Wnt/Notum Spatial Feedback Inhibition Controls Neoblast Differentiation to Regulate Reversible Growth of the Planarian Brain Eric M

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STEM CELLS AND REGENERATION RESEARCH ARTICLE Wnt/Notum spatial feedback inhibition controls neoblast differentiation to regulate reversible growth of the planarian brain Eric M. Hill1 and Christian P. Petersen1,2,*

ABSTRACT 2013; Wills et al., 2008a,b); hence, they represent model systems Mechanisms determining final organ size are poorly understood. well suited for investigating the developmental mechanisms that Animals undergoing regeneration or ongoing adult growth are likely to underlie the extent of growth and attainment of size (Baguñà and require sustained and robust mechanisms to achieve and maintain Romero, 1981; Oviedo et al., 2003; Wada et al., 2013). appropriate sizes. Planarians, well known for their ability to undergo Planarians are flatworms that continue to undergo significant whole-body regeneration using pluripotent adult stem cells of the tissue turnover throughout adulthood and exhibit an extreme neoblast population, can reversibly scale body size over an order of capacity for growth control that allows reversible alteration of magnitude by controlling cell number. Using quantitative analysis, we body and organ size over an order of magnitude through the showed that after injury planarians perfectly restored brain:body regulation of cell number. Nutrient uptake leads to an increase in ‘ ’ proportion by increasing brain cell number through epimorphosis body size, whereas prolonged starvation results in degrowth ,a or decreasing brain cell number through tissue remodeling reduction of body size and cell number without an alteration in (morphallaxis), as appropriate. We identified a pathway controlling a relative tissue proportions or function (Baguñà and Romero, 1981; brain size set-point that involves feedback inhibition between wnt11- Morgan, 1898; Oviedo et al., 2003; Romero and Baguñà, 1991; 6/wntA/wnt4a and notum, encoding conserved antagonistic signaling Takeda et al., 2009). Additionally, planarians exhibit robustness in factors expressed at opposite brain poles. wnt11-6/wntA/wnt4a growth control through the process of regeneration. Planarians do undergoes feedback inhibition through canonical Wnt signaling not appreciably increase in size during regeneration, so small but is likely to regulate brain size in a non-canonical pathway amputated tissue fragments ultimately become small but well- independently of beta-catenin-1 and APC. Wnt/Notum signaling formed individuals, in which overt body proportionality appears tunes numbers of differentiated brain cells in regenerative growth and broadly restored (Morgan, 1898). Planarians accomplish tissue remodeling by influencing the abundance of brain progenitors regeneration both by the production of missing structures in a descended from pluripotent stem cells, as opposed to regulating cell regeneration blastema (termed epimorphosis) and through death. These results suggest that the attainment of final organ size remodeling of pre-existing tissues [originally termed morphallaxis might be accomplished by achieving a balance of positional signaling (Morgan, 1898)]. The term morphallaxis has also been used to inputs that regulate the rates of tissue production. describe putative regeneration mechanisms that might occur independently of cell proliferation (Morgan, 1901); therefore, for KEY WORDS: Wnt signaling, Notum, Organ size, Planaria, clarity we will describe the process of injury-induced alterations to Regeneration, Tissue remodeling pre-existing tissues as ‘tissue remodeling’ (Forsthoefel et al., 2011; Oviedo et al., 2003). Planarians reproduce asexually by fission, so INTRODUCTION they are a suitable model to study the mechanisms of natural Most animal species possess stereotyped body forms, in which proportion regulation and organ sizing through control of cell organs and appendages grow to defined proportions with respect to number (Oviedo et al., 2003). total body size. Mutations that alter organ proportion can underlie The relationships between epimorphosis and tissue remodeling evolutionary changes (Abzhanov et al., 2004; Jones et al., 2012) and are not fully understood, but several cellular events and molecular result in human developmental disorders, such as microcephaly pathways have been identified as contributing to each of these (Mochida and Walsh, 2001). Many molecular pathways have been processes. Growth from feeding or epimorphic regeneration described as contributing to growth regulation (Conlon and Raff, depends on parenchymal cells termed neoblasts that are the only 1999; Lander, 2011; Schwank and Basler, 2010; Tumaneng et al., proliferating cells in adult planarians (Baguñà, 1976; Newmark and 2012), primarily through genetic studies of species that cease or Sánchez Alvarado, 2000; Reddien et al., 2005b). Neoblasts are a largely dampen growth at the end of embryogenesis, but despite heterogeneous population containing both adult pluripotent stem considerable interest, the developmental mechanisms explaining cells (Wagner et al., 2011) and more lineage-restricted dividing cells size attainment largely remain a mystery. By contrast, species that (Adler et al., 2014; Cowles et al., 2013; Currie and Pearson, 2013; undergo regeneration and ongoing growth throughout adulthood Forsthoefel et al., 2012; Lapan and Reddien, 2012; Marz et al., must possess robust mechanisms to control animal form and 2013; Scimone et al., 2014a,b, 2011; van Wolfswinkel et al., 2014; proportion actively (Elliott and Sánchez Alvarado, 2013; Rink, Vásquez-Doorman and Petersen, 2014; Vogg et al., 2014; Wenemoser et al., 2012). Tissue removal results in proliferative 1Department of Molecular Biosciences, Northwestern University, Evanston, activation of neoblasts thought to be necessary for production of IL 60208, USA. 2Robert Lurie Comprehensive Cancer Center, Northwestern missing tissue types within the regeneration blastema. By contrast, University, Evanston, IL 60208, USA. the cellular basis for regenerative tissue remodeling is less well *Author for correspondence ([email protected]) understood but has been proposed to involve a wave of systemic injury-induced cell death proportional to the extent of tissue

Received 23 February 2015; Accepted 27 October 2015 removed by injury (Pellettieri et al., 2010). Within the intestine, DEVELOPMENT

4217 STEM CELLS AND REGENERATION Development (2015) 142, 4217-4229 doi:10.1242/dev.123612 regeneration involves both substantial differentiation of new cells numbers in decapitated animals forming a new brain through and incorporation of pre-existing cells, suggesting a complexity of epimorphosis (Fig. 1D, dotted lines; Fig. 1E, lower) or amputated tissue additions and alterations during regeneration (Forsthoefel head fragments undergoing brain remodeling (Fig. 1D, solid lines; et al., 2011). As planarians can perform tissue remodeling and Fig. 1E, upper). In order to allow evaluation of regenerative progress blastema formation through periods of starvation, it is possible that with respect to the complete body plan and across replicates of regulated autophagy could contribute to these processes (González- varying sizes, neural cell numbers were normalized to the total Estévez, 2009; González-Estévez et al., 2007). Perturbation of fragment length to give a value for brain:body proportion at each several molecular processes can influence body and organ size in time in regeneration (Fig. 1D). Relative brain size during planarians, including bioelectric signaling (Beane et al., 2012), FGF regeneration was compared with values interpolated from a power signaling (Cebrià et al., 2002a), JNK signaling (Almuedo-Castillo law regression analysis of brain cell number versus body length in et al., 2014), TORC1 signaling (González-Estévez et al., 2012b; uninjured animals to determine when appropriate organ size was Peiris et al., 2012; Tu et al., 2012) and insulin-like peptide signaling achieved (Fig. 1D, dashed black lines). In animals remodeling a pre- (Miller and Newmark, 2012). However, there is still a limited existing organ, brain:body proportion decreased rapidly during the understanding of the developmental and molecular events that 72 h following decapitation and continued to decrease until underlie the attainment and maintenance of appropriate organ size stabilizing around day 9. In epimorphic growth, cintillo+ and gad+ in planarians. neurons emerged around day 3, coincident with appearance of the We investigated the planarian brain as a model for regenerative brain primordia (Cebrià, 2007), and brain:body proportion organ size control, because it undergoes prominent and easily continued to increase until stabilizing by day 9. Throughout either identifiable changes in cell number (Oviedo et al., 2003). Here, we regeneration scenario, brains had a constant ratio of neuronal cell show that planarians completely restore appropriate brain:body numbers to brain length (Fig. S1F), indicating that proper scale proportions through either epimorphic growth or tissue remodeling. within the brain is established early and maintained through periods We find that the secreted Wnt inhibitor notum (Petersen and of organ size change (Takeda et al., 2009), whereas the proportion Reddien, 2011) is expressed in anterior brain neurons and promotes of the organ with respect to the body is subject to regulation. brain size in regeneration, remodeling and homeostasis. notum Although remodeling and epimorphosis ultimately achieved similar (RNAi) brain phenotypes are suppressed by inhibition of wnt11-6/ brain:body proportions, the absolute brain sizes of these fragments wntA/wnt4a (Gurley et al., 2010; Kobayashi et al., 2007; Riddiford were significantly different (Fig. S1G), indicating that whole-body and Olson, 2011), hereafter referred to as wnt11-6, an inhibitor of regeneration in planarians is not the exact replacement of cell brain size expressed in the posterior brain (Adell et al., 2009; numbers but rather the restoration of appropriate body form. Kobayashi et al., 2007). wnt11-6 signaling through beta-catenin-1 Therefore, regeneration can achieve correct brain proportions is required for notum expression in the brain, but is likely to inhibit through either epimorphosis or remodeling programs, suggesting brain size through β-catenin-independent signaling. This Wnt/ the existence of mechanisms that actively control organ notum negative feedback loop regulates brain:body proportion proportionality. through control of neoblast differentiation, rather than cell death, in both epimorphosis and remodeling. The ability of the planarian notum and wnt11-6 are expressed in neurons at opposite to restore organ proportionality provides a unique system to study poles of the brain organ size determination and control of the extent of regeneration. We took a candidate approach to identify molecules that control brain:body proportion in planarians, reasoning that mechanisms RESULTS underlying robust size attainment would involve organ-specific Planarians robustly restore brain:body proportionality secreted signals. Notum proteins are secreted lipases that deacylate through regeneration or remodeling Wnt ligands and prevent binding to Frizzled receptors (Kakugawa We first sought to clarify the precise relationships between brain cell et al., 2015; Zhang et al., 2015), thus inhibiting Wnt signaling in number, brain size and body size. We examined uninjured adult many animals, including planarians, fruit flies, zebrafish and planarians across a range of overall sizes using multiplex mammals (Flowers et al., 2012; Gerlitz and Basler, 2002; Giráldez fluorescence in situ hybridizations (FISH) to measure numbers of et al., 2002; Petersen and Reddien, 2011; Traister et al., 2008). abundant neurons [cholinergic neurons expressing choline Planarians have a single notum homolog, and in uninjured animals, acetyltransferase (chat) (Nishimura et al., 2010)] and more rare notum is expressed in the head at the anterior body pole and the cell types of the brain [putative chemosensory neurons expressing a anterior brain commissure (Fig. 2A, cyan) (Petersen and Reddien, degenerin homolog cintillo (Oviedo et al., 2003) and GABAergic 2011). Double FISH determined that notum expression at the neurons expressing glutamine decarboxylase (gad) (Nishimura anterior pole is within collagen+ body-wall muscle cells (Witchley et al., 2008); Fig. 1A,B]. Populations of all three neuronal cell types et al., 2013) (Fig. 2B, white arrows; 85.5% of notum+ anterior pole were correlated strongly with brain length (Fig. S1B,C) and body cells were collagen+), whereas notum expression at the anterior size (Fig. 1C; Fig. S1D), consistent with previous analyses (Oviedo brain commissure is within chat+ cholinergic neurons (90.1% of et al., 2003; Takeda et al., 2009). Neuronal cell size, as measured by notum+ cells at the anterior commissure were chat+; Fig. 2C, white in situ hybridization signal area for cintillo+ and gad+ neurons arrows). Notum proteins can inhibit Wnt signaling, so we sought to (labeling the cell body), did not vary with animal size, confirming identify a planarian Wnt gene expressed near the brain. Among the previous analyses indicating that control of cell number is a nine planarian Wnt genes, wnt11-6 has prominent expression in the principal method of body and organ size regulation in planarians posterior brain (Fig. 2A, magenta) in addition to dispersed cells (Fig. S1E) (Baguñà and Romero, 1981; Oviedo et al., 2003). Thus, throughout the body (Adell et al., 2009; Gurley et al., 2010; adult planarians maintain a specific number of brain-related cells Kobayashi et al., 2007). Double FISH determined that a majority of with respect to body size. wnt11-6 cells associated with the brain (51.3%) are expressed in We next examined whether brain:body proportionality is chat+ neurons (Fig. 2D, white arrows). notum and wnt11-6 are precisely restored through regeneration by measuring brain cell expressed at opposite ends of the brain in distinct expression DEVELOPMENT

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Fig. 1. Planarian brain:body proportion is restored through regeneration by either increasing or decreasing brain cell number as necessary. (A) FISH detecting chat expression in intact animals 2-8 mm in length. (B) Triple FISH detects chemosensory neurons (expressing cintillo, magenta), GABAergic neurons (expressing gad, cyan) and cholinergic neurons (expressing chat, gray). (C) Numbers of neuronal subpopulations (cintillo+, magenta; gad+, cyan) from differently sized uninjured animals plotted against body length. (D) Average numbers of cintillo+ neurons (top, magenta) and gad+ neurons (bottom, cyan) normalized to animal length during epimorphic regeneration of a new brain (dotted lines) or remodeling of the pre-existing brain (solid lines; averages of n≥5 samples; bars, s.d.) measured by FISH. Black dashed lines in D indicate interpolated brain proportion based on analysis of intact brain proportions (see Materials and Methods). (E-G) WISH showing cintillo expression in intact animals (E), head fragments remodeling a pre-existing brain (F) and trunk fragments regenerating a new brain through epimorphosis (G). (F,G) Black dashed lines indicate amputation plane; bottom panels show higher magnification view of top panels. Scale bars: 300 µm in A,B,E and F,G top panels; 150 µm in F,G bottom panels. Anterior, top. d, day. domains that are maintained and restored during brain remodeling (Fig. S3A), length of the pharynx neuropile at the distal end of and epimorphic brain growth, respectively, and we were not able to that organ (Fig. S3B) or total body length (Fig. S3C). Finally, identify cells that coexpress notum and wnt11-6 in any conditions inhibition of notum did not significantly alter the density of neurons (Fig. 2A,E). within the brain (Fig. S3D) or average neuronal cell size (Fig. S3E), indicating that notum is likely to regulate brain:body proportion notum and wnt11-6 are required for proper control of brain principally through the regulation of brain cell number. proportion Furthermore, although previous work identified notum as Given its regionalized expression within the brain, we next sought controlling anterior pole formation during head regeneration to identify putative functions of notum in the control of brain (Petersen and Reddien, 2011), inhibition of notum in head proportion. Inhibition of notum caused a reduction of cintillo+ and fragments did not eliminate expression of sFRP-1, a marker of the gad+ brain cell proportions in regenerating head fragments anterior pole (Fig. S4A). Therefore, the role of notum in brain sizing compared with control animals (Fig. 3A,B). Therefore, notum has is likely to be independent of anterior pole formation. Collectively, a role in promoting brain cell numbers during conditions of brain these results demonstrate a specific function for notum in promoting cell loss through tissue remodeling. notum(RNAi) regenerating head appropriate brain size in regenerative degrowth. fragments possessed normal regionalized expression of three notum(RNAi) head fragments undergoing brain remodeling homeotic transcription factors [orthopedia (otp) (Umesono et al., additionally formed an ectopic set of photoreceptors within the 1997), orthodenticle B (otxB) and orthodenticle A (otxA) (Umesono head tip anterior to the original photoreceptors (19 of 21 worms; et al., 1999)] and a brain branch marker [G protein alpha subunit Fig. S4B). Such animals had anterior chat+ neural tissue associated (gpas) (Cebrià et al., 2002b); Fig. S2], suggesting that notum with the ectopic photoreceptors and a more anterior placement of primarily affects size but not pattern of the brain. The size reduction the posterior brain boundary (Fig. 3A). We suggest that brain size phenotype appeared to be specific to the brain, because notum reduction through notum RNAi might change the position of an eye

RNAi did not change relative pharynx-to-body proportion field that could be set up through underlying tissue of the brain. DEVELOPMENT

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Fig. 2. notum and wnt11-6 are expressed in neurons at opposite poles of the brain. (A) Double FISH to detect notum (cyan), wnt11-6 (magenta) and chat (gray) expression in uninjured animals. notum is expressed at the anterior pole and in the brain commissure, whereas wnt11-6 is expressed at the posterior of each brain lobe (arrows). (B,C) Double FISH detecting expression of notum and either collagen (B) or chat (C). Of the notum+ cells at the anterior pole, 85.5±4.6% express collagen, marking musculature (out of 131 cells counted in five animals), whereas 90.1±4.4% of notum+ cells at the anterior commissure express chat, marking neurons (out of 81 notum+ cells counted in five animals; white arrows, double positive cells; yellow arrows, cells that express only notum). (D) Of the wnt11-6+ cells near the posterior brain, 51.3±0.7% express chat (out of 2362 wnt11-6+ cells counted in four animals). (E) Double FISH detecting expression of notum (cyan) and wnt11-6 (magenta) in head fragments undergoing brain remodeling (top panels) and trunk fragments forming a new brain through epimorphosis (bottom panels). Scale bars: 50 µm in A-D; 150 µm in E. Anterior, top. d, day.

Alternatively, notum could have functions in eye placement that also indicating that, like notum, wnt11-6 specifically regulates the affect brain size. We examined the relationship between the small relationship between brain cell number and body size. Simultaneous brain and ectopic eye phenotypes by inhibiting ovo, a transcription inhibition of notum and wnt11-6 in amputated head fragments factor required for production of photoreceptors (Lapan and undergoing brain remodeling completely suppressed the ectopic Reddien, 2012). Dual inhibition of notum and ovo caused a photoreceptor and small brain phenotype caused by notum reduction in brain size and absence of ectopic anterior inhibition (Fig. S5A; 65 of 67 animals) and instead resulted in an photoreceptors (Fig. S4C,D). Additionally, prep(RNAi) increased brain size similar to wnt11-6(RNAi) animals (Fig. 3A,B). homeostasis animals that form ectopic anterior photoreceptors qPCR confirmed that the suppressive effects of wnt11-6 dsRNA on (Fig. S4E) (Felix and Aboobaker, 2010), similar to notum(RNAi) the notum RNAi phenotype were not caused by alteration of notum animals, have normal numbers of cintillo+ cells (Fig. S4F), RNAi efficiency (Fig. S5B). We conclude that wnt11-6 is required suggesting a potential separation in requirements for brain sizing for the notum(RNAi) brain size phenotype during remodeling, and eye placement. We conclude that eye placement functions for consistent with a mechanism in which notum inhibits wnt11-6, notum are not required for its control of brain size and we did not which in turn normally suppresses brain size. investigate them further. We next tested whether notum and wnt11-6 also control brain size wnt11-6(RNAi) planarians undergo brain expansion (Adell et al., during formation of a new head through epimorphic regeneration. 2009; Kobayashi et al., 2007) and in the planarian Dugesia japonica After decapitation, notum is expressed by 18 h near the anterior- form ectopic posterior photoreceptors (Kobayashi et al., 2007), facing wound site, and subsequently by 48-72 h in the new anterior suggestive of opposing functions to notum in brain size control. We pole (Petersen and Reddien, 2011). Administration of notum therefore tested possible functional interactions between notum and dsRNA prior to injury results in a range of defects, including wnt11-6 in Schmidtea mediterranea using double RNAi. wnt11-6 head/tail polarity transformations or defective head regeneration (RNAi) head fragments had increased brain:body proportions (Petersen and Reddien, 2011). To examine functions for notum compared with control animals (Fig. 3A,B) but no defect in specifically in brain growth, we delivered notum dsRNA to animals photoreceptor number (Fig. S5A; 49 of 51 animals). wnt11-6(RNAi) 24 h after head amputation, reasoning that establishment of pole animals also had no defects in mediolateral brain organization (Fig. identity is likely to precede head and brain formation (Fig. S6A). S2) or significant changes in pharynx proportion, pharynx neuropile Such animals succeeded in regenerating a head and forming an size, body size, neuron density or neuron cell size (Fig. S3), anterior pole (Fig. S6B,C) but formed elongated or supernumerary DEVELOPMENT

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Fig. 3. notum inhibits wnt11-6 to control brain size in regenerative degrowth and growth. (A) Day 21 regenerating head fragments undergoing brain remodeling stained for expression of chat, gad and cintillo after indicated RNAi treatments. (B) Brain:body proportion [cintillo+ (magenta) or gad+ (cyan) cell numbers divided by body length and normalized to control treatments] from animals treated as in A, n≥18 worms per condition. (C,D) Animals were injected with indicated dsRNA 24 h after amputation of heads and tails, then fixed 21 days after amputation (21dR) and stained by multiplex FISH for chat (C) to show brain morphology and for cintillo (D; magenta) and gad (D; cyan) to quantify brain size normalized to body length; n≥18 animals per condition. (E) Animals undergoing epimorphosis (dashed lines) or remodeling (solid lines) were stained for expression of cintillo to measure brain proportion (cell number divided by body length) following administration of control (gray), notum dsRNA (left, red) or wnt11-6 dsRNA (right, blue); n≥4 animals per time point. All error bars indicate s.d. *P<0.05, **P<0.005, two-tailed t-test. Scale bars: 300 µm. Anterior, top. photoreceptors (Fig. S6B; 8 of 12 worms) reminiscent of the inhibits wnt11-6 function to control the relationship between brain photoreceptor phenotype in notum(RNAi) regenerating head and body size and suggest that this regulation could occur through a fragments (Fig. S4B). notum(RNAi)-epimorphosis animals process common to both epimorphosis and remodeling. exhibited reduced brain proportions (Fig. 3C,D). These We then investigated the dynamic emergence of brain proportion phenotypes were also dependent upon wnt11-6,asnotum(RNAi); phenotypes after notum and wnt11-6 RNAi in epimorphosis and wnt11-6(RNAi)-epimorphosis animals lacked photoreceptor defects remodeling (Fig. 3E). notum or wnt11-6 inhibition caused (Fig. S6D; 31 of 34 worms) and had increased brain proportions progressive defects in brain:body proportion (Fig. 3E) and in (Fig. 3C,D). The effect of Wnt signaling on brain proportion absolute neuronal cell number (Fig. S7A) that were stronger at later appears to be specific to wnt11-6, because inhibition of wnt1, times during regeneration (after 4 days). notum/wnt11-6 signaling is another planarian Wnt gene whose activity is known to be affected therefore unlikely to affect general processes intrinsic to early by notum (Petersen and Reddien, 2009a, 2011), did not significantly wound-induced signaling or all instances of neurogenesis but rather affect brain size or suppress the notum(RNAi) small brain phenotype has a function specific to brain size attainment. Notably, inhibition (Fig. S6E). notum and/or wnt11-6 inhibition in trunk fragments did of notum and wnt11-6 each caused an identical change in ultimate not alter pharynx proportion or total body size, further indicating brain:body proportion achieved through both brain epimorphosis that the scaling functions of these genes are specific for the brain and brain remodeling (Fig. 3E, day 21). Consistent with this

(Fig. S6F,G). These results support the hypothesis that notum interpretation, prolonged inhibition of notum or wnt11-6 in the DEVELOPMENT

4221 STEM CELLS AND REGENERATION Development (2015) 142, 4217-4229 doi:10.1242/dev.123612 absence of injury caused effects on brain size similar to those that other or redundantly acting Wnts might participate in these observed through regeneration (Fig. S7B,C). Together, these results processes. Inhibition of beta-catenin-1 decreased notum+ cell suggest that notum/wnt11-6 signaling might specifically influence numbers in the anterior commissure and anterior pole and, by target brain size. We conclude that notum and wnt11-6 antagonism contrast, inhibition of APC (encoding a component of the β-catenin acts in an essentially constitutive process to control brain cell destruction complex) increased the numbers of these notum+ cells number, potentially through the determination and maintenance of a (Fig. 4A,B). Therefore, transcriptional activation of notum in the size set-point for the planarian brain. anterior brain is likely to occur through canonical Wnt signaling. We additionally found that the notum+ cells of the brain commissure wnt11-6 regulates notum expression at the anterior brain express four of the nine planarian frizzled genes [ frizzled-5/8-2, commissure to form an inhibitory spatial feedback loop frizzled-5/8-3, frizzled-5/8-4 and frizzled-1/2/7 (Liu et al., 2013)], Homeostatic mechanisms, such as set-points, commonly rely on suggesting that they should be capable of receiving Wnt signals feedback inhibition to provide output stabilization (Stanger, 2011). (Fig. S8C). As notum and wnt11-6 were not coexpressed in any cells Notum acts as a feedback inhibitor of Wnt signaling in multiple during regeneration (Fig. 2E), we conclude that wnt11-6 acts either animal species (Flowers et al., 2012; Gerlitz and Basler, 2002; at a distance or indirectly to activate notum expression. Together, Giráldez et al., 2002; Petersen and Reddien, 2011); therefore, we these results indicate that wnt11-6 is required for the expression of examined the potential requirement of wnt11-6 for notum its own secreted inhibitor, notum, within the brain to establish a expression. Inhibition of wnt11-6 reduced numbers of notum- negative feedback loop across the organ axis. expressing cells in the anterior brain in conditions of homeostasis and remodeling (Fig. 4A,B; Fig. S8A). This function for wnt11-6 wnt11-6 is likely to suppress brain growth through non- was specific for the brain, because wnt11-6 RNAi did not noticeably canonical Wnt signaling reduce numbers of notum+ cells at the anterior pole (Fig. 4A,B; Wnt family ligands can signal through either canonical, β-catenin- Fig. S8A) or induced early after amputation (Fig. S8B), suggesting dependent pathways or non-canonical, β-catenin-independent

Fig. 4. wnt11-6 activates anterior brain notum expression through canonical Wnt signaling to form a negative feedback loop. (A) notum expression (red) in uninjured control, wnt11-6(RNAi), beta-catenin-1(RNAi) and APC(RNAi) animals fixed after 14 days of RNAi feeding (14dF). Arrows indicate normal (white), reduced (yellow) or increased (green) notum+ cell numbers. (B) Quantification of notum+ cell numbers at the brain commissure and anterior body pole from A(n=5 animals per condition). (C,D) Day 14 regenerating trunk fragments (C) or day 21 regenerating head fragments (D) stained for cintillo expression after the indicated RNAi treatments [upper, animal images; lower, quantifications of cintillo+ cell number per animal length normalized to control animals. n≥9 (C) or n≥5 (D) animals per condition]. (E) Animals were fed dsRNA every 3 days for 14 (beta-catenin-1) or 22 days (control, wnt11-6, Dvl1;Dvl2) then fixed and analyzed for brain:body proportion as measured by cintillo+ cell numbers per animal length normalized to control animals (n≥5 animals per condition). All error bars indicate s.d. Scale bars: 100 µm in A; 150 µm in C,D. *P<0.05, **P<0.005, n.s. P>0.05, two-tailed t-test. Anterior, top. DEVELOPMENT

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(Fig. 4D). Furthermore, simultaneous inhibition of APC and notum resulted in small-brained animals (Fig. 4D), confirming that the large brain APC(RNAi) phenotype depends on notum. The simplest interpretation of these results is that wnt11-6 activates notum through canonical Wnt signaling but wnt11-6 suppresses brain growth through non-canonical, beta-catenin-1-independent signaling (Fig. 4E). Inhibition of Dishevelled (Dvl) homologs Dvl-1 and Dvl-2, genes that function in both canonical and non-canonical Wnt signaling pathways, caused brain enlargement similar to wnt11-6 RNAi and unlike beta-catenin-1 RNAi (Fig. 4F), consistent with this interpretation.

notum and wnt11-6 regulate neoblast differentiation to influence brain size We then sought to identify the cellular mechanisms regulated by notum and wnt11-6 in control of brain size and hypothesized that they are likely to be common to epimorphosis and tissue remodeling. We first tested for candidate functions for notum and wnt11-6 in apoptosis because of their ability to control cell number and their activation during tissue remodeling (Pellettieri et al., 2010). We measured the role of notum and wnt11-6 in directing cell death using whole-mount terminal uridine nick-end labeling (TUNEL) in fixed tissue fragments undergoing brain remodeling (Fig. 5A,B) (Pellettieri et al., 2010). Both notum(RNAi) and wnt11- 6(RNAi) fragments were able to activate cell death by 3 days post- amputation and return to basal levels around day 14 or 15 (Fig. 5A). Furthermore, these treatments did not significantly alter numbers of TUNEL+ cells throughout the animal at any regenerative time point (Fig. 5B). Therefore, we conclude that notum and wnt11-6 are unlikely to act through injury-induced cell death to direct a change in brain size. Ultimate organ size is likely to result from a balance between cell loss and cell production. Planarians use neoblasts, a population that includes pluripotent stem cells, for regeneration, growth and homeostatic cell replacement (Reddien et al., 2005b; Wagner et al., 2011). Therefore, we next investigated whether notum or wnt11-6 regulates neoblast-dependent tissue production to control + Fig. 5. notum and wnt11-6 do not control injury-induced cell death. brain size. To test this hypothesis, we measured cintillo cell number (A,B) Cell death during brain remodeling in regenerating head fragments was in lethally irradiated animals injected with control, notum or wnt11-6 assayed by TUNEL staining over a time series of 21 days. (A) Animals were dsRNA 2 days prior to head removal (Fig. 6A). As expected, treated with control, notum or wnt11-6 dsRNA by injection (three injections over decapitated irradiated trunk fragments did not produce a head 3 days), amputated pre- and post-pharyngeally, then TUNEL stained. blastema or cintillo+ cells (zero cells in three animals examined for (B) TUNEL+ cell numbers per fragment area were quantified for all conditions and time points (n≥5 animals per time point; n.s. P>0.05, two-tailed t-test). All each RNAi condition). Surprisingly, control RNAi irradiated head error bars indicate s.d. Scale bars: 300 µm. Anterior, top. d, day. fragments undergoing brain remodeling lost an excess number of cintillo+ cells compared with non-irradiated control head fragments, pathways (Nusse, 2012). We examined intracellular components of indicating that normal tissue remodeling involves a neoblast- canonical Wnt signaling and their relationships to notum and wnt11- dependent activity that promotes brain cell number. notum(RNAi) 6 to clarify the signaling pathways used in control of brain size. non-irradiated or irradiated head fragments all attained a number RNAi of beta-catenin-1 [at a time of regeneration prior to body- of cintillo+ cells similar to irradiated control RNAi head fragments, wide formation of ectopic anterior central nervous system structures indicating that notum requires neoblasts to promote brain size (Gurley et al., 2008; Iglesias et al., 2008; Petersen and Reddien, during remodeling (Fig. 6A). Additionally, irradiation completely 2008)] caused a decrease in brain size in heads regenerated from suppressed the large brain phenotype observed with wnt11-6 RNAi, anterior-facing wounds (Fig. 4C), opposite to the wnt11-6 RNAi suggesting that wnt11-6 also requires neoblasts to affect brain size in phenotype and consistent with previous findings (Owen et al., all regenerative contexts (Fig. 6A). The simplest interpretation of 2015). wnt11-6(RNAi);beta-catenin-1(RNAi) animals had large these results is that remodeling involves the production of new brain brains similar to wnt11-6 inhibition alone (Fig. 4C), suggesting cells in a manner regulated by notum and wnt11-6. To test this that wnt11-6 might act downstream or in parallel to beta-catenin-1 hypothesis, we measured the ability of notum(RNAi) or wnt11-6 to control brain size. We inhibited APC to overactivate canonical (RNAi) animals undergoing brain remodeling to produce new chat+ Wnt signaling in order to test the hypothesis that beta-catenin-1 cells of the brain after a pulse of bromodeoxyuridine (BrdU) on day 9 promotes brain size mainly through activating notum expression. and before fixation on day 13. notum RNAi caused a reduction in In head fragments undergoing brain remodeling, APC RNAi relative numbers of BrdU+chat+ cells, whereas wnt11-6 RNAi phenocopied wnt11-6 RNAi to produce an enlarged brain increased their numbers (Fig. 6B). Together, these results strongly DEVELOPMENT

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Fig. 6. notum and wnt11-6 regulate formation of new brain tissue in epimorphosis and in remodeling. (A) Non-irradiated or lethally irradiated (6000 rad) animals were injected with control, notum or wnt11-6 dsRNA three times over 60 h. Heads were removed, fixed 7 days later, and numbers of cintillo+ cells quantified by FISH. (B) Regenerating head fragments undergoing RNAi were injected with BrdU at day 9, fixed at day 13 and stained for chat by FISH (green) and BrdU (red) by immunofluorescence. Right, quantification of BrdU+chat+ cells from the posterior brain, normalized to animal size and control animal values. (C,D) Mitotic activity in tail fragments (C) and head fragments (D) was assessed by immunostaining for phospho-Ser10 of histone H3 (H3P) over a regeneration time series and quantified as average H3P+ cell number normalized to total fragment area (n≥5 animals per time point per condition; see Fig. S9 for representative images). Error bars, s.d. in A-D. *P<0.05, **P<0.005, n.s. P>0.05, two-tailed t-test. Scale bars: 50 µm. suggest that new brain cell production contributes to attainment of Neoblasts are a heterogeneous population that includes both appropriate proportion during regenerative tissue remodeling and pluripotent cells and distinct subpopulations specified for that notum and wnt11-6 are likely to control brain size by influencing production of multiple differentiated tissues (Adler et al., 2014; the formation of new brain cells. Cowles et al., 2013; Currie and Pearson, 2013; Forsthoefel et al., We tested for possible functions of wnt11-6 on global 2012; Lapan and Reddien, 2012; Marz et al., 2013; Scimone et al., proliferation, because neoblasts are the only proliferating cells in 2011, 2014a,b; van Wolfswinkel et al., 2014; Vásquez-Doorman planarians (Baguñà, 1976; Newmark and Sánchez Alvarado, 2000; and Petersen, 2014; Vogg et al., 2014; Wenemoser et al., 2012). We Reddien et al., 2005b). We measured global mitotic index in control, hypothesized that notum and wnt11-6 might regulate brain cell notum(RNAi) and wnt11-6(RNAi) head or tail fragments during brain production by controlling numbers of brain cell progenitors marked remodeling or epimorphosis by staining with anti-phophoSer10- by expression of lineage-specific transcription factors. We first histone H3 (Wenemoser and Reddien, 2010). In both remodeling and examined ap2, which is required for production of trpA+ brain epimorphosis, notum(RNAi) and wnt11-6(RNAi) animals had neurons and expressed in nearby smedwi-1+ neoblasts (Wenemoser broadly similar global proliferative activities (Fig. 6C,D; Fig. S9), et al., 2012). Like all other neuronal populations tested, trpA+ brain with differences at selected times (elevated after wnt11-6 RNAi in cell numbers increased after wnt11-6 RNAi and decreased after day 3 tail fragments and day 13 head fragments and reduced after notum RNAi (Fig. S10A,B). As hypothesized, ap2+ smedwi-1+ cell notum RNAi in day 7 in tail fragments). Thus, wnt11-6 and notum do numbers were elevated after wnt11-6 RNAi and reduced after notum not appear to control cell division globally throughout regeneration RNAi both in formation of a new brain through epimorphosis as would be expected for regulation that constitutively maintains (Fig. 7A) and in alteration of a pre-existing brain through appropriate brain size, although we cannot rule out the possibility remodeling (Fig. 7B). We examined two additional neoblast that they control proliferation of neoblast subpopulations at isolated subpopulations that contribute to the brain, lhx1/5-1+smedwi-1+ + windows in regeneration. cells that form serT serotonergic neurons of the brain (Currie and DEVELOPMENT

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Fig. 7. notum and wnt11-6 regulate numbers of brain cell progenitors. (A,B) Double FISH to detect coexpression of smedwi-1 (green) and ap2, lhx1/5-1 or coe (magenta) in (A) trunk fragments regenerating a new brain or (B) head fragments undergoing brain remodeling 4 days after amputation and treated with indicated dsRNAs. Upper panels, images are representative planes from a confocal stack of the region indicated by the cartoon (white arrows, double positive cells; yellow insets show magnified representative double positive cells). Lower panels, graphs show quantifications of progenitor cell numbers (see Materials and Methods for details; n=4 animals per condition). Error bars indicate s.d. *P<0.05, **P<0.005, n.s. P>0.05, two-tailed t-test. Scale bars: 100 µm. Anterior, left in A; top in B.

Pearson, 2013) (Fig. S10C,D) and coe+smedwi-1+ cells that form a system for dissection of this process. Our studies support a model in variety of neuronal subtypes [including cpp-1+, chat+, gad+, tph+, which negative feedback signaling directs the size of the planarian th+ and tbh+ neurons (Cowles et al., 2013)]. In all cases, notum brain through reversible regenerative growth (Fig. 8). We propose (RNAi) animals had fewer brain progenitors and wnt11-6(RNAi) that wnt11-6 from the posterior brain inhibits the formation or animals had more brain progenitors (Fig. 7A,B). Taken together, we division of brain progenitors to modify the rate of brain cell suggest that notum inhibits wnt11-6 activity, which in turn limits production. Through the canonical Wnt signaling pathway, wnt11-6 production of differentiated neurons through control of neoblast activates the expression of its own secreted inhibitor, notum, at the differentiation but not cell death. These results suggest that opposite end of the organ. In turn, notum promotes brain growth appropriate size attainment in regeneration involves regulation of through its inhibition of non-canonical wnt11-6 activity. This the rates or extents of tissue production through organ-specific process operates to maintain brain proportionality in uninjured regionalized signaling. animals and restore appropriate brain:body proportion in animals either growing a new brain through blastema formation or shrinking DISCUSSION a pre-existing brain through remodeling. The notum/wnt11-6 The mechanisms underlying size attainment in development and feedback loop does not appear to control the ability to respond to regeneration are poorly understood, and planarians offer a powerful injury or significantly affect the early rates of brain size increase in DEVELOPMENT

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proliferation and cell death (Conlon and Raff, 1999). Tissue remodeling activated by injury coincides with waves of early wound-induced and later systemic cell death dependent on the extent of missing tissue (Pellettieri et al., 2010). We observed no role for notum/wnt11-6 signaling in programmed cell death during brain remodeling (Fig. 5) and instead found that notum and wnt11-6 controlled the size of the pool of brain progenitors descended from pluripotent neoblasts (Fig. 7). Additionally, depletion of stem cells by irradiation and notum RNAi caused identical excesses of cell loss to brains undergoing tissue remodeling, and perturbation of wnt11-6 and notum had opposite effects on the rate of neoblast differentiation to brain cells in remodeling (Fig. 6A,B). Together, these results Fig. 8. notum/wnt11-6 feedback regulation dampens brain cell differentiation to achieve proper brain:body scaling. (Left) Cartoon suggest that an important component of size regulation through depicting planarian brain with neurons (black), wnt11-6-expressing neurons remodeling is the control of neoblast activity. Likewise, starvation- (red with black outline) and other brain-associated cell types (red with white induced degrowth that reduces size globally involves body-wide outline) in the posterior brain region (red), notum-expressing neurons at the reduction of differentiation (González-Estévez et al., 2012a), and anterior commissure (green with black outline) and neoblasts that surround the intestinal remodeling coincides with a significant amount of new brain (blue, medial neoblasts shown). (Right) Model for regulatory pathway cell production in that organ (Forsthoefel et al., 2011). We suggest influencing brain size. wnt11-6 inhibits neoblast production of differentiated + + + + that the wnt11-6/notum signaling system can tune the size of the brain cells (including cintillo , gad , trpA and tph neurons) by suppressing formation or division of neural progenitors (ap2+smedwi-1+, coe+smedwi-1+ brain in contexts of regenerative growth and degrowth and in the and lhx1/5-1+smedwi-1+ cells) from pluripotent neoblasts. wnt11-6 signals absence of injury to allow alteration of the fraction of neoblasts directly or indirectly through β-catenin-dependent canonical Wnt signaling to specified to brain cell fates. Lineage-committed progenitor cell activate expression of notum in neurons at the anterior brain commissure. number during embryogenesis of several vertebrate model systems wnt11-6 is likely to signal independently of beta-catenin-1 in control of has been shown to be correlated with final organ size (Kicheva et al., neoblasts to suppress brain size. notum encodes a secreted protein that 2014; Stanger et al., 2007), suggesting that the regulation of inhibits wnt11-6 function to promote ongoing synthesis of brain cells from neoblasts. Levels of wnt11-6 and notum signaling control numbers of progenitor numbers could be an ancient and conserved mechanism neoblasts committing to brain cell fates to influence the size of the brain. for organ size control. The use of Wnt signaling for organ size control and anterior epimorphosis or decrease in brain remodeling, but rather controls a patterning is also widespread (Petersen and Reddien, 2009b). brain size set-point. The ability to perform large-scale unbiased Production of the Wnt inhibitor Dkk through cell differentiation RNAi screens in planarians, the unique ability to achieve organ regulates the size of zebrafish mechanosensory organs by proportions by either increases or decreases in cell number and to counteracting Wnt signals required for progenitor proliferation measure these processes quantitatively in whole animals will enable (Wada et al., 2013). Overactivation of β-catenin increases the size this system to be used to identify the genetic architecture underlying of the mammalian brain through cell cycle regulation of neural attainment of organ size. progenitors (Chenn and Walsh, 2002, 2003), a phenotype broadly Our studies highlight that whole-body regeneration acts primarily similar to planarian APC RNAi (Fig. 4D). Mammalian wnt1, to restore form rather than simply to replace missing tissues. For wnt3a and wnt8 participate in brain patterning and proliferation example, decapitation of animals with an average of 45 cintillo+ (Erter et al., 2001; Lee et al., 2000; McMahon and Bradley, 1990; cells caused regeneration of only 30 new cintillo+ cells from Thomas and Capecchi, 1990), but the precise mechanisms that regenerating trunk fragments (Fig. S1G), but this number perfectly relate Wnt ligands and secreted inhibitors for control of brain size restored the proportion of cintillo+ cells to body size (Fig. 1D). remain unclear in vertebrates, as do their relationships with Therefore, planarian regeneration does not replace an absolute additional regulatory pathways. Perturbation of planarian insulin number of cells removed by injury. Likewise, amputated head (Miller and Newmark, 2012) or hippo signaling (Lin and Pearson, fragments starting with 45 cintillo+ cells lost 20 of these cells 2014) causes body-wide defects in neoblast proliferation, through tissue remodeling (Fig. S1G), and this reduction also suggesting that alternative pathways are used for control of brain restored brain proportion (Fig. 1D). Given that uninjured planarians size. Our results reveal a broadly conserved relationship between do not possess a fixed size (Reddien and Sánchez Alvarado, 2004), Wnt activity and brain size and suggest that the planarian brain is we argue that restoration of proportion is central to the completion a tractable system for discovery of additional size regulatory of regeneration and might be a requirement to ensure fidelity in pathways. successive rounds of asexual reproduction or a property intrinsic to Growth inhibitors produced as a consequence of differentiation whole-body regeneration. additionally have broad use in the control of organ size We find unexpected similarities between the mechanisms of (Gamer et al., 2003; Gomer, 2001; Lander et al., 2009; Stanger, growth control involved in epimorphosis, tissue remodeling and 2008). As a negative regulator of brain growth expressed in neurons homeostatic maintenance. After or in parallel to early injury- of the brain and predicted to encode a secreted protein, wnt11-6 induced proliferation (Wenemoser and Reddien, 2010), tissue shares similarity with the action of molecules such as myostatin polarization (Petersen and Reddien, 2009a, 2011) and injury- (McPherron and Lee, 1997; McPherron et al., 1997) and gdf11 (Wu induced signaling (Gavino et al., 2013; Wenemoser et al., 2012), et al., 2003), proposed chalones that limit cell numbers within the production of the regeneration blastema might involve an extreme tissue from which they are expressed, the mammalian muscle and form of tissue turnover shared among aspects of tissue remodeling olfactory epithelium, respectively. Our studies demonstrate an and normal maintenance, similar to the concept of homeostatic additional layer of regulation in regenerative organ size control, in regeneration (Wills et al., 2008a,b). The control of organ size is which the action of a putative chalone (wnt11-6) is tuned through frequently described as the result of a balance between cell the use of a feedback inhibitor (notum). In principle, restoration and DEVELOPMENT

4226 STEM CELLS AND REGENERATION Development (2015) 142, 4217-4229 doi:10.1242/dev.123612 maintenance of organ proportions could occur by achieving and Terminal uridine nick-end labeling (TUNEL) preserving maximal activity of growth inhibitors in order to halt cell TUNEL was performed as described by Pellettieri et al. (2010), with differentiation at an appropriate endpoint. However, it is unlikely modifications. Animals were sacrificed in 5% N-acetyl-cysteine in 1× PBS, that wnt11-6 activity levels are maximally high or low at the fixed in 4% formaldehyde in 1× PBSTx, and bleached overnight in 6% endpoint of regeneration, because perturbation of wnt11-6 or notum hydrogen peroxide in 1× PBSTx. Samples were labeled with DIG-11-dUTP (Roche) by terminal deoxyuridine transferase (TdT) reaction (Fermentas) at can reversibly affect brain size during homeostatic conditions in 37°C for 2 h, then blocked and incubated overnight in anti-DIG-POD animals starting with optimal brain:body proportions (Fig. S7B,C). (Roche; 1:2000 in 10% horse serum in 1× PBSTx) prior to tyramide Instead, maintenance of ongoing growth and regeneration abilities development (Invitrogen). might require constitutively expressed dampening mechanisms (Reddien, 2011), such as wnt11-6 feedback inhibition through Image analysis notum, to prevent cessation of growth regulation. Spatial regulatory Imaging modules that achieve a sustained balance between growth inhibitors Imaging was performed with a Leica M210F dissecting scope with a Leica and activators could be essential for regenerative abilities, DFC295 camera, a Leica DM5500B compound microscope with Optigrid, proportional growth and defining target organ size. Leica SP5 or Leica TCS SPE confocal compound microscopes. Fluorescent images collected by compound microscopy are maximal projections of a z- MATERIALS AND METHODS stack and adjusted for brightness and contrast using Adobe Photoshop. Planarian culture and irradiation treatments Asexual Schmidtea mediterranea (CIW4) were maintained in 1× Montjuic Cell counting salts between 18 and 20°C. Gamma irradiation (6000 rad) was performed cintillo+ cells and gad+ or trpA+ cells in the medial brain region were with a cesium-137 source irradiator at least 24 h prior to amputation to counted manually. chat+ cells from one lobe of each animal were counted eliminate all dividing cells. from a z-series of images using three-dimensional segmentation software in Imaris. Animal lengths were measured with ImageJ (National Institutes of Whole-mount in situ hybridization Health) as visualized with Hoechst. Relative brain length was measured Animals were fixed and stained as described previously (Pearson et al., from the most posterior brain branch to the most anterior brain branch as 2009). Antibodies were used in MABT containing 10% horse serum for visualized by chat FISH signal or Hoechst. Samples from similar fragments FISH [anti-DIG-POD 1:2000 (Roche), anti-FL-POD 1:1000 (Roche), anti- and time points were averaged and significant differences determined by DNP-POD 1:500 (PerkinElmer)] or NBT/BCIP in situ hybridization [(anti- two-tailed Student’s t-tests. Cells coexpressing wnt11-6 and chat, notum and DIG-AP 1:4000 (Roche)]. For multiplex FISH, peroxidase activity was chat or notum and collagen were counted manually from z-stack confocal quenched between tyramide reactions using 4% formaldehyde (Pearson images (0.5-1 μm thick) using ImageJ. For notum coexpression, images et al., 2009) or 100 mM sodium azide (King and Newmark, 2013) for at least were taken of the head region containing both the anterior body pole and 1 h at room temperature. Nuclear counterstaining was performed using anterior brain commissure. For wnt11-6, images were taken of the posterior 1:1000 Hoechst 33342 (Invitrogen) in PBSTx (1× phosphate buffered saline of brain. Cells labeled with BrdU and chat or expressing both smedwi1 and with 0.1% Triton X-100). either ap2, coe or lhx1/5 during regeneration were manually blind-counted from z-stack images near the posterior cephalic ganglia (BrdU), anterior- RNA interference facing wound site (epimorphosis) or between the lobes of the cephalic RNA interference by feeding was performed using E. coli HT115 cultures ganglia (remodeling) by manual electronic labeling in using ImageJ and checking for consistency by comparing neighboring planes. H3P+ and expressing dsRNA from cDNA cloned into pPR244 (Gurley et al., 2008; + Reddien et al., 2005a). For regeneration experiments, animals were fed liver- TUNEL cell numbers were quantified using CellProfiler (Jones et al., bacteria mixture four times over 9 days. For long-term homeostasis 2008; Lamprecht et al., 2007) or ImageJ. experiments, animals were fed RNAi bacterial food every 3 days. dsRNA targeting C. elegans unc-22 was used as a negative control. For RNAi by Regression analysis + + injection, dsRNA was synthesized by in vitro transcription and diluted to Lines of best fit for cintillo and gad compared with either body (Table S1) 2000 ng/μl, then administered to animal fragments by microinjection or brain length (Table S2) in uninjured animals were determined by 2 (Drummond Scientific). For double RNAi, liver-bacteria mixtures or in vitro regression analysis in Microsoft Excel. Equations and r values are shown in transcribed dsRNA were mixed in equal volumes prior to administration, Tables S1 and S2. Estimates of intact proportions, shown on graphs of with single gene inhibition controls normalized with control dsRNA. notum, regenerative time courses by a dashed black line, are averages of values 2 beta-catenin-1, APC, Dvl-1 and Dvl-2 plasmids were described previously interpolated from power regression (the analysis type with highest r values) (Gurley et al., 2008; Petersen and Reddien, 2011). wnt11-6 was cloned using body or brain length measurements of animals on day 15 of using primers 5′-TCGCATACAGCTTCAATCACA-3′ and 5′-AATGAT- remodeling or epimorphosis. TTTGTGCCATACGAA-3′. Acknowledgements We thank the Pearson Laboratory (UToronto) for kindly sharing their TUNEL protocol BrdU labeling and Dr Erik Andersen, Adam Hockenberry and all past and present members of the Day 9 head fragments from RNAi-fed animals were injected with 5 mg/ml Petersen laboratory for helpful discussions of the manuscript. BrdU (Sigma-Aldrich) in water, then 4 days later fixed and stained as previously described (Vásquez-Doorman and Petersen, 2014). Competing interests The authors declare no competing or financial interests. Whole-mount immunostaining Animals were sacrificed in 0.75 M HCl, then fixed with Carnoy’s solution Author contributions (60% ethanol, 30% chloroform and 10% acetic acid) and bleached overnight Conceived and designed the experiments: E.M.H., C.P.P. Performed the with 6% hydrogen peroxide in methanol. Animals were blocked for 6 h in experiments: E.M.H. Analyzed the data: E.M.H., C.P.P. Contributed reagents/ materials/analysis tools: E.M.H., C.P.P. Wrote the paper: E.M.H., C.P.P. 1× PBSTB (1x phosphate buffered saline, 0.3% Triton X-100, 0.25% bovine serum albumin) and primary and secondary antibody incubations Funding performed overnight using rabbit anti-phospho-Histone H3 Ser10 (Cell The authors acknowledge support from a National Institutes of Health institutional Signaling; 1:3000 in 1× PBSTB) followed by anti-rabbit horseradish predoctoral training program [Cellular and Molecular Basis of Disease Training peroxidase conjugate (Invitrogen; 1:1000 in 1× PBSTB) and Alexa568- Program 2T32GM008061-31 to E.M.H.] and an Ellison Medical Foundation New tyramide amplification (1:150; Invitrogen). Scholar in Aging Research Award [AG-NS-0835-11 to C.P.P.], a National Institutes DEVELOPMENT

4227 STEM CELLS AND REGENERATION Development (2015) 142, 4217-4229 doi:10.1242/dev.123612 of Health Director’s New Innovator Award [1DP2DE024365-01 to C.P.P.] and an Gaviño, M. A., Wenemoser, D., Wang, I. E. and Reddien, P. W. (2013). Tissue American Cancer Society institutional research grant [ACS-IRG 93-037-15 to absence initiates regeneration through Follistatin-mediated inhibition of Activin C.P.P.]. The funders had no role in study design, data collection and analysis, signaling. eLife 2, e00247. decision to publish or preparation of the manuscript. Deposited in PMC for release Gerlitz, O. and Basler, K. (2002). Wingful, an extracellular feedback inhibitor of after 12 months. Wingless. Genes Dev. 16, 1055-1059. Giráldez, A. J., Copley, R. R. and Cohen, S. M. (2002). HSPG modification by the Supplementary information secreted enzyme Notum shapes the Wingless morphogen gradient. Dev. Cell 2, 667-676. Supplementary information available online at Gomer, R. H. (2001). 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