Stem cell systems and regeneration in planaria
TL;DR: Planarians as an experimental system provide unique opportunities for addressing a spectrum of current problems in stem cell research, including the evolutionary conservation of pluripotency, the dynamic organization of differentiation lineages and the mechanisms underlying organismal stem cell homeostasis.
Abstract: Planarians are members of the Platyhelminthes (flatworms). These animals have evolved a remarkable stem cell system. A single pluripotent adult stem cell type (“neoblast”) gives rise to the entire range of cell types and organs in the planarian body plan, including a brain, digestive-, excretory-, sensory- and reproductive systems. Neoblasts are abundantly present throughout the mesenchyme and divide continuously. The resulting stream of progenitors and turnover of differentiated cells drive the rapid self-renewal of the entire animal within a matter of weeks. Planarians grow and literally de-grow (“shrink”) by the food supply-dependent adjustment of organismal turnover rates, scaling body plan proportions over as much as a 50-fold size range. Their dynamic body architecture further allows astonishing regenerative abilities, including the regeneration of complete and perfectly proportioned animals even from tiny tissue remnants. Planarians as an experimental system, therefore, provide unique opportunities for addressing a spectrum of current problems in stem cell research, including the evolutionary conservation of pluripotency, the dynamic organization of differentiation lineages and the mechanisms underlying organismal stem cell homeostasis. The first part of this review focuses on the molecular biology of neoblasts as pluripotent stem cells. The second part examines the fascinating mechanistic and conceptual challenges posed by a stem cell system that epitomizes a universal design principle of biological systems: the dynamic steady state.
Citations
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01 May 2011
TL;DR: In this article, the authors used ionizing radiation and single-cell transplantation to identify clonogenic neoblasts that can form large descendant-cell colonies in vivo, and concluded that broadly distributed, adult pluripotent stem cells underlie the remarkable regenerative abilities of planarians.
Abstract: Pluripotent cells in the embryo can generate all cell types, but lineage-restricted cells are generally thought to replenish adult tissues. Planarians are flatworms and regenerate from tiny body fragments, a process requiring a population of proliferating cells (neoblasts). Whether regeneration is accomplished by pluripotent cells or by the collective activity of multiple lineage-restricted cell types is unknown. We used ionizing radiation and single-cell transplantation to identify neoblasts that can form large descendant-cell colonies in vivo. These clonogenic neoblasts (cNeoblasts) produce cells that differentiate into neuronal, intestinal, and other known postmitotic cell types and are distributed throughout the body. Single transplanted cNeoblasts restored regeneration in lethally irradiated hosts. We conclude that broadly distributed, adult pluripotent stem cells underlie the remarkable regenerative abilities of planarians.
443 citations
TL;DR: It is shown that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH and that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments.
Abstract: The freshwater planarian Schmidtea mediterranea has emerged as a powerful model for studies of regenerative, stem cell, and germ cell biology Whole-mount in situ hybridization (WISH) and whole-mount fluorescent in situ hybridization (FISH) are critical methods for determining gene expression patterns in planarians While expression patterns for a number of genes have been elucidated using established protocols, determining the expression patterns for particularly low-abundance transcripts remains a challenge We show here that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH To further improve signal sensitivity we optimized blocking conditions for multiple anti-hapten antibodies, developed a copper sulfate quenching step that virtually eliminates autofluorescence, and enhanced signal intensity through iterative rounds of tyramide signal amplification For FISH on regenerating planarians, we employed a heat-induced antigen retrieval step that provides a better balance between permeabilization of mature tissues and preservation of regenerating tissues We also show that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments Finally, we apply these modifications to elucidate the expression patterns of a few low-abundance transcripts The modifications we present here provide significant improvements in signal intensity and signal sensitivity for WISH and FISH in planarians Additionally, these modifications might be of widespread utility for whole-mount FISH in other model organisms
254 citations
Cites background from "Stem cell systems and regeneration ..."
...After captivating scientists with their remarkable regenerative capacity for over a century [2], significant progress is being made in understanding how planarians reestablish axial polarity following injury [3-5], how their stem cells regulate choices between self-renewal and differentiation [6,7], and how their organ systems,...
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TL;DR: RNA interference is the only technique available to assess gene function in planarians in which it has been induced by means of injection of double‐stranded RNA, soaking, or ingestion of bacteria expressing dsRNA.
Abstract: Background
The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA.
Results
We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery.
Conclusions
This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. Developmental Dynamics 242:718–730, 2013. © 2013 Wiley Periodicals, Inc.
189 citations
TL;DR: The genome assembly of S. mediterranea is reported, using long-read sequencing and a de novo assembler enhanced for low-complexity reads to provide a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms.
Abstract: The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea, using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms.
168 citations
TL;DR: The availability of D. lacteum as a regeneration-impaired planarian model species provides a first step towards elucidating the evolutionary mechanisms that ultimately determine why some animals regenerate and others do not.
Abstract: Species capable of regenerating lost body parts occur throughout the animal kingdom, yet close relatives are often regeneration incompetent. Why in the face of 'survival of the fittest' some animals regenerate but others do not remains a fascinating question. Planarian flatworms are well known and studied for their ability to regenerate from minute tissue pieces, yet species with limited regeneration abilities have been described even amongst planarians. Here we report the characterization of the regeneration defect in the planarian Dendrocoelum lacteum and its successful rescue. Tissue fragments cut from the posterior half of the body of this species are unable to regenerate a head and ultimately die. We find that this defect originates during the early stages of head specification, which require inhibition of canonical Wnt signalling in other planarian species. Notably, RNA interference (RNAi)-mediated knockdown of Dlac-β-catenin-1, the Wnt signal transducer, restored the regeneration of fully functional heads on tail pieces, rescuing D. lacteum's regeneration defect. Our results demonstrate the utility of comparative studies towards the reactivation of regenerative abilities in regeneration-deficient animals. Furthermore, the availability of D. lacteum as a regeneration-impaired planarian model species provides a first step towards elucidating the evolutionary mechanisms that ultimately determine why some animals regenerate and others do not.
149 citations
References
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TL;DR: Induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions is demonstrated and iPS cells, designated iPS, exhibit the morphology and growth properties of ES cells and express ES cell marker genes.
23,959 citations
"Stem cell systems and regeneration ..." refers background in this paper
...However, only a small fraction of cells overexpressing all reprogramming factors ever become pluripotent (Takahashi and Yamanaka 2006)....
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...…discovery that overexpressing a cocktail of transcription factors can convert terminally differentiated cells back into a pluripotent state has placed dramatic emphasis on the importance of transcriptional mechanisms in the establishment andmaintenance of pluripotency (Takahashi and Yamanaka 2006)....
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...However, the discovery that overexpressing a cocktail of transcription factors can convert terminally differentiated cells back into a pluripotent state has placed dramatic emphasis on the importance of transcriptional mechanisms in the establishment andmaintenance of pluripotency (Takahashi and Yamanaka 2006)....
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...…pluripotent stem cells, but pluripotent cells can be derived either from early embryos (embryonic stem [ES] cells; Weissman 2000) or via the recently discovered “reprogramming” of somatic cells back into a quasi-embryonic state (induced pluripotent stem [IPS] cells; Takahashi and Yamanaka 2006)....
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TL;DR: An update of the core Wnt/β-catenin signaling pathway is provided, how its various components contribute to disease, and outstanding questions to be addressed in the future are discussed.
4,561 citations
"Stem cell systems and regeneration ..." refers background in this paper
...β-Catenin is the intracellular effector of canonical Wnt signals (Clevers and Nusse 2012)....
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...Mechanistic detail is once again lacking, but the profound effects of altered Smedβ-catenin-1 activity are conceptually important. β-Catenin is the intracellular effector of canonical Wnt signals (Clevers and Nusse 2012)....
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TL;DR: Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt.
Abstract: In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing.
3,641 citations
"Stem cell systems and regeneration ..." refers background in this paper
...The TOR pathway, which has diverse functions in cell growth and metabolism (Zoncu et al. 2011), recently emerged as an important component in orchestrating the Neoblast response to wounding and regeneration (Peiris et al....
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...The TOR pathway, which has diverse functions in cell growth and metabolism (Zoncu et al. 2011), recently emerged as an important component in orchestrating the Neoblast response to wounding and regeneration (Peiris et al. 2012; Tu et al. 2012; González-Estévez et al. 2012b)....
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TL;DR: It is shown that relatively simple molecular mechanisms based on auto- and cross catalysis can account for a primary pattern of morphogens to determine pattern formation of the tissue, and the theory is applied to quantitative data on hydra and is shown to account for activation and inhibition of secondary head formation.
Abstract: One of the elementary processes in morphogenesis is the formation of a spatial pattern of tissue structures, starting from almost homogeneous tissue. It will be shown that relatively simple molecular mechanisms based on auto- and cross catalysis can account for a primary pattern of morphogens to determine pattern formation of the tissue. The theory is based on short range activation, long range inhibition, and a distinction between activator and inhibitor concentrations on one hand, and the densities of their sources on the other. While source density is expected to change slowly, e.g. as an effect of cell differentiation, the concentration of activators and inhibitors can change rapidly to establish the primary pattern; this results from auto- and cross catalytic effects on the sources, spreading by diffusion or other mechanisms, and degradation. Employing an approximative equation, a criterium is derived for models, which lead to a striking pattern, starting from an even distribution of morphogens, and assuming a shallow source gradient. The polarity of the pattern depends on the direction of the source gradient, but can be rather independent of other features of source distribution. Models are proposed which explain size regulation (constant proportion of the parts of the pattern irrespective of total size). Depending on the choice of constants, aperiodic patterns, implying a one-to-one correlation between morphogen concentration and position in the tissue, or nearly periodic patterns can be obtained. The theory can be applied not only to multicellular tissues, but also to intracellular differentiation, e.g. of polar cells. The theory permits various molecular interpretations. One of the simplest models involves bimolecular activation and monomolecular inhibition. Source gradients may be substituted by, or added to, sink gradients, e.g. of degrading enzymes. Inhibitors can be substituted by substances required for, and depleted by activation. Sources may be either synthesizing systems or particulate structures releasing activators and inhibitors. Calculations by computer are presented to exemplify the main features of the theory proposed. The theory is applied to quantitative data on hydra — a suitable one-dimensional model for pattern formation — and is shown to account for activation and inhibition of secondary head formation.
2,832 citations
"Stem cell systems and regeneration ..." refers background in this paper
...Pattern formation, the analysis of collective properties arising from dynamic interactions (Bois et al. 2011; Gierer and Meinhardt 1972; Howard et al. 2011), provides an appealing conceptual framework for rationalizing de novo organ regeneration....
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TL;DR: The review begins with a detailed examination of hematopoi-via the umbilical vein to the fetal liver between dpc 8.5 etic (blood-forming) stem cells, and the transcription pro-The earliest stem cells in ontogeny are totipotent, ex- files of each of these populations are quite distinct.
1,890 citations
"Stem cell systems and regeneration ..." refers background in this paper
...PCNA and H3P as stem cell markers? In other systems, true stem cells capable of self-renewal and the production of multiple progeny are generally rare amongst the cycling cells within a tissue (Weissman 2000)....
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...In other systems, true stem cells capable of self-renewal and the production of multiple progeny are generally rare amongst the cycling cells within a tissue (Weissman 2000)....
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...Stem cell homoeostasis has to balance the two foundations of stemness, self renewal and differentiation into multiple progeny (Weissman 2000)....
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...Adult vertebrates do not harbor pluripotent stem cells, but pluripotent cells can be derived either from early embryos (embryonic stem [ES] cells; Weissman 2000) or via the recently discovered “reprogramming” of somatic cells back into a quasi-embryonic state (induced pluripotent stem [IPS] cells;…...
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