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
Source: PubMed

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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    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.
    Gene 10/2013; DOI:10.1016/j.gene.2013.09.101 · 2.08 Impact Factor
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    ABSTRACT: COVER PHOTOGRAPH: Low magnification image of planarian flatworms (Schmidtea mediterranea) fed with liver paste containing either red, green, yellow, or blue food dye. Uptake of colored liver paste containing in vitro synthesized double-stranded RNA is readily visualized in the planarian gut. Ingestion and processing of double-stranded RNA in the planarian gut leads to systemic RNA-interference. Image credit: Labib Rouhana and Phillip A. Newmark, Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign. From RNA Interference by feeding in vitro-synthesized double-stranded RNA to planarians, Labib Rouhana, Jennifer A. Weiss, David J. Forsthoefel, Hayoung Lee, Ryan S. King, Takeshi Inoue, Norito Shibata, Kiyokazu Agata, and Phillip A. Newmark, Developmental Dynamics 242:718-730, 2013.
    Developmental Dynamics 06/2013; 242(6):C1. DOI:10.1002/dvdy.23982 · 2.67 Impact Factor
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    ABSTRACT: BACKGROUND: 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. RESULTS: 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 show 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. CONCLUSION: 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.
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