Developmental Cell, Vol. 8, 635–649, May, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.devcel.2005.02.014 Identification of Genes Needed for Regeneration, Stem Cell Function, and Tissue Homeostasis by Systematic Gene Perturbation in Planaria Peter W. Reddien, Adam L. Bermange,1,2 number of attributes not manifested by current ecdyso- Kenneth J. Murfitt,1 Joya R. Jennings, zoan model systems (e.g., C. elegans and Drosophila), and Alejandro Sánchez Alvarado* such as regeneration and adult somatic stem cells. Department of Neurobiology and Anatomy Therefore, studies of planarian biology will help the un- University of Utah School of Medicine derstanding of processes relevant to human develop- 401 MREB, 20N 1900E ment and health not easily studied in current inverte- Salt Lake City, Utah 84132 brate genetic systems. Neoblasts are the only known proliferating cells in adult planarians and reside in the parenchyma. Follow- Summary ing injury, a neoblast proliferative response is triggered, generating a regeneration blastema consisting of ini- Planarians have been a classic model system for the tially undifferentiated cells covered by epidermal cells. study of regeneration, tissue homeostasis, and stem Moreover, essentially all tissues in adult planarians turn cell biology for over a century, but they have not his- over and are replaced by neoblast progeny. Although torically been accessible to extensive genetic ma- the characteristics and diversity of the neoblasts still nipulation. Here we utilize RNA-mediated genetic await careful molecular elucidation, neoblasts may be interference (RNAi) to introduce large-scale gene in- totipotent stem cells (Reddien and Sánchez Alvarado, hibition studies to the classic planarian system. 1065 2004). The use of stem cells to replace aged or dam- genes were screened. Phenotypes associated with aged cells is important to the life of most metazoans, the RNAi of 240 genes identify many specific defects including humans, but poorly understood mechanisti- cally (Weissman, 2000). Thus, planarians provide are- in the process of regeneration and define the major nas for mechanistic investigation of in vivo stem cell categories of defects planarians display following regulation that are likely to inform the functioning of gene perturbations. We assessed the effects of inhib- stem cells in all animals. iting genes with RNAi on tissue homeostasis in intact How can the function of genes regulating planarian animals and stem cell (neoblast) proliferation in am- biology be explored? One approach that has been piv- putated animals identifying candidate stem cell, re- otal in understanding the biology of multiple metazo- generation, and homeostasis regulators. Our study ans, including D. melanogaster (Nusslein-Volhard and demonstrates the great potential of RNAi for the sys- Wieschaus, 1980), C. elegans (Brenner, 1974), and tematic exploration of gene function in understudied Danio rerio (Driever et al., 1996; Haffter et al., 1996), organisms and establishes planarians as a powerful involves large-scale functional genetic surveys or mu- model for the molecular genetic study of stem cells, tant screens. Such an approach has historically been regeneration, and tissue homeostasis. precluded by planarian life cycles. The development of dsRNA-mediated genetic interference (RNAi) (Fire et al., Introduction 1998) and the application of RNAi to systematic studies of gene function (Fraser et al., 2000; Gonczy et al., Planarians are bilaterally symmetric metazoans re- 2000) has opened the door for a new generation of ge- nowned for their regenerative capacities, extensive tis- netic manipulations. Since RNAi is an effective method- sue turnover as part of their normal homeostasis, and ology for perturbing planarian gene function (Sánchez the presence of a pluripotent adult stem cell population Alvarado and Newmark, 1999), the in vivo activities of known as the neoblasts (Reddien and Sánchez Alva- planarian genes can now be addressed for the first rado, 2004). Planarians have derivatives of all three time. We developed an RNAi-based screening strategy germ layers, bilobed cephalic ganglia, photoreceptors and performed the first large-scale survey of gene func- and other sensory cell types, cilia and a muscular sys- tion in planarian biology, with an emphasis on the pro- tem for locomotion and negotiating objects, a gastro- cesses of tissue homeostasis and regeneration. We vascular system for digestion, a muscular pharynx, an selected 1065 genes, intended to be a representative excretory system, and an epidermis (Hyman, 1951). sampling of the planarian S. mediterranea genome, and Surrounding the branched gastrovascular system is a found that RNAi of 240 of these genes generated phe- mesenchymal tissue known as the parenchyma (Hy- notypes. Inherently potent in the RNAi-screening strat- man, 1951). Planarians are currently viewed as mem- egy is the fact that known gene sequences allow bers of the Lophotrochozoa, which are one of the three immediate association of phenotypes with predicted major phyletic groupings of bilaterally symmetric ani- biochemical function(s). Since this screen is the first mals (Adoutte et al., 2000). The Lophotrochozoa are systematic investigation of gene function in planarians, comprised of a diverse set of animals that display a essentially all of the RNAi phenotypes and associated genes are new. Additional experiments involving the study of gene function in homeostasis, neoblast prolif- *Correspondence: [email protected] eration, and blastema differentiation and patterning al- 1These authors contributed equally to this work. 2 Present address: London Research Institute, Lincoln’s Inn Fields lowed grouping of genes into functional categories. To- Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3PX, United gether, our data present a broad survey of the genetic Kingdom. control of planarian biology and identify genes with Developmental Cell 636 candidate functions in the sequential events of regener- gories. There is, however, a practical problem involving ation. the comparisons of phenotypes that are descriptive. Because essentially all of the phenotypes reported in Results this manuscript are new, we have devised a nomencla- ture system that allows common usage of descriptive An RNAi Screen in S. mediterranea terms for different phenotypes and comparison of dif- RNAi has been demonstrated to result in specific and ferent regeneration defects. To assist the reader, major near complete elimination of detectable S. mediterra- terms used to define phenotypes are listed in Figure nea mRNA and proteins (Newmark et al., 2003; Sánchez 1E; planarian body regions related to locations where Alvarado and Newmark, 1999; data not shown). None- defects were observed, and the terms used in pheno- theless, since it is impractical to monitor protein levels type descriptions to identify these regions, are pre- for the products of each gene in an RNAi screen, some sented in Figure 1F. phenotypes resulting from dsRNA treatment could re- flect incomplete gene loss of function. The RNAi by 240 Genes for which RNAi Generates a Phenotype feeding methodology used for our screen involves ex- Have Been Identified pressing dsRNA from a planarian gene in bacteria and Of the 1065 genes perturbed by RNAi, 240 (22.5%) con- suspending those bacteria with blended liver and agar- ferred specific phenotypes when perturbed (Tables 1 ose (Newmark et al., 2003). We optimized the feeding and 2; see Supplemental Table S1 available with this method and protocol used in this manuscript (Figure 1, article online). A sampling of the spectrum of pheno- see Experimental Procedures) through extensive exper- types observed can be found in Table 1 and Figures imentation (data not shown). We generated an RNAi 2A–2J. Genes are identified in the tables and figures vector (pDONRdT7) that contains two T7 RNA polymer- with an RNAi clone identification name in which the let- ase promoters flanked by two class I T7 transcriptional ters “H” and “NB” refer to genes from the head- and terminators (Figure 1A) that results in more effective neoblast-enriched cDNA libraries, respectively, “E” in- RNAi than does the conventional vector (data not dicates that clones are “entry” clones in the Gateway shown). pDONRdT7 utilizes a modified Gateway clon- naming system, and the alphanumerical code refers to ing strategy (Invitrogen) to facilitate cDNA transfer. 96-well plate coordinates. Many phenotypic categories S. mediterranea cDNAs randomly selected from two were uncovered, including the inability to regenerate cDNA libraries were inserted into pDONRdT7 and intro- (Figure 2B). Blastema-sized abnormalities have been duced into the RNaseIII-deficient bacterial strain HT115 categorized on a scale from 0 to 3, with “BLST(0)” refer- (Timmons et al., 2001). The two cDNA libraries were de- ring to no regeneration and “BLST(3)” referring to nor- rived from a neoblast-enriched cell population and ani- mal regeneration (Figures 1E and 2B). Other major phe- mal heads; all cDNAs used define unique genes (see notypic categories include ventral curling (Figure 2B), Experimental Procedures). 1065 genes were inhibited blastema shape and morphology abnormalities (Figure using RNAi by feeding. Given the fact that the S. medi- 2C), a variety of photoreceptor abnormalities (Figure terranea genome sequence is currently incomplete, it 2D), behavioral defects (Table 2; Supplemental Table is unknown what percentage of the total number of S1), tissue regression (Figure