of EflMsiA mRNA and protein. In Type I (M)-archeocytes, presumably archeocytes undergoing self-renewal, the expression levels of EflMsiA mRNA and protein were high. In Type II (M)-archeocytes, presumably archeocytes committed to differentiate (committed archeocytes), the expression levels of EflMsiA mRNA and protein were about 60% and 30% lower than those in Type I (M)-archeocytes. From these results, archeocytes can be molecularly defined for the first time as EflMsiA-mRNA-expressing cells. Furthermore, these findings shed light on the mode of cell division of archeocytes and suggest that archeocytes divide symmetrically for both self-renewal and differentiation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Fluorescence-activated cell sorting (FACS) is a useful method for stem cell biology, which enables us to isolate the living stem cells of interest from mixture of a variety of cells. In general, the target cells for FACS need to be labeled for various cell surface markers. However, in non-model organisms, we usually do not have specific labels for such cell surface markers. Here, we describe a method for isolating stem cells from non-model organisms, such as planarians, based on physiological and morphological properties of the stem cells. This method may also be applicable to other non-model animals.

The growth in size of freshwater planarians in response to nutrient intake is limited by the eventual separation of tail and body fragments in a process called fission. The resulting tail fragment regenerates the entire body as an artificially amputated tail fragment would do, and the body fragment regenerates a tail, resulting in two whole planarians. This regenerative ability is supported by pluripotent somatic stem cells, called neoblasts, which are distributed throughout almost the entire body of the planarian. Neoblasts are the only planarian cells with the ability to continuously proliferate and give rise to all types of cells during regeneration, asexual reproduction, homeostasis, and growth. In order to investigate the molecular characteristics of neoblasts, we conducted an extensive search for neoblast-specific genes using the High Coverage Expression Profiling (HiCEP) method, and tested the function of the resulting candidates by RNAi. Disruption of the expression of one candidate gene, DjP2X-A (Dugesia japonica membrane protein P2X homologue), resulted in a unique phenotype. DjP2X-A RNAi leads to an increase of fission events upon feeding. We confirmed by immunohistochemistry that DjP2X-A is a membrane protein, and elucidated its role in regulating neoblast proliferation, thereby explaining its unique phenotype. We found that DjP2X-A decreases the burst of neoblast proliferation that normally occurs after feeding. We also found that DjP2X-A is required for normal proliferation in starved animals. We propose that DjP2X-A modulates stem cell proliferation in response to the nutritional condition.

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.

Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA-expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU-incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell-lineage-specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell-lineage-specific markers during archeocyte differentiation into other cell types. Choanocytes are food-entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell-like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.

To achieve an integrated understanding of the stem cell system of planarians at both the cellular and molecular levels, we developed a new method by combining "fluorescent activated cell sorting (FACS) index sorting" analysis and single-cell reverse transcription-polymerase chain reaction (RT-PCR) to detect the gene expression and cell cycle state of stem cells simultaneously. Single cells were collected using FACS, and cDNAs of each cell were used for semi-quantitative RT-PCR. The results were plotted on the FACS sorting profile using the "index sorting" function, which enabled us to analyze the gene expression in combination with cell biological data (such as cell cycle phase) for each cell. Here we investigated the adult stem cells of planarians using this method and obtained findings suggesting that the stem cells might undergo commitment during S to G2/M phase. This method could be a powerful and straightforward tool for examining the stem cell biology of not only planarians but also other organisms, including vertebrates.

Planarians change in body size depending upon whether they are in feeding or starving conditions. To investigate how planarians regulate this flexible system, the numbers of total cells and specific cell types were counted and compared among worms 2 mm to 9 mm in body length. The total cell number increased linearly with increasing body length, but the ratio of cell numbers between the head and the trunk portion was constant (1:3). Interestingly, counting the numbers of specific neurons in the eye and brain after immunostaining using cell type-specific antibodies revealed that the ratio between different neuron types was constant regardless of the brain and body size. These results suggest that planarians can maintain proportionality while changing their body size by maintaining a constant ratio of different cell types. To understand this system and reveal how planarians restore the original ratio during eye and brain regeneration, the numbers of specialized cells were Investigated during regeneration. The results further substantiate the existence of some form of "counting mechanism" that has the ability to regulate both the absolute and relative numbers of different cell types in complex organs such as the brain during cell turnover, starvation, and regeneration.

Planarians have regenerative ability made possible by pluripotent stem cells referred to as neoblasts. Classical ultrastructural studies have indicated that stem cells can be distinguished by a unique cytoplasmic structure known as the chromatoid body and their undifferentiated features, and they are specifically eliminated by X-ray irradiation. Recently, by using fluorescence activated cell sorting (FACS), planarian cells were separated into two X-ray-sensitive fractions (X1 and X2) and an X-ray-insensitive fraction (XIS) according to DNA content and cytoplasmic size. Here we analyzed the fractionated cells by transmission electron microscopy (TEM). First, we found that both undifferentiated cells (stem cells) and regenerative cells (differentiating cells) were concentrated in the X1 fraction containing the S/G2/M phase cells. The regenerative cells were considered to be committed stem cells or progenitor cells, suggesting that some stem cells may maintain proliferative ability even after cell fate-commitment. Second, we succeeded in identifying a new type of stem cells, which were small in size with few chromatoid bodies and a heterochromatin-rich nucleus. Interestingly, they were concentrated in the X2 fraction, containing G0/G1 phase cells. These results suggest that planarian stem cells are not homogeneous, but may consist of heterogeneous populations, like mammalian stem cells.

The planarian's remarkable regenerative ability is thought to be supported by the stem cells (neoblasts) found throughout its body. Here we report the identification of a subpopulation of neoblasts, which was revealed by the expression of the nanos-related gene of the planarian Dugesia japonica, termed Djnos. Djnos-expressing cells in the asexual planarian were distributed to the prospective ovary or testes forming region in the sexual planarian. During sexualization, Djnos-expressing cells produce germ cells, suggesting that in the asexual state these cells were kept as germline stem cells for the oogonia and spermatogonia. Interestingly, the germline stem cells were indistinguishable from the neoblasts by morphology and X-ray sensitivity and did not seem to contribute to the regeneration at all. Germline stem cells initially appear in the growing infant planarian, suggesting that germline stem cells are separated from somatic stem cells in the planarian. Thus, planarian neoblasts can be classified into two groups; somatic stem cells for regeneration and tissue renewal, and germline stem cells for production of germ cells during sexualization. However, Djnos-positive cells appeared in the newly formed trunk region from the head piece, suggesting that somatic stem cells can convert to germline stem cells.

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.

Sponges (phylum Porifera) have remarkable regenerative and reconstitutive abilities and represent evolutionarily the oldest metazoans. To investigate sponge stem cell differentiation, we have focused on the asexual reproductive system in the freshwater sponge Ephydatia fluviatilis. During germination, thousands of stem cells proliferate and differentiate to form a fully functional sponge. As an initial step of our investigation of stem cell (archeocyte) differentiation, we isolated molecular markers for two differentiated cell types: spicule-making sclerocyte cells, and cells involved in innate immunity. Sclerocyte lineage-specific Ef silicatein shares 45% to 62% identity with other sponge silicateins. As in situ hybridization of Ef silicatein specifically detects archeocytes possibly committed to sclerocytes, as well as sclerocytes with an immature or mature spicule, therefore covering all the developmental stages, we conclude that Ef silicatein is a suitable sclerocyte lineage marker. Ef lectin, a marker for the cell type involved in innate immunity, shares 59% to 65% identity with the marine sponge Suberites domuncula galactose-binding protein (Sd GBP) and horseshoe crab Tachypleus tridentatus tachylectin1/lectinL6. Since Sd GBP and tachylectin1 are known to bind to bacterial lipopolysaccharides and inhibit the growth of bacteria, Ef lectin may have a similar function and be expressed in a specialized type of cell involved in defense against invading bacteria. Ef lectin mRNA and protein are not expressed in early stages of development, but are detected in late stages. Therefore, Ef lectin may be specifically expressed in differentiating and/or differentiated cells. We suggest Ef lectin as a marker for cells that assume innate immunity in freshwater sponges.

In order to investigate the cellular system of the freshwater sponge, Ephydatia fluviatilis, we isolated a molecular marker for the most prominent cell type, the choanocyte. After feeding sponge with fluorescent beads, fluorescent-labeled choanocytes were collected by fluorescence activated cell sorting (FACS). By protein profiling choanocyte and archeocyte (stem cell)-rich fractions, proteins characteristic of choanocyte were identified. The partial amino-acid sequence of one of the proteins characteristic of choanocyte matches the deduced amino-acid sequence of sponge expression tag (EST) clones and mouse annexin VII. These EST clones overlap and encode a protein, designated Ef annexin, which includes four annexin domains. Whole mount in situ hybridization shows Ef annexin expression in chamber-forming choanocytes in 7-day-old sponge, leading us to conclude that Ef annexin can be used as a choanocyte marker. In the early development stage, Ef annexin expression can be detected in both large single cells, characteristic of archeocytes, and cells forming 2-, 4- and multiple-cell clusters. These results indicate that Ef annexin is initially expressed in the choanocyte-committed archeocyte which then undergoes several mitotic cell divisions to form a choanocyte chamber. This suggests that the single choanocyte chamber essentially originates from a single archeocyte.

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 migration of adult stem cells in vivo is an important issue, but the complex tissue structures involved, and limited accessibility of the cells hinder a detailed investigation. To overcome these problems, the freshwater planarian Dugesia japonica was used because it has a simple body plan and abundant adult pluripotent stem cells (neoblasts) distributed uniformly throughout its body. To investigate the migratory mechanisms of neoblasts, two planarian homologous genes of metastatic tumor antigen (MTA‐A and MTA‐B), a protein involved in cancer metastasis that functions through histone deacetylation, were identified, and their function was analyzed using RNA interference (RNAi). MTA‐A or MTA‐B knockdown disrupted homeostatic tissue turnover and regeneration in planarians. Whereas neoblasts in MTA‐A (RNAi) and MTA‐B (RNAi) animals were maintained, neoblast differentiation was inhibited. Furthermore, the normal uniform neoblast distribution pattern changed to a branch‐like pattern in MTA‐A (RNAi) and MTA‐B (RNAi) animals. To examine the neoblast migratory ability, a partial X‐ray irradiation assay was performed in D. japonica. Using this assay system, the MTA‐A knockdown neoblasts migrated collectively in a branch‐like pattern, and the MTA‐B knockdown neoblasts were not able to migrate. These results indicated that MTA‐A was required for the exit of neoblasts from the branch‐like region, and that MTA‐B was required for neoblast migration. Thus, the migration mediated by MTA‐A and MTA‐B enabled uniform neoblast distribution and was required for neoblast differentiation to achieve tissue homeostasis and regeneration. [ABSTRACT FROM AUTHOR]

Adult planarians possess somatic pluripotent stem cells called neoblasts that give rise to all missing cell types during regeneration and homeostasis. Recent studies revealed that the Yorkie (Yki)/Yes-associated protein ( YAP) transcriptional coactivator family plays an important role in the regulation of tissue growth during development and regeneration, and therefore we investigated the role of a planarian yki-related gene (termed Djyki) during regeneration and homeostasis of the freshwater planarian Dugesia japonica. We found that knockdown of the function of Djyki by RNA interference ( RNAi) downregulated neoblast proliferation and caused regeneration defects after amputation. In addition, Djyki RNAi caused edema during homeostasis. These seemingly distinct defects induced by Djyki RNAi were rescued by simultaneous RNAi of a planarian mats-related gene (termed Djmats), suggesting an important role of Djmats in the negative regulation of Djyki, in accordance with the conservation of the functional relationship of these two genes during the course of evolution. Interestingly, Djyki RNAi did not prevent normal protonephridial structure, suggesting that Djyki RNAi induced the edema phenotype without affecting the excretory system. Further analyses revealed that increased expression of the D. japonica gene DjaquaporinA ( DjaqpA), which belongs to a large gene family that encodes a water channel protein for the regulation of transcellular water flow, promoted the induction of edema, but not defects in neoblast dynamics, in Djyki( RNAi) animals. Thus, we conclude that Djyki plays two distinct roles in the regulation of active proliferation of stem cells and in osmotic water transport across the body surface in D. japonica. [ABSTRACT FROM AUTHOR]

Freshwater planarians, Plathelminthes, have been an intriguing model animal of regeneration studies for more than 100 years. Their robust regenerative ability is one of asexual reproductive capacity, in which complete animals develop from tiny body fragments within a week. Pluripotent adult somatic stem cells, called neoblasts, assure this regenerative ability. Neoblasts give rise to not only all types of somatic cells, but also germline cells. During the last decade, several experimental techniques for the analysis of planarian neoblasts at the molecular level, such as in situ hybridization, RNAi and fluorescence activated cell sorting, have been established. Moreover, information about genes involved in maintenance and differentiation of neoblasts has been accumulated. One of the molecular features of neoblasts is the expression of many RNA regulators, which are involved in germline development in other animals, such as vasa and piwi family genes. In this review, we introduce physiological and molecular features of the neoblast, and discuss how germline genes regulate planarian neoblasts and what differences exist between neoblasts and germline cells. [ABSTRACT FROM AUTHOR]

Abstract: We propose two major evolutionary origins of stem cell systems in the animal kingdom. Adult pluripotent stem cell systems are found in many invertebrates and probably evolved as components of asexual reproduction. Lineage-specific stem cell systems probably evolved later and include neural and hematopoietic stem cell types. We propose that these two types of stem cell systems evolved independently. The vasa-like genes regulate reproductive stem cells, but not lineage-specific stem cells, which may be regulated by gcm genes. Here, we review the evidence for the molecular basis for the evolutionary origin of these two different stem cell systems. [Copyright &y& Elsevier]

In order to investigate the cellular system of the freshwater sponge,Ephydatia fluviatilis, we isolated a molecular marker for the most prominent cell type, the choanocyte. After feeding sponge with fluorescent beads, fluorescent-labeled choanocytes were collected by fluorescence activated cell sorting (FACS). By protein profiling choanocyte and archeocyte (stem cell)-rich fractions, proteins characteristic of choanocyte were identified. The partial amino-acid sequence of one of the proteins characteristic of choanocyte matches the deduced amino-acid sequence of sponge expression tag (EST) clones and mouse annexin VII. These EST clones overlap and encode a protein, designated Ef annexin, which includes four annexin domains. Whole mountin situhybridization showsEf annexinexpression in chamber-forming choanocytes in 7-day-old sponge, leading us to conclude thatEf annexincan be used as a choanocyte marker. In the early development stage,Ef annexinexpression can be detected in both large single cells, characteristic of archeocytes, and cells forming 2-, 4- and multiple-cell clusters. These results indicate thatEf annexinis initially expressed in the choanocyte-committed archeocyte which then undergoes several mitotic cell divisions to form a choanocyte chamber. This suggests that the single choanocyte chamber essentially originates from a single archeocyte. [ABSTRACT FROM AUTHOR]

Planarians can regenerate using a pluripotent stem cell system. This phenomenon provides a unique opportunity to understand gene regulation in the process of differentiation from pluripotent stem cells. Recent studies have made significant advances in our understanding of the pluripotent stem cell system in this model. In particular, a gene knockdown method by RNA interference enabled great progress in identifying genes involved in regeneration and stem cell regulation. [Copyright &y& Elsevier]

Presents a study that identified planarian stem cells through the use of molecular markers to examine planarian regeneration. Background on planarians; Analysis of the cellular events involved in the process of planarian regeneration; Information on the evolution of the cell-type complexity of neural cells.

The coincidence of cell cycle exit and differentiation has been described in a wide variety of stem cells and organisms for decades, but the causal relationship is still unclear due to the complicated regulation of the cell cycle. Here, we used the planarian Dugesia japonica since they may possess a simple cell cycle regulation in which Cdh1 is one of the factors responsible for exiting the cell cycle. When cdh1 was functionally inhibited, the planarians could not maintain their tissue homeostasis and could not regenerate their missing body parts. While the knockdown of cdh1 caused pronounced accumulation of the stem cells, the progenitor and differentiated cells were decreased. Further analyses indicated that the stem cells with cdh1 knockdown did not undergo differentiation even though they received ERK signaling activation as an induction signal. These results suggested that stem cells could not acquire differentiation competence without cell cycle exit. Thus, we propose that cell cycle regulation determines the differentiation competence and that cell cycle exit to G0 enables stem cells to undergo differentiation. [Display omitted] • As a homolog causing cell cycle exit, cdh1 was found but not CKIs in planarians. • The knockdown of cdh1 caused disruption of tissue homeostasis and regeneration. • The stem cells with cdh1 knockdown could not undergo differentiation. • Stem cells with cell cycle progression may persist their undifferentiated state. • Cell cycle exit to G0 may dictate stem cells to undergo differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Planarian regeneration depends on the presence and precise regulation of pluripotent adult somatic stem cells named neoblasts, which differentiate to replace cells of any missing tissue. A characteristic feature of neoblasts is the presence of large perinuclear nonmembranous organelles named “chromatoid bodies”, which are comparable to ribonucleoprotein structures found in germ cells of organisms across different phyla. In order to better understand regulation of gene expression in neoblasts, and potentially the function and composition of chromatoid bodies, we characterized homologues to known germ and soma ribonucleoprotein granule components from other organisms and analyzed their function during regeneration of the planarian Dugesia japonica. Expression in neoblasts was detected for 49 of 55 analyzed genes, highlighting the prevalence of post-transcriptional regulation in planarian stem cells. RNAi-mediated knockdown of two factors [ago-2 and bruli] lead to loss of neoblasts, and consequently loss of regeneration, corroborating with results previously reported for a bruli ortholog in the planarian Schmidtea mediterranea (Guo et al., 2006). Conversely, depletion mRNA turnover factors [edc-4 or upf-1], exoribonucleases [xrn-1 or xrn-2], or DEAD box RNA helicases [Djcbc-1 or vas-1] inhibited planarian regeneration, but did not reduce neoblast proliferation or abundance. We also found that depletion of cap-dependent translation initiation factors eIF-3A or eIF-2A interrupted cell cycle progression outside the M-phase of mitosis. Our results show that a set of post-transcriptional regulators is required to maintain the stem cell identity in neoblasts, while another facilitates proper differentiation. We propose that planarian neoblasts maintain pluripotency by employing mechanisms of post-transcriptional regulation exhibited in germ cells and early development of most metazoans. [Copyright &y& Elsevier]

Abstract: The remarkable regenerative ability of planarians is made possible by a system of pluripotent stem cells. Recent molecular biological and ultrastructural studies have revealed that planarian stem cells consist of heterogeneous populations, which can be classified into several subsets according to their differential expression of RNA binding protein genes. In this study, we focused on planarian musashi family genes. Musashi encodes an evolutionarily conserved RNA binding protein known to be expressed in neural lineage cells, including neural stem cells, in many animals. Here, we investigated whether planarian musashi-like genes can be used as markers for detecting neural fate-restricted cells. Three musashi family genes, DjmlgA, DjmlgB and DjmlgC (Dugesia japonica musashi-like gene A, B, C), and Djdmlg (Dugesia japonica DAZAP-like/musashi-like gene) were obtained by searching a planarian EST database and 5′ RACE, and each was found to have two RNA recognition motifs. We analyzed the types of cells expressing DjmlgA, DjmlgB, DjmlgC and Djdmlg by in situ hybridization, RT-PCR and single-cell RT-PCR analysis. Although Djdmlg was expressed in X-ray-sensitive stem cells and various types of differentiated cells, expression of the other three musashi-like genes was restricted to neural cells, as we expected. Further detailed analyses yielded the unexpected finding that these three planarian musashi family genes were predominantly expressed in X-ray-resistant differentiated neurons, but not in X-ray-sensitive stem cells. RNAi experiments suggested that these planarian musashi family genes might be involved in neural cell differentiation after neural cell-fate commitment. [Copyright &y& Elsevier]