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An RNAi Screen Reveals Intestinal Regulators of Branching Morphogenesis, Differentiation, and Stem Cell Proliferation in Planarians

Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Developmental Cell (Impact Factor: 10.37). 10/2012; 23(4):691-704. DOI: 10.1016/j.devcel.2012.09.008
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ABSTRACT Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of postmitotic tissues. Understanding how these processes are orchestrated requires characterizing cell-type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling postembryonic organogenesis.

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    • "Indeed, in schistosomes RNAi lasts up to 40 days, and in O. viverrini for at least nine days [33]. Furthermore, because feeding is an efficient RNAi delivery method in planarians for genes expressed in diverse tissues and locations, this would indicate that free living flatworms are able to incorporate RNAi inducers and transport the signal [58] [59]. Likewise our confocal results suggest that diffusion of the silencing signal also in F. hepatica. "
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    ABSTRACT: In trematodes RNA interference is the current tool of choice for functional analysis of genes since classical reverse genetic approaches remain unavailable. Whereas this approach has been optimized in schistosomes, few reports are available for other trematodes, likely reflecting the difficulties in the establishment of the technology. Here we standardized conditions for RNAi in the liver fluke Fasciola hepatica, the causative agent of fasciolosis, one of the most problematic infections affecting livestock worldwide. Targeting a single copy gene, encoding leucine aminopeptidase (LAP) as a model, we refined delivery conditions which identified electro-soaking, i.e. electroporation and subsequent incubation as efficient for introduction of small RNAs into the fluke. Knock down of LAP was achieved with as little as 2.5μg/ml dsRNA concentrations, which may reduce or obviate off-target effects. However, at these concentrations, tracking incorporation by fluorescent labeling was difficult. While both long dsRNA and short interfering RNA (siRNA) are equally effective at inducing a short-term knock down, dsRNA induced persistent silencing up to 21 days after treatment, suggesting that mechanisms of amplification of the interfering signal can be present in this pathogen. Persistent silencing of the invasive stage for up to 3 weeks (close to what it takes for the fluke to reach the liver) opens the possibility of using RNAi for the validation of putative therapeutic targets.
    Molecular and Biochemical Parasitology 10/2014; 197(1-2). DOI:10.1016/j.molbiopara.2014.10.001 · 2.24 Impact Factor
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    • "As shown in Fig. 2, the PIC enabled high quality in vivo imaging in multiple channels and at different magnifications (ranging from 4× to 40×). We imaged stained cell nuclei (Fig. 2 (A, B)) and mitochondria (Fig. 2 (C)) and the planarian gut (Fig. 2 (D)) several hours after the worm had been fed organic beef liver laced with several microliters of fluorescent dye (see Methods and Ref.38). Notably, we obtained quality images at high magnification of the ocelli region (Fig. 2 (B)), which contains the photoreceptors and is therefore the most light sensitive region in the animal at the wavelengths used15. "
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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.
    Scientific Reports 09/2014; 4:6388. DOI:10.1038/srep06388 · 5.58 Impact Factor
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    • "In the case of the epithelial cells lining the cavity and external wall of the pharynx, the absence of staining is consistent with the fact that their nuclei , where the bulk of the SL RNA signal accumulates (see Section 3.4), are located in the cell bodies sunken into the parenchyma (Ishii, 1964, 1966). Similarly, the gut epithelium is formed by cells with an extremely high cytoplasm/nuclear volume ratio (Forsthoefel et al., 2012) and this may therefore give the impression of lack of SL1 expression. Whether SL1 transcription and SL1 trans-splicing occur at some level in the gastrodermis remains to be determined using more sensitive methodologies . "
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    ABSTRACT: Spliced leader (SL) trans-splicing is a biological phenomenon, common among many metazoan taxa, consisting in the transfer of a short leader sequence from a small SL RNA to the 5' end of a subset of pre-mRNAs. While knowledge of the biochemical mechanisms driving this process has accumulated over the years, the functional consequences of such post-transcriptional event at the organismal level remain unclear. In addition, the fact that functional analyses have been undertaken mainly in trypanosomes and nematodes, leaves a somehow fragmented picture of the possible biological significance and evolution of SL trans-splicing in eukaryotes. Here, we analyzed the spatial expression of SL RNAs in the planarian flatworm Schmidtea mediterranea, with the goal of identifying novel developmental paradigms for the study of trans-splicing in metazoans. Besides the previously identified SL1 and SL2, S. mediterranea expresses a third SL RNA described here as SL3. While, SL1 and SL2 are collectively expressed in a broad range of planarian cell types, SL3 is highly enriched in a subset of the planarian stem cells engaged in regenerative responses. Our findings provide new opportunities to study how trans-splicing may regulate the phenotype of a cell.
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