Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians

Abstract

Freshwater planarian flatworms are capable of regenerating complete organisms from tiny fragments of their bodies; the basis for this regenerative prowess is an experimentally accessible stem cell population that is present in the adult planarian. The study of these organisms, classic experimental models for investigating metazoan regeneration, has been revitalized by the application of modern molecular biological approaches. The identification of thousands of unique planarian ESTs, coupled with large-scale whole-mount in situ hybridization screens, and the ability to inhibit planarian gene expression through double-stranded RNA-mediated genetic interference, provide a wealth of tools for studying the molecular mechanisms that regulate tissue regeneration and stem cell biology in these organisms. Here we show that, as in Caenorhabditis elegans, ingestion of bacterially expressed double-stranded RNA can inhibit gene expression in planarians. This inhibition persists throughout the process of regeneration, allowing phenotypes with disrupted regenerative patterning to be identified. These results pave the way for large-scale screens for genes involved in regenerative processes.

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

“…Although most cells possess the basic RNAi core machinery, some cell types have the intriguing ability to naturally take up exogenous dsRNA and use it to initiate RNAi silencing 2,3,[5][6][7] . Furthermore, some organisms, such as plants, C. elegans and planaria (Girardia tigrina) can transmit the RNA silencing signal from cell to cell, resulting in the systemic spread of the RNAi response 8,26,29,30 . It is currently believed that insects lack a pathway for the systemic spreading of RNAi.…”

“…Thus, the basic machinery that mediates dsRNA upake in Drosophila is also required for systemic spread of the RNAi signal in C. elegans, suggesting that the dsRNA entry pathway is evolutionarily conserved and functionally relevant in intact organisms. 8,26,29,30 . It is currently believed that insects lack a pathway for the systemic spreading of RNAi.…”

BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration

“…Animals were fixed as previously described (Newmark et al, 2003;Sánchez Alvarado and Newmark, 1999). Following rehydration animals were blocked for 6 hours at room temperature (RT) in PBTxB (PBS+0.3% Triton X-100+0.25% BSA), then incubated overnight with 1:5000 VC-1 (kind gift of Dr K. Agata, RIKEN, Kobe, Japan), 1:133 anti-Synaptotagmin (kind gift of Dr K. Agata), 1:400 anti-acetylated tubulin .…”

“…How do wounded planarian tissues specify what to regenerate, and what rules govern the rearrangement of body coordinates? Recent technological developments (Newmark and Sánchez Alvarado, 2000;Newmark et al, 2003;Reddien et al, 2005a;Sánchez Alvarado and Newmark, 1999) make it now possible to begin to address these long-standing questions.…”

Entre homéostasie et développement, quelles stratégies pour régénérer ?

“…En particulier dans les deux modèles, il est possible d'éteindre spécifiquement mais transitoirement l'expression de gènes grâce à l'ARN interférence, obtenue en nourrissant les animaux avec des bactéries qui produisent des ARNs double-brin (Newmark et al, 2003;Reddien et al, 2005;Chera et al, 2006;Buzgariu et al, 2008). Dans les deux systèmes, le séquençage de collections d'ADNc et de séquences génomiques a mis en évidence la grande conservation des cascades de signalisation au sein des eumétazoaires (Guder et al, 2006;Sanchez Alvarado & Tsonis, 2006;Chapman et al, 2010).…”