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: 10.47). 03/2012; 13(3):R19. DOI: 10.1186/gb-2012-13-3-r19
Source: PubMed

ABSTRACT 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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    • "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.
    Molecular Reproduction and Development 05/2014; 81(5). DOI:10.1002/mrd.22308 · 2.68 Impact Factor
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    • "Although egr-4 was not expressed in neoblasts, we investigated whether these defects in regeneration were due to a non-cellautonomous effect of egr-4 RNAi on neoblast maintenance and/or proliferation. However, no differences in neoblast number or distribution were observed after immunostaining and in situ hybridization for the neoblast-specific markers anti-SMEDWI-1 antibody and Smed-histone-2B, respectively (Fig. 2B) (Reddien et al., 2005; Guo et al., 2006; Solana et al., 2012). Thus, neoblasts were normally found below the wound epithelium on day 3. Eight 1836 RESEARCH ARTICLE Development (2014) 141, 1835-1847 doi:10.1242/dev.101345 "
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    ABSTRACT: During the regeneration of freshwater planarians, polarity and patterning programs play essential roles in determining whether a head or a tail regenerates at anterior or posterior-facing wounds. This decision is made very soon after amputation. The pivotal role of the Wnt/β-catenin and Hh signaling pathways in re-establishing anterior-posterior (AP) polarity has been well documented. However, the mechanisms that control the growth and differentiation of the blastema in accordance with its AP identity are less well understood. Previous studies have described a role of Smed-egfr-3, a planarian epidermal growth factor receptor, in blastema growth and differentiation. Here, we identify Smed-egr-4, a zinc-finger transcription factor belonging to the early growth response gene family, as a putative downstream target of Smed-egfr-3. Smed-egr-4 is mainly expressed in the central nervous system and its silencing inhibits anterior regeneration without affecting the regeneration of posterior regions. Single and combinatorial RNA interference to target different elements of the Wnt/β-catenin pathway, together with expression analysis of brain- and anterior-specific markers, revealed that Smed-egr-4: (1) is expressed in two phases - an early Smed-egfr-3-independent phase and a late Smed-egfr-3-dependent phase; (2) is necessary for the differentiation of the brain primordia in the early stages of regeneration; and (3) that it appears to antagonize the activity of the Wnt/β-catenin pathway to allow head regeneration. These results suggest that a conserved EGFR/egr pathway plays an important role in cell differentiation during planarian regeneration and indicate an association between early brain differentiation and the proper progression of head regeneration.
    Development 04/2014; 141(9). DOI:10.1242/dev.101345 · 6.27 Impact Factor
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    • "Whether such genomic organization reflects on complex but specific regulatory programs modulating the temporal and/or spatial regulation of histones in planarians remains to be determined. However, it is known that specific histone variants are primarily expressed in planarian neoblasts, in particular H4 (Rouhana, Vieira, Roberts- Galbraith, & Newmark, 2012) and H2B (Solana et al., 2012). "
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    ABSTRACT: The freshwater planarian Schmidtea mediterranea has emerged as a powerful model system for studying regeneration and adult stem cell (ASC) biology. This is largely due to the developmental plasticity of these organisms and the abundant distribution and experimental accessibility of their ASCs. Techniques such as whole mount in situ hybridization, dsRNA-mediated interference, halogenated thymidine analogs for defining cell lineages, and fluorescence-activated cell sorting among other methods, have allowed researchers to interrogate the biology and attendant pluripotent stem cells of these animals in great detail. Therefore, it has now become possible to interrogate and define the roles that epigenetic states may play in regulating ASCs, and by extension, regeneration proper. Here, we provide a primer on the types and number of histone families found in S. mediterranea, known as epigenetic marks of these molecules and a survey of epigenetic modifying enzymes encoded by the planarian genome. We also review experimental evidence indicating that such modifications may in fact play key roles in determining the activities of planarian stem cells.
    Current Topics in Developmental Biology 01/2014; 108:71-93. DOI:10.1016/B978-0-12-391498-9.00004-8 · 4.21 Impact Factor
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