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A mex3 homolog is required for differentiation during planarian stem cell lineage development

ABSTRACT: Neoblasts are adult stem cells (ASCs) in planarians which sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving the postmitotic fate remain poorly defined. Here we used transcriptional profiling of irradiation-sensitive and -insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers, which distinguish two epithelial progenitor populations, and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 is required for generating progenitors of epithelial, neural, eye, pharyngeal, and protonephridial lineages, and restricting expansion of the stem cell compartment. We also demonstrate the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results show that mex3-1 promotes differentiation in multiple, if not all, lineages, and maintains the balance between ASC self-renewal and commitment. Sorted and irradiated animals were collected as previously described (Labbe, et al., 2012) and RNAi conditions were triplicated, RNA was purified independently, then each replicate was pooled in equal amounts for sequencing at the timepoint: 1 feed day 12 for control RNAi and mex3-1 RNAi.

ORGANISM(S): Schmidtea mediterranea

SUBMITTER: Bret Pearson

PROVIDER: E-GEOD-68581 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A mex3 homolog is required for differentiation during planarian stem cell lineage development

Project description:Neoblasts are adult stem cells (ASCs) in planarians which sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving the postmitotic fate remain poorly defined. Here we used transcriptional profiling of irradiation-sensitive and -insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers, which distinguish two epithelial progenitor populations, and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 is required for generating progenitors of epithelial, neural, eye, pharyngeal, and protonephridial lineages, and restricting expansion of the stem cell compartment. We also demonstrate the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results show that mex3-1 promotes differentiation in multiple, if not all, lineages, and maintains the balance between ASC self-renewal and commitment.

2015-06-15 | GSE68581 | GEO

Planarian stem cell transcriptome

Project description:Many long-lived species of animals require the function of adult stem cells throughout their lives. However, the transcriptomes of stem cells in invertebrates and vertebrates have not been compared, and consequently ancestral regulatory circuits that control stem cell populations remain poorly defined. In this study, we have used data from high-throughput RNA sequencing (RNA-Seq) to compare the transcriptomes of pluripotent adult stem cells from planarians with the transcriptomes of human and mouse pluripotent embryonic stem cells. From a stringently-defined set of 4,432 orthologs shared between planarians, mice and humans, we identified 123 conserved genes that are ≥ 5-fold differentially expressed in stem cells from all three species. Guided by this gene set, we used RNAi screening in adult planarians to discover novel stem cell regulators, which we found to affect the stem cell-associated functions of tissue homeostasis, regeneration, and stem cell maintenance. Examples of genes that disrupted these processes included the orthologs of TBL3, PSD12, TTC27, and RACK1. From these analyses, we concluded that by comparing stem cell transcriptomes from diverse species, it is possible to uncover conserved factors that function in stem cell biology. These results provide insights into which genes composed the ancestral circuitry underlying the control of stem cell self-renewal and pluripotency. Three planarian tissues were analyzed: stem cells, stem cell progeny, and differentiated tissues. For the manuscript: Labbe, RM, et. al., 2012, Stem Cells

2012-06-01 | E-GEOD-37910 | biostudies-arrayexpress

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

Single-cell analysis reveals prominent classes within the planarian stem cell compartment

Project description:Single cell qPCR analysis identified two prominent expression signatures within the planarian stem cells, the neoblasts. RNAi against zinc finger protein zfp-1 results in elimination of one of the two classes. Worms were treated with RNAi by feeding. X1 or X2 cells were isolated by FACS, or blastemas were dissected. mRNA libraries were sequenced.

2014-09-30 | E-GEOD-58057 | biostudies-arrayexpress

RNAseq in whole freshwater planarians, Schmidtea mediterranea, following RNAi of the transcription factors lhx1/5-1 and pitx.

Project description:Freshwater planarians can be used as a model to investigate neuronal function and maintenance in adults in vivo. We knocked down expression for 2 transcription factors, then sequenced resulting animals to examine transcriptional changes that may have occurred. There are 3 triplicated conditions: control(RNAi) using the unc22 gene from C. elegans, lhx1/5-1(RNAi), and pitx(RNAi). Worms were fed RNAi for each gene 5 times over 12 days then were collected 12 days later. Approximately 50 million single-end reads were performed on each sample.

2013-05-31 | E-GEOD-47348 | biostudies-arrayexpress

Smed-myb-1 specifies early temporal identity during planarian epidermal differentiation

Project description:The planarian epidermis provides an excellent model to explore adult stem cell (ASC) lineage development due to well-characterized and distinct spatiotemporal phases during lineage progression. Using flow cytometry-isolated cells enriched in epidermal progenitors, we performed transcriptional profiling and RNAi screening to uncover regulators of epidermal differentiation. We identified a MYB-type transcription factor (Smed-myb-1) required for specification of the first temporal phase of post-mitotic maturation. Knockdown of myb-1 abolished the early progenitor phase of differentiation without ceasing production of subsequent epidermal progenitors or homeostatic turnover and regeneration of the epidermis. Further examination revealed accelerated maturation of ASC descendants, with premature entry into subsequent progeny phases and ultimately the epidermis. These results demonstrate that a spatiotemporal shift in lineage progression occurs in the absence of the early progenitor state after myb-1 RNAi, and identify myb-1 as a critical regulator of the early temporal window in the step-wise specification during planarian epidermal differentiation.

2018-08-18 | GSE118674 | GEO

Canonical and early lineage-specific stem cell types identified in planarian SirNeoblasts

Project description:The pluripotent stem cells in planarians, a model for tissue and cellular regeneration, have yet to be definitively identified. We recently developed a method to enrich piwi-1+ cells in Schmidtea mediterranea, by staining cells with SiR-DNA and Cell Tracker Green, named SirNeoblasts that permits their propagation and subsequent functional study in vivo. Since traditional enrichment for planarian neoblasts by Hoechst 33342 staining, to generate X1 cells, blocks the cell cycle, inducing cytotoxicity, this method represents a substantive technological advance for functional investigation of cell fate and regeneration in basic and applied research questions. However, the similarities in heterogeneity of cell subtypes between SirNeoblasts and X1 remain unknown. In this work, we performed single cell RNA sequencing for SirNeoblasts for comparison with differential expression patterns in a publicly available X1 single cell RNA sequencing data. We found first that all of the lineage-specific progenitor cells in X1 were present in comparable proportions in SirNeoblasts. In addition, SirNeoblasts contain an early muscle progenitor unreported in X1. Analysis of new markers for putative pluripotent stem cells identified here, with subsequent sub-clustering analysis revealed earlier lineages of epidermal, muscular, intestinal, and pharyngeal progenitors than have been observed in X1. Using the ski-3 and GCM markers, we also identified a pluripotent cell population at higher resolution than that provided by tgs-1. The use of SirNeoblast will enable broad experimental advances in regeneration and cell fate specification, given the possibility for propagation and transplantation of recombinant and mutagenized pluripotent stem cells not previously afforded to this rapid and versatile model system.

2021-03-23 | GSE158706 | 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

RNA Seq analysis of Schmidtea mediterranea treated with RNAi against chd4, p53, or unc22 to identify factors involved in neoblast differentiation.

Project description:Adult stem cells are tissue-specific cells with the capacity to self-renew and differentiate to continually replace cells lost to normal physiological turnover or injury. Neoblasts, the planarian stem cells, are widely distributed throughout the body mesenchyme, driving constitutive renewal of tissues during homeostasis and endowing planarians with the remarkable capacity to regenerate wholly from tiny tissue fragments. Neoblasts are the only dividing cells in planarians and are believed to be collectively comprised of both a heterogeneous population of pluripotent cells with broad differentiation potential and also lineage-committed progenitor cells that give rise to specific tissues. Recent work has identified a prominent class of neoblasts referred to as zeta-class, which express the zinc-finger gene zfp-1. Zfp-1, along with the chromatin remodeling factor chd4, the tumor suppressor gene p53, and most recently an RNA-binding protein mex3-1, have all been shown to be required for maintenance of two related post-mitotic, sub-epidermal cell populations expressing the specific marker genes prog-1 and AGAT-1, which have been widely used to asses neoblast differentiation. Given that zeta-class neoblasts are required for the generation of prog-1 and AGAT-1 positive cells and other markers of epidermal cell types, prog-1 and AGAT-1 likely mark two major populations of epidermal progeny cells. To understand the molecular mechanisms underlying neoblast differentiation and how they give rise to multiple cell types, we devised a strategy to identify critical factors enriched in the AGAT-1 positive cell population required for epidermal lineage progression. We performed RNA-Seq analysis of chd4 and p53 RNAi animals and characterized additional markers enriched in AGAT-1 positive and related post-mitotic cells.

2015-10-21 | GSE72389 | GEO

Jiao2018 - Feedback regulation in a stem cell model with acute myeloid leukaemia

Project description:This is a mathematical model describing the hematopoietic lineages with leukemia lineages, as controlled by end-product negative feedback inhibition. Variables include hematopoietic stem cells, progenitor cells, terminally differentiated HSCs, leukemia stem cells, and terminally differentiated leukemia stem cells.

2024-09-02 | BIOMD0000000898 | BioModels

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