DjPum, a homologue of Drosophila Pumilio, is essential to planarian stem cell maintenance

As stem cells are rare and difficult to study in vivo in adults, the use of classical models of regeneration to address fundamental aspects of the stem cell biology is emerging. Planarian regeneration, which is based upon totipotent stem cells present in the adult--the so-called neoblasts--provides a unique opportunity to study in vivo the molecular program that defines a stem cell. The choice of a stem cell to self-renew or differentiate involves regulatory molecules that also operate as translational repressors, such as members of PUF proteins. In this study, we identified a homologue of the Drosophila PUF gene Pumilio (DjPum) in the planarian Dugesia japonica, with an expression pattern preferentially restricted to neoblasts. Through RNA interference (RNAi), we demonstrate that gene silencing of DjPum dramatically reduces the number of neoblasts, thus supporting the intriguing hypothesis that stem cell maintenance may be an ancestral function of PUF proteins.     

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.     

MicroRNAs from the Planarian Schmidtea mediterranea: a model system for stem cell biology

MicroRNAs (miRNAs) are approximately 22-nt RNA molecules that typically bind to the 3' untranslated regions of target mRNAs and function to either induce mRNA degradation or repress translation. miRNAs have been shown to play important roles in the function of stem cells and cell lineage decisions in a variety of organisms, including humans. Planarians are bilaterally symmetric metazoans that have the unique ability to completely regenerate lost tissues or organs. This regenerative capacity is facilitated by a population of stem cells known as neoblasts. Planarians are therefore an excellent model system for studying many aspects of stem cell biology. Here we report the cloning and initial characterization of 71 miRNAs from the planarian Schmidtea mediterranea. While several of the S. mediterranea miRNAs are members of miRNA families identified in other species, we also identified a number of planarian-specific miRNAs. This work lays the foundation for functional studies aimed at addressing the role of these miRNAs in regeneration, cell lineage decisions, and basic stem cell biology.     

A small scale expression screen identifies tissue specific markers in the Dugesia japonica strain Pek-1

Freshwater planaria has tremendous capacity to reform the missing part of the body and therefore is considered as one of the most important model organism for regeneration study.At present,Schmidtea mediterranea and Dugesia japonica are the two major species utilized for laboratory manipulations.Dugesia japonica flatworms are widely distributed in the Far East including Cherry Valley region in the north-west area of Beijing,China.We reported here the establishment of an asexual Dugesia japonica strain Pek-1,as a suitable system for regeneration study.Using morphological,karyotypical as well as phyiogenetic analyses,we confirmed that these flatworms indeed belonged to Dugesia japonica.We went on to show that the commonly used in situ probes and immunohistochemistry reagents and protocols were applicable to the Pek-1 strain.Using this strain,we carried out small scale analysis on EST,RNAi and gene expression.We identified 193 unique EST sequences and 65 of them had not been reported in planarian.By RNAi analysis,we showed that 48 genes,when down-regulated individually,had no effect on regeneration.Furthermore,we identified 3 groups of tissue specific expressing genes that were useful for cell lineage analysis.We concluded that the Dugesia japonica Pek-1 swain could be another suitable animal model to regeneration research.     

Identification and origin of the germline stem cells as revealed by the expression of nanos-related gene in planarians

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.     

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.     

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.     

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.     

p53同系物Djp53在涡虫胚基早期发育阶段的组织特异性表达及其在再生中的作用

为了探索东亚三角涡虫Djp53基因在涡虫组织中的表达和功能,利用整体原位杂交、RT-PCR技术,检测了涡虫Djp53基因在组织中的表达分布特点,结果表明,Djp53基因在再生1、3、5天的胚基中具有较强的阳性信号,且3天的表达量最高;而在再生7、10天和成虫的实质组织中表达较弱．RNA干扰后的RT-PCR检测显示,Djp53表达量显著下降,涡虫不能正常再生或出现眼点缺陷．由此推断,东亚三角涡虫Djp53基因在早期胚基发育阶段,通过调节多功能干细胞的迁移和增殖分化影响早期胚基的形成,是涡虫早期胚基发育必不可少的一个基因,并且在涡虫成体和再生后期对多功能干细胞的维持具有重要的作用.     

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.     

SPORTS1.0: A Tool for Annotating and Profiling Non-coding RNAs Optimized for rRNA- and tRNA-derived Small RNAs

High-throughput RNA-seq has revolutionized the process of small RNA (sRNA) discovery, leading to a rapid expansion of sRNA categories. In addition to the previously well-characterized sRNAs such as microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and small nucleolar RNA (snoRNAs), recent emerging studies have spotlighted on tRNA-derived sRNAs (tsRNAs) and rRNA-derived sRNAs (rsRNAs) as new categories of sRNAs that bear versatile functions. Since existing software and pipelines for sRNA annotation are mostly focused on analyzing miRNAs or piRNAs, here we developed the sRNA annotation pipelineoptimized for rRNA- and tRNA-derived sRNAs (SPORTS1.0). SPORTS1.0 is optimized for analyzing tsRNAs and rsRNAs from sRNA-seq data, in addition to its capacity to annotate canonical sRNAs such as miRNAs and piRNAs. Moreover, SPORTS1.0 can predict potential RNA modification sites based on nucleotide mismatches within sRNAs. SPORTS1.0 is precompiled to annotate sRNAs for a wide range of 68 species across bacteria, yeast, plant, and animal kingdoms, while additional species for analyses could be readily expanded upon end users’ input. For demonstration, by analyzing sRNA datasets using SPORTS1.0, we reveal that distinct signatures are present in tsRNAs and rsRNAs from different mouse cell types. We also find that compared to other sRNA species, tsRNAs bear the highest mismatch rate, which is consistent with their highly modified nature. SPORTS1.0 is an open-source software and can be publically accessed at https://github.com/junchaoshi/sports1.0.     

Presence of rhodopsin-like proteins in the planarian head

The presence of rhodopsin-like protein was detected in the head of the freshwater planarian Dugesia japonica japonica Ichikawa et Kawakatsu by use of anti-frog-rhodopsin rabbit IgG. Two membrane proteins of molecular weights 65000 and 62000 were separated by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis and found to react with the anti-rhodopsin IgG. The antibody may be useful for monitoring regeneration of the planarian eye.     

The body margin of the planarian Dugesia japonica: characterization by the expression of an intermediate filament gene

We have cloned and sequenced a cDNA encoding an intermediate filament protein (IF) from the planarian Dugesia japonica named DjIFb. The deduced amino acid sequence of DjIFb has similarity to those of protostomic IFs and lamins, supporting a previous hypothesis that the protostomic IFs, including DjIFb, are evolutionarily closer to lamins than to vertebrate cytoplasmic IFs. In addition, analysis of the exon/intron organization revealed that 8 out of 10 introns of DjIFb were coincident in their position, even in the codon phase, with those of the non-neuronal IF of the snail Helix aspersa. This suggests that the Platyhelminthes are not the most primitive Bilateria but instead are evolutionarily close to the Mollusca. The DjIFb gene was expressed in particular cells, probably a kind of adhesive gland cell, which were present in the marginal region encircling the planarian body. The localization of DjIFb protein suggests that it plays an important role in the secretion of an adhesive substance. The specific expression pattern of the DjIFb gene enabled us to monitor how the body margin forms during planarian regeneration.     

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

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

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 and functional analysis of musashi-like genes in planarian CNS regeneration

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.