Defining the molecular profile of planarian pluripotent stem cells using a combinatorial RNA-seq, RNAi and irradiation approach

Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, UK.
Genome biology (Impact Factor: 10.81). 03/2012; 13(3):R19. DOI: 10.1186/gb-2012-13-3-r19
Source: PubMed


Planarian stem cells, or neoblasts, drive the almost unlimited regeneration capacities of freshwater planarians. Neoblasts are traditionally described by their morphological features and by the fact that they are the only proliferative cell type in asexual planarians. Therefore, they can be specifically eliminated by irradiation. Irradiation, however, is likely to induce transcriptome-wide changes in gene expression that are not associated with neoblast ablation. This has affected the accurate description of their specific transcriptomic profile.
We introduce the use of Smed-histone-2B RNA interference (RNAi) for genetic ablation of neoblast cells in Schmidtea mediterranea as an alternative to irradiation. We characterize the rapid, neoblast-specific phenotype induced by Smed-histone-2B RNAi, resulting in neoblast ablation. We compare and triangulate RNA-seq data after using both irradiation and Smed-histone-2B RNAi over a time course as means of neoblast ablation. Our analyses show that Smed-histone-2B RNAi eliminates neoblast gene expression with high specificity and discrimination from gene expression in other cellular compartments. We compile a high confidence list of genes downregulated by both irradiation and Smed-histone-2B RNAi and validate their expression in neoblast cells. Lastly, we analyze the overall expression profile of neoblast cells.
Our list of neoblast genes parallels their morphological features and is highly enriched for nuclear components, chromatin remodeling factors, RNA splicing factors, RNA granule components and the machinery of cell division. Our data reveal that the regulation of planarian stem cells relies on posttranscriptional regulatory mechanisms and suggest that planarians are an ideal model for this understudied aspect of stem cell biology.

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    • "Freshwater planarians are a popular model organism for studying stem cell biology and regeneration1234 because of their large population of pluripotent adult stem cells, strong regenerative capabilities, and ease of breeding and manipulation. In addition, the development of molecular biology techniques for planarians, such as RNA interference (RNAi)5, immunohistochemistry467, RNA sequencing8910, EST11, and genome databases12, has allowed researchers to undertake detailed studies of gene function during regeneration91314. One limitation to current planarian research, however, is a lack of effective techniques for live imaging. "
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    ABSTRACT: Planarians are an important model organism for regeneration and stem cell research. A complete understanding of stem cell and regeneration dynamics in these animals requires time-lapse imaging in vivo, which has been difficult to achieve due to a lack of tissue-specific markers and the strong negative phototaxis of planarians. We have developed the Planarian Immobilization Chip (PIC) for rapid, stable immobilization of planarians for in vivo imaging without injury or biochemical alteration. The chip is easy and inexpensive to fabricate, and worms can be mounted for and removed after imaging within minutes. We show that the PIC enables significantly higher-stability immobilization than can be achieved with standard techniques, allowing for imaging of planarians at sub-cellular resolution in vivo using brightfield and fluorescence microscopy. We validate the performance of the PIC by performing time-lapse imaging of planarian wound closure and sequential imaging over days of head regeneration. We further show that the device can be used to immobilize Hydra, another photophobic regenerative model organism. The simple fabrication, low cost, ease of use, and enhanced specimen stability of the PIC should enable its broad application to in vivo studies of stem cell and regeneration dynamics in planarians and Hydra.
    Full-text · Article · Sep 2014 · Scientific Reports
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    • "To find dynamin homologs in S. mediterranea, we performed extensive reciprocal BLAST analyses using fly and mouse dynamin protein sequences against the fully sequenced and assembled planarian genome and various available transcriptomes (Sánchez Alvarado et al., 2002; Sandmann et al., 2011; Labbé et al., 2012; Solana et al., 2012). From this, we identified a total of six predicted planarian dynamins, which we cloned using 3′ RACE. "
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    ABSTRACT: Dynamins are GTPases that are required for separation of vesicles from the plasma membrane and thus are key regulators of endocytosis in eukaryotic cells. This role for dynamin proteins is especially crucial for the proper function of neurons, where they ensure that synaptic vesicles and their neurotransmitter cargo are recycled in the presynaptic cell. Here we have characterized the dynamin protein family in the freshwater planarian Schmidtea mediterranea and showed that it possesses six dynamins with tissue specific expression profiles. Of these six planarian homologs, two are necessary for normal tissue homeostasis, and the loss of another, Smed-dynA-1, leads to an abnormal behavioral phenotype, which we have quantified using automated center of mass tracking. Smed-dynA-1 is primarily expressed in the planarian nervous system and is a functional homolog of the mammalian Dynamin I. The distinct expression profiles of the six dynamin genes makes planarians an interesting new system to reveal novel dynamin functions, which may be determined by their differential tissue localization. The observed complexity of neurotransmitter regulation combined with the tools of quantitative behavioral assays as a functional readout for neuronal activity, renders planarians an ideal system for studying how the nervous system controls behavior.
    Full-text · Article · Jun 2014 · Biology Open
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    • "transcriptome analyses indicating a neoblast-specific expression of the rad51 homologue gene in S. mediterranea ( € Onal et al., 2012; Solana et al., 2012). Studies in vertebrate cells have suggested an essential role for Rad51 in the repair of spontaneously occurring chromosome breaks during cell proliferation (Sonoda et al., 1998), and the knockout of Rad51 in mice is embryonically lethal (Lim and Hasty, 1996; Tsuzuki et al., 1996). "
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    ABSTRACT: Rad51, a conserved eukaryotic protein, mediates the homologous-recombination repair of DNA double-strand breaks that occur during both mitosis and meiosis. During prophase I of meiosis, homologous recombination enhances the linkage between homologous chromosomes to increase the accuracy of segregation at anaphase I. In polyploidy situtations, however, difficulties with homologous chromosome segregation often disrupt meiosis. Yet, triploid individuals of the planarian Dugesia ryukyuensis are able to produce functional gametes through a specialized form of meiosis. To shed light on the molecular mechanisms that promote successful meiosis in triploid D. ryukyuensis, we investigated rad51 gene function. We isolated three genes of the Rad51 family, the Rad51 homolog Dr-rad51 and the Rad51 paralogs Dr-rad51B and Dr-rad51C. Dr-rad51 was expressed in germ-line and presumably in somatic stem cells, but was not necessary for the regeneration of somatic tissue. RNA-interference (RNAi) depletion of Dr-rad51 during sexualization did not affect chromosome behavior in zygotene oocytes, but did result in the loss of chiasmata at the diplotene stage. Thus, homologous recombination does not appear to be necessary for synapsis, but is needed for crossover and proper segregation in D. ryukyuensis. Mol. Reprod. Dev. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · May 2014 · Molecular Reproduction and Development
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