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Digital Gene Expression profiling of Schmidtea mediterranea stem cells.

ABSTRACT: The freshwater planarian Schmidtea mediterranea is well known by its amazing regeneration capabilities thanks to the presence of adult stem cells, the neoblasts, the only proliferative cells and responsible for the differentiation in all the cell types of the organism. This study involves the creation of separated transcript libraries from both, isolated neoblasts and differentiated cells, as well as their posterior sequencing and quantification through Digital Gene Expression (DGE) for the characterization of the neoblast transcriptome. Three DGE libraries were produced from FACS isolated cell populations X1 (proliferating stem cells, S/G2/M), X2 (a mix of proliferating stem cells and stem cell progeny, G0/G1) and Xin (differentiated cells, G0/G1). Cells were isolated from a pool of 32 regenerating animals two days after being cut off pre- and post-pharingeally to trigger regeneration and neoblast proliferation.

ORGANISM(S): Schmidtea mediterranea

SUBMITTER: Gustavo Rodriguez-Esteban

PROVIDER: E-GEOD-51681 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Digital Gene Expression profiling of Schmidtea mediterranea stem cells.

Project description:The freshwater planarian Schmidtea mediterranea is well known by its amazing regeneration capabilities thanks to the presence of adult stem cells, the neoblasts, the only proliferative cells and responsible for the differentiation in all the cell types of the organism. This study involves the creation of separated transcript libraries from both, isolated neoblasts and differentiated cells, as well as their posterior sequencing and quantification through Digital Gene Expression (DGE) for the characterization of the neoblast transcriptome.

2015-05-07 | GSE51681 | GEO

Identification of TOR-responsive slow-cycling neoblasts in planarians

Project description:Epimorphic regeneration commonly relies on the activation of quiescent stem cells to drive new cell production. The planarian Schmidtea mediterranea is among the best regenerators in nature, thanks to its large population of adult stem cells, called neoblasts. While neoblasts have long been known to drive regeneration, whether a subset of neoblasts is reserved for this purpose is unknown. Here, we revisited the idea of reserved neoblasts by approaching neoblast heterogeneity from a regulatory perspective. By implementing a new fluorescence-activated cell sorting strategy in planarians, we identified a population of neoblasts defined by low transcriptional activity. These RNAlow neoblasts are relatively slow-cycling at homeostasis and undergo a morphological regeneration response characterized by cell growth at 48 hours post-amputation. At this time, RNAlow neoblasts proliferated in a TOR-dependent manner. Additionally, knockdown of the tumour suppressor Lrig-1, which is enriched in RNAlow neoblasts, resulted in RNAlow neoblast growth and hyperproliferation at homeostasis, and ultimately delayed regeneration. We propose that slow-cycling RNAlow neoblasts represent a regeneration-reserved neoblast population.

2020-12-31 | GSE157860 | GEO

Egf signaling directs neoblast repopulation by regulating asymmetric cell division in planarians

Project description:The goal of this experiment is to identify target genes of Egf signaling which regulate recovery of the neoblast population following exposure to a sublethal dose of radiation. A large population of highly proliferative stem cells (neoblasts) is required for physiological tissue homeostasis and regeneration after injury in planarians. Recent studies indicate that survival of a few neoblasts after sublethal irradiation results in the clonal expansion of the surviving stem cells and the eventual restoration of tissue homeostasis and regenerative capacity. Many genes are known to be required for the normal function of neoblasts, but the precise mechanisms regulating the population dynamics of these adult pluripotent stem cells remain largely unknown. By coupling RNAi screening and sublethal irradiation, we uncovered a central role for Epidermal Growth Factor (EGF) signaling during in vivo neoblast expansion mediated by Smed-egfr-3 (egfr-3) and its putative ligand Smed-neuregulin-7 (nrg-7). Furthermore, the EGFR-3 protein localizes asymmetrically on the cytoplasmic membrane of neoblasts and the ratio of asymmetric to symmetric cell divisions decreases significantly in egfr-3(RNAi) worms. Our results not only provide the first molecular evidence of asymmetric stem cell divisions in planarian, but also demonstrate that EGF signaling likely functions as an essential regulator of neoblast clonal expansion.

2016-08-18 | GSE84025 | GEO

A tetraspanin family member functionally resolves and facilitates the purification of adult pluripotent stem cells used for whole-body regeneration (Bulk)

Project description:Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) capable of sustaining tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single cell transplantation to address this long-standing issue. Large-scale scRNA-seq of fluorescently sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein, and marked by a conserved cell-surface protein coding gene, tetraspanin 1 (tspan1). tspan1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody rescued with high efficiency the viability of lethally irradiated animals after single-cell transplantation. Our work demonstrates the first prospective isolation of an adult PSC, bridges a conceptual dichotomy between operationally and molecularly defined neoblasts, and sheds light on the mechanisms governing in vivo pluripotency and the source of regeneration in planarians.

2018-06-14 | GSE107874 | GEO

Tetraspanin family member functionally resolves and facilitates the purification of adult pluripotent stem cells used for whole-body regeneration (Single)

2018-06-14 | GSE107873 | GEO

RNA Seq analysis of gene expression during Schmidtea mediterranea embryogenesis, including yolk and adult samples

Project description:Planarian neoblasts are pluripotent, adult somatic stem cells and lineage-primed progenitors required for the production and maintenance of all differentiated cell types, including the germline. Neoblasts, originally defined by Harriet Randolph as undifferentiated, embryonic-like cells residing in the adult parenchyma, are frequently compared to embryonic stem cells yet their developmental origin remains obscure. We investigated the provenance of neoblasts during S. mediterranea embryogenesis, and report that neoblasts arise from an anarchic, cycling piwi-1+ population that is wholly responsible for production of all temporary and definitive organs during embryogenesis. Early embryonic piwi-1+ cells are molecularly and functionally distinct from neoblasts: they express unique cohorts of early embryo enriched transcripts and the cells behave differently than neoblasts in cell transplantation assays. Neoblast lineages, established as organogenesis commences, persist into adulthood, where they act as agents of tissue homeostasis and regeneration.

2016-12-01 | GSE82280 | GEO

Planarians recruit piRNAs for mRNA turnover in adult stem cells

Project description:PIWI proteins utilize small RNAs called piRNAs to silence transposable elements, thereby protecting germline integrity. In planarian flatworms, PIWI proteins are essential for regeneration, which requires adult stem cells termed neoblasts. Here, we characterize planarian piRNAs and examine the roles of PIWI proteins in neoblast biology. We find that the planarian PIWI proteins SMEDWI-2 and SMEDWI-3 cooperate to degrade active transposons via the ping-pong cycle. Unexpectedly, we discover that SMEDWI-3 plays an additional role in planarian mRNA surveillance. While SMEDWI-3 degrades numerous neoblast mRNAs in a homotypic ping-pong cycle, it is also guided to another subset of neoblast mRNAs by antisense piRNAs and binds these without degrading them. Mechanistically, the distinct activities of SMEDWI-3 are primarily dictated by the degree of complementarity between target mRNAs and antisense piRNAs. Planarian PIWI proteins thus act as both nucleases and RNA-binding proteins, enabling planarians to repurpose piRNAs for critical roles in neoblast mRNA turnover.

2019-09-04 | GSE122199 | GEO

High throughput expression-based phenotyping and RNAi screening reveals novel regulators of planarian stem cells

Project description:The complexity of cell types and states revealed by scRNAseq cell atlases presents a challenge for the systematic analysis of fate determinants using traditional screening methodologies. Differentiation in the planarian Schmidtea mediterranea exemplifies this problem, as these animals continuously produce over 100 differentiated cell types for homeostasis and regeneration using adult pluripotent stem cells termed neoblasts. The signaling factors enabling neoblast self-renewal and selective differentiation of these many fates are still incompletely understood. We developed a method using high-throughput expression profiling by qPCR and whole-animal RNAseq to simultaneously assess numerous cell fate markers as the phenotypic readout in large-scale RNAi screens. Applying this method, we performed an RNAi screen of 400 kinases, receptors, and other regulatory molecules to reveal specific functions for 30 previously unknown factors in neoblast biology. 17 genes were required for neoblast maintenance, including factors likely involved in cell-cycle regulation, nutrient sensing, and chromatin modification. Multidimensional expression information additionally revealed several specific regulators of other neoblast activities, including a mink1 kinase regulating global neoblast differentiation, the energy responsive kinase adenylate kinase-2 regulating intestine specification within the neoblast population, an RNA acetyl transferase nat10 regulating epidermal differentiation, and a pak1 kinase that restricts neoblast localization to prevent tissue outgrowths. These results identify several new regulators of neoblast activities and demonstrate the applicability of expression-based screening for systematic analysis of stem cell phenotypes in whole animals.

2023-08-31 | GSE212136 | GEO

Cell cycle-dependent resolution of DNA double-strand breaks repair

Project description:Damage to genomic DNA, especially as DNA double strand breaks (DSB), elicits prompt activation of DNA damage response (DDR) which arrest cell-cycle either G1/S or G2/M to avoid entering S and M phase with DNA damage. In mammalian organs cells are in both proliferating and quiescent states. Quiescent cells are already arrested in G0, therefore there may be fundamental difference in DDR between proliferating and quiescent cells. To address these differences we studied recruitment of DSB repair factors and resolution of DNA lesions induced at site-specific DSBs occurring at different cell cycle phases, i.e. in asynchronously proliferating, G0, and G1 arrested cells. Strikingly, DSBs occurring in G0 quiescent cells are irreparable with a sustained activation of p53-pathway. Conversely, reentry of G0-damaged cells into cell cycle progression, show a delayed clearance of recruited DNA repair factors bound at DSBs, indicating an inefficient repair when compared to DSBs induced in asynchronously proliferating or G1 cells. Moreover, we found that initial recognition of DSBs and assembly of DSB factors is largely similar at different cell cycle phases. Our study thereby demonstrates the crucial role of cell cycle phases in repair and resolution of DSBs.

2015-12-30 | GSE71447 | GEO

454 Life Sciences/Roche and Illumina sequencing of small RNAs from planarian samples

Project description:Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for piRNA expression in planarians. Nevertheless, the full complement of piRNAs, and microRNAs (miRNAs) in this organism has yet to be defined. Here, we report on the large scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs uncovered ten miRNAs enriched in planarian stem cells. These miRNAs are down-regulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals. Small RNAs were sequenced from FACS-sorted neoblast stem cells, untreated planarians and irradiated planarians, depleted of neoblasts

2009-06-30 | E-GEOD-16159 | biostudies-arrayexpress

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