RNAi screening strategy. (A) S. mediterranea cDNAs were transferred into pDONRdT7, which contains two T7 promoters and terminators, using a single-step Gateway (Invitrogen) reaction (see methods). (B) Screening procedure (see methods for details). (C) Animals with a phenotype were labelled with αH3P (mitotic neoblasts) and VC-1 (photoreceptor neurons). Animals with no phenotype were labelled with VC-1 and screened for defects. (D) 143 genes that conferred phenotypes following RNAi and amputation were inhibited with five dsRNA feedings, left intact, and observed for six weeks. Presence and capacity to divide of neoblasts was assessed by amputation, fixation, and labeling with αH3P (see methods). (E) Systematic phenotype nomenclature: uppercase “phenotype terms” with lowercase “descriptors” in parentheses, with lowercase “modifiers” in brackets. Blastema size descriptors refer to mean animal blastema size, ranging from 0 (none) to 3 (normal). e.g., BLST(0.5) indicates half of the animals had no detectable blastema and half of the animals had a size “1” blastema. This glossary can be used to examine phenotypes shown in Tables 2, S1, S4, Figure 2, Figure 6 and supplemental tables. (F) Planarian anatomy and terminology for body regions used to describe phenotypes. Wild-type dorsal surfaces, clonal line CIW-4 asexual S. mediterranea animal (left) and regenerating animal (right) face the viewer. Anterior, top. Bar, 0.1 mm. Photoreceptors (PR) sense light. The brain region (BRN) lies posterior and ventral to the PR. The pharynx (PHX) controls feeding and defecation. Head and tail were removed from the regenerating animal (right). Nine days of regeneration have occurred, and blastemas (BLST) are visible (unpigmented regions).

Identification of genes with candidate functions in planarian regeneration. (A) Planarian regeneration is divided into seven stages, “I” through “VII”. Anterior region of a decapitated planarian is shown, facing up. Wound healing involves epidermal cell spreading. The blastema is anterior and white. Stages correspond to numbered text sections in which phenotype categories are discussed. Neoblasts are depicted only when a process involving their function is described. V, pharynx formation occurs in pre-existing tissue of a fragment lacking original pharynx. (B) Gene groups sharing phenotype profiles are summarized. Profiles identify genes with predicted functions corresponding to regeneration stages in (A). Some genes are found in multiple categories. LYS, lysis. Reg, regeneration (blastema formation); “abort”, too small or no blastema. CRL, curling. BLST, blastema. VC-1, abnormal photoreceptors (see text, Table S3). PHX, pharynx regeneration in tail fragments. RGRS, tissue regression. BHV, behavior abnormal. H3P categorization as in Figure 4, Table S3. LES; lesions. Novel, no predicted function. Specific, if predicted to encode proteins involved in signal transduction, transcription, cell adhesion, neuronal functions, disease, RNA binding, channels/transporter function, cytoskeletal regulation. Basal, if predicted to encode proteins involved in translation, metabolism, RNA splicing, proteolysis, protein folding, vesicle trafficking, cell cycle, or cytoskeleton machinery.

Analyses of blastema differentiation and pattern formation in animals with screen phenotypes. (A–N) Anterior, left. (A) Photoreceptor system defect terminology. EXTNT, photoreceptor regeneration extent abnormal; descriptors: nopr, no photoreceptors; trce, trace development; ltd, limited development; sqish, slightly underdeveloped. (B) Normal photoreceptors. oc, optic chiasmata. cb, cell bodies. (C–N) Representative defects. Bar, 0.1 mm. Arrowheads, abnormalities. PRCELLS, photoreceptor cell bodies abnormal. Descriptors: wd, photoreceptors wide; difus, diffuse clustering; asym, asymmetry; trs, tears, ectopic neurons posterior to cluster; ecto, ectopic photoreceptor. DISORG, axon disorganization. Descriptors: straightoc, oc straight; splitoc, axons fail to cross midline; fwdproj, cell body projections toward anterior tip; ectoax, extra projections. H.68.4A RNAi cephalic ganglia were also labeled with α-synaptotagmin. (O) Cellular defect categorization. Left, each square represents mean phenotype of animals with a single gene inhibited. Y, blastema sizes: "0" (none) and "3" (normal). (P) X, Y scatter-plot coordinates for "phenotype profiles" are listed. Genes categorized as in 3B. Complete listing of data is in Table S3.

Representative phenotypes from the RNAi screen. Phenotype nomenclature and homologies are in Table 2. White arrowheads, defects. Anterior, left. v, ventral surface. Bar, 0.2 mm. (A) Control, unc-22 (a C. elegans gene) RNAi animal. Irradiation at 6000rad blocked regeneration (BLST(0), 8d) and caused curling (CRL, 15d). Black arrowhead, photoreceptor. P, pharynx. Brackets, blastema (unpigmented). (B) Reduced regeneration, curling, and caudal regeneration defects. (C) Pointed, wide, and indented blastemas. (D) Diffuse, faint, and asymmetric photoreceptors. (E) Regression of the anterior tip and between the photoreceptors. (F) Lesions and lysis. (G) Bloated and blistered. (H) Sticking and stretching and hourglass postures. (I) Spots and freckles. (J) Growth and bump.

Representative mitotic defects in amputated animals. Anterior, left. (A) αH3P-labeling examples from the RNAi of 140 genes (see text). 14d, 14 days. Bar, 1 mm. Irradiated animals, 6000 rads. Control unc-22 RNAi animals had an average of 212±37 labeled cells/mm length (from photoreceptors to tail). Defects were categorized as LOW(v), LOW, LOW(s), normal, HIGH(s), HIGH, and HIGH(v) (“v”, very; “s”, slightly). LOW(s) threshold is set at control mean less 2× standard deviation (sd). This absolute value was divided into three equal ranges to set LOW and LOW(v). The same ranges added to the mean plus 2× sd set the high ranges. For those within 2× sd but visually abnormal, data were considered significant if P<0.01 (t-test). (B) Genes are characterized as in Figure 3B. (C) αH3P-labeling examples of RNAi animals fixed 16 or 24h, +/− feeding, after amputation (see text). Animals were fixed after the first or second amputation as appropriate. Control animals were fixed at 16h or 24h. Bar, 1 mm. Data categorized as in (A) above; numerator=number of genes in a particular category, 139=total number of genes analyzed. Complete table of results are in Table S4. (D) Each square represents mean phenotype of animals in which a single gene was inhibited by RNAi. Y axis, blastema size with 3=normal and 0=no regeneration. X axis, number of cells labelled with αH3P (see C). Colors represent whether RNAi of genes caused curling following amputation.

Homeostasis defects in dsRNA-fed animals. (A–D), Arrowheads, defects. v, ventral. Bar, 0.4 mm. Anatomy and nomenclature, Figure 1E, F. Additional terms: all, entire animal; ant, anterior half or the anterior end of a region; int, gastrovascular system. (A) unc-22 RNAi, negative control. Irradiation at 6000 rads caused tissue regression (8d) and curling (15d). (B) Regression. (C) Curling. (D) Lesions and lysis. (E–G), Each square represents mean phenotype with a single gene inhibited by RNAi. Square location for a given gene is the same in each panel. Y axis, blastema size with 3=normal and 0=no regeneration. X axis, number of cells labelled with αH3P (see Figure 4C). (E) Colors represent defects in intact RNAi animals. (F) Colors represent regression and curling defects in intact RNAi animals. (G) Colors represent lesion formation in intact RNAi animals.