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: 9.71). 10/2012; 23(4):691-704. DOI: 10.1016/j.devcel.2012.09.008
Source: PubMed


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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    • "Future experiments should determine if wwp1 also functions together with a Krüppel - like factor to regulate longevity in S . mediterranea or if a novel mechanism exists in planarians . In addition , wwp1 ( RNAi ) animals showed a decrease in the neoblast population . Recent evidence suggests that the intes - tine might serve as a neoblast niche ( Forsthoefel et al . , 2012 ) ; thus , inhibition of wwp1 function in the intestine might also underlie neoblast regulation defects ."
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    ABSTRACT: E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation. Copyright © 2015. Published by Elsevier Inc.
    Developmental Biology 05/2015; 404(2). DOI:10.1016/j.ydbio.2015.04.021 · 3.55 Impact Factor
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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 · 1.79 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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