STEM CELLS AND REGENERATION RESEARCH ARTICLE

Wnt/Notum spatial feedback inhibition controls neoblastdifferentiation to regulate reversible growth of the planarian brainEric M. Hill1 and Christian P. Petersen1,2,*

ABSTRACTMechanisms determining final organ size are poorly understood.Animals undergoing regeneration or ongoing adult growth are likely torequire sustained and robust mechanisms to achieve and maintainappropriate sizes. Planarians, well known for their ability to undergowhole-body regeneration using pluripotent adult stem cells of theneoblast population, can reversibly scale body size over an order ofmagnitude by controlling cell number. Using quantitative analysis, weshowed that after injury planarians perfectly restored brain:bodyproportion by increasing brain cell number through epimorphosisor decreasing brain cell number through tissue remodeling(morphallaxis), as appropriate. We identified a pathway controlling abrain size set-point that involves feedback inhibition between wnt11-6/wntA/wnt4a and notum, encoding conserved antagonistic signalingfactors expressed at opposite brain poles. wnt11-6/wntA/wnt4aundergoes feedback inhibition through canonical Wnt signalingbut is likely to regulate brain size in a non-canonical pathwayindependently of beta-catenin-1 and APC. Wnt/Notum signalingtunes numbers of differentiated brain cells in regenerative growth andtissue remodeling by influencing the abundance of brain progenitorsdescended from pluripotent stem cells, as opposed to regulating celldeath. These results suggest that the attainment of final organ sizemight be accomplished by achieving a balance of positional signalinginputs that regulate the rates of tissue production.

KEY WORDS: Wnt signaling, Notum, Organ size, Planaria,Regeneration, Tissue remodeling

INTRODUCTIONMost animal species possess stereotyped body forms, in whichorgans and appendages grow to defined proportions with respect tototal body size. Mutations that alter organ proportion can underlieevolutionary changes (Abzhanov et al., 2004; Jones et al., 2012) andresult in human developmental disorders, such as microcephaly(Mochida and Walsh, 2001). Many molecular pathways have beendescribed as contributing to growth regulation (Conlon and Raff,1999; Lander, 2011; Schwank and Basler, 2010; Tumaneng et al.,2012), primarily through genetic studies of species that cease orlargely dampen growth at the end of embryogenesis, but despiteconsiderable interest, the developmental mechanisms explainingsize attainment largely remain a mystery. By contrast, species thatundergo regeneration and ongoing growth throughout adulthoodmust possess robust mechanisms to control animal form andproportion actively (Elliott and Sánchez Alvarado, 2013; Rink,

2013; Wills et al., 2008a,b); hence, they represent model systemswell suited for investigating the developmental mechanisms thatunderlie the extent of growth and attainment of size (Baguñà andRomero, 1981; Oviedo et al., 2003; Wada et al., 2013).

Planarians are flatworms that continue to undergo significanttissue turnover throughout adulthood and exhibit an extremecapacity for growth control that allows reversible alteration ofbody and organ size over an order of magnitude through theregulation of cell number. Nutrient uptake leads to an increase inbody size, whereas prolonged starvation results in ‘degrowth’, areduction of body size and cell number without an alteration inrelative tissue proportions or function (Baguñà and Romero, 1981;Morgan, 1898; Oviedo et al., 2003; Romero and Baguñà, 1991;Takeda et al., 2009). Additionally, planarians exhibit robustness ingrowth control through the process of regeneration. Planarians donot appreciably increase in size during regeneration, so smallamputated tissue fragments ultimately become small but well-formed individuals, in which overt body proportionality appearsbroadly restored (Morgan, 1898). Planarians accomplishregeneration both by the production of missing structures in aregeneration blastema (termed epimorphosis) and throughremodeling of pre-existing tissues [originally termed morphallaxis(Morgan, 1898)]. The term morphallaxis has also been used todescribe putative regeneration mechanisms that might occurindependently of cell proliferation (Morgan, 1901); therefore, forclarity we will describe the process of injury-induced alterations topre-existing tissues as ‘tissue remodeling’ (Forsthoefel et al., 2011;Oviedo et al., 2003). Planarians reproduce asexually by fission, sothey are a suitable model to study the mechanisms of naturalproportion regulation and organ sizing through control of cellnumber (Oviedo et al., 2003).

The relationships between epimorphosis and tissue remodelingare not fully understood, but several cellular events and molecularpathways have been identified as contributing to each of theseprocesses. Growth from feeding or epimorphic regenerationdepends on parenchymal cells termed neoblasts that are the onlyproliferating cells in adult planarians (Baguñà, 1976; Newmark andSánchez Alvarado, 2000; Reddien et al., 2005b). Neoblasts are aheterogeneous population containing both adult pluripotent stemcells (Wagner et al., 2011) and more lineage-restricted dividing cells(Adler et al., 2014; Cowles et al., 2013; Currie and Pearson, 2013;Forsthoefel et al., 2012; Lapan and Reddien, 2012; Marz et al.,2013; Scimone et al., 2014a,b, 2011; van Wolfswinkel et al., 2014;Vásquez-Doorman and Petersen, 2014; Vogg et al., 2014;Wenemoser et al., 2012). Tissue removal results in proliferativeactivation of neoblasts thought to be necessary for production ofmissing tissue types within the regeneration blastema. By contrast,the cellular basis for regenerative tissue remodeling is less wellunderstood but has been proposed to involve a wave of systemicinjury-induced cell death proportional to the extent of tissueremoved by injury (Pellettieri et al., 2010). Within the intestine,Received 23 February 2015; Accepted 27 October 2015

1Department of Molecular Biosciences, Northwestern University, Evanston,IL 60208, USA. 2Robert Lurie Comprehensive Cancer Center, NorthwesternUniversity, Evanston, IL 60208, USA.

*Author for correspondence (christian-p-petersen@northwestern.edu)

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regeneration involves both substantial differentiation of new cellsand incorporation of pre-existing cells, suggesting a complexity oftissue additions and alterations during regeneration (Forsthoefelet al., 2011). As planarians can perform tissue remodeling andblastema formation through periods of starvation, it is possible thatregulated autophagy could contribute to these processes (González-Estévez, 2009; González-Estévez et al., 2007). Perturbation ofseveral molecular processes can influence body and organ size inplanarians, including bioelectric signaling (Beane et al., 2012), FGFsignaling (Cebrià et al., 2002a), JNK signaling (Almuedo-Castilloet al., 2014), TORC1 signaling (González-Estévez et al., 2012b;Peiris et al., 2012; Tu et al., 2012) and insulin-like peptide signaling(Miller and Newmark, 2012). However, there is still a limitedunderstanding of the developmental and molecular events thatunderlie the attainment and maintenance of appropriate organ sizein planarians.We investigated the planarian brain as a model for regenerative

organ size control, because it undergoes prominent and easilyidentifiable changes in cell number (Oviedo et al., 2003). Here, weshow that planarians completely restore appropriate brain:bodyproportions through either epimorphic growth or tissue remodeling.We find that the secreted Wnt inhibitor notum (Petersen andReddien, 2011) is expressed in anterior brain neurons and promotesbrain size in regeneration, remodeling and homeostasis. notum(RNAi) brain phenotypes are suppressed by inhibition of wnt11-6/wntA/wnt4a (Gurley et al., 2010; Kobayashi et al., 2007; Riddifordand Olson, 2011), hereafter referred to as wnt11-6, an inhibitor ofbrain size expressed in the posterior brain (Adell et al., 2009;Kobayashi et al., 2007). wnt11-6 signaling through beta-catenin-1is required for notum expression in the brain, but is likely to inhibitbrain size through β-catenin-independent signaling. This Wnt/notum negative feedback loop regulates brain:body proportionthrough control of neoblast differentiation, rather than cell death,in both epimorphosis and remodeling. The ability of the planarianto restore organ proportionality provides a unique system to studyorgan size determination and control of the extent of regeneration.

RESULTSPlanarians robustly restore brain:body proportionalitythrough regeneration or remodelingWe first sought to clarify the precise relationships between brain cellnumber, brain size and body size. We examined uninjured adultplanarians across a range of overall sizes using multiplexfluorescence in situ hybridizations (FISH) to measure numbers ofabundant neurons [cholinergic neurons expressing cholineacetyltransferase (chat) (Nishimura et al., 2010)] and more rarecell types of the brain [putative chemosensory neurons expressing adegenerin homolog cintillo (Oviedo et al., 2003) and GABAergicneurons expressing glutamine decarboxylase (gad) (Nishimuraet al., 2008); Fig. 1A,B]. Populations of all three neuronal cell typeswere correlated strongly with brain length (Fig. S1B,C) and bodysize (Fig. 1C; Fig. S1D), consistent with previous analyses (Oviedoet al., 2003; Takeda et al., 2009). Neuronal cell size, as measured byin situ hybridization signal area for cintillo+ and gad+ neurons(labeling the cell body), did not vary with animal size, confirmingprevious analyses indicating that control of cell number is aprincipal method of body and organ size regulation in planarians(Fig. S1E) (Baguñà and Romero, 1981; Oviedo et al., 2003). Thus,adult planarians maintain a specific number of brain-related cellswith respect to body size.We next examined whether brain:body proportionality is

precisely restored through regeneration by measuring brain cell

numbers in decapitated animals forming a new brain throughepimorphosis (Fig. 1D, dotted lines; Fig. 1E, lower) or amputatedhead fragments undergoing brain remodeling (Fig. 1D, solid lines;Fig. 1E, upper). In order to allow evaluation of regenerative progresswith respect to the complete body plan and across replicates ofvarying sizes, neural cell numbers were normalized to the totalfragment length to give a value for brain:body proportion at eachtime in regeneration (Fig. 1D). Relative brain size duringregeneration was compared with values interpolated from a powerlaw regression analysis of brain cell number versus body length inuninjured animals to determine when appropriate organ size wasachieved (Fig. 1D, dashed black lines). In animals remodeling a pre-existing organ, brain:body proportion decreased rapidly during the72 h following decapitation and continued to decrease untilstabilizing around day 9. In epimorphic growth, cintillo+ and gad+

neurons emerged around day 3, coincident with appearance of thebrain primordia (Cebrià, 2007), and brain:body proportioncontinued to increase until stabilizing by day 9. Throughout eitherregeneration scenario, brains had a constant ratio of neuronal cellnumbers to brain length (Fig. S1F), indicating that proper scalewithin the brain is established early and maintained through periodsof organ size change (Takeda et al., 2009), whereas the proportionof the organ with respect to the body is subject to regulation.Although remodeling and epimorphosis ultimately achieved similarbrain:body proportions, the absolute brain sizes of these fragmentswere significantly different (Fig. S1G), indicating that whole-bodyregeneration in planarians is not the exact replacement of cellnumbers but rather the restoration of appropriate body form.Therefore, regeneration can achieve correct brain proportionsthrough either epimorphosis or remodeling programs, suggestingthe existence of mechanisms that actively control organproportionality.

notum and wnt11-6 are expressed in neurons at oppositepoles of the brainWe took a candidate approach to identify molecules that controlbrain:body proportion in planarians, reasoning that mechanismsunderlying robust size attainment would involve organ-specificsecreted signals. Notum proteins are secreted lipases that deacylateWnt ligands and prevent binding to Frizzled receptors (Kakugawaet al., 2015; Zhang et al., 2015), thus inhibiting Wnt signaling inmany animals, including planarians, fruit flies, zebrafish andmammals (Flowers et al., 2012; Gerlitz and Basler, 2002; Giráldezet al., 2002; Petersen and Reddien, 2011; Traister et al., 2008).Planarians have a single notum homolog, and in uninjured animals,notum is expressed in the head at the anterior body pole and theanterior brain commissure (Fig. 2A, cyan) (Petersen and Reddien,2011). Double FISH determined that notum expression at theanterior pole is within collagen+ body-wall muscle cells (Witchleyet al., 2013) (Fig. 2B, white arrows; 85.5% of notum+ anterior polecells were collagen+), whereas notum expression at the anteriorbrain commissure is within chat+ cholinergic neurons (90.1% ofnotum+ cells at the anterior commissure were chat+; Fig. 2C, whitearrows). Notum proteins can inhibit Wnt signaling, so we sought toidentify a planarian Wnt gene expressed near the brain. Among thenine planarian Wnt genes, wnt11-6 has prominent expression in theposterior brain (Fig. 2A, magenta) in addition to dispersed cellsthroughout the body (Adell et al., 2009; Gurley et al., 2010;Kobayashi et al., 2007). Double FISH determined that a majority ofwnt11-6 cells associated with the brain (51.3%) are expressed inchat+ neurons (Fig. 2D, white arrows). notum and wnt11-6 areexpressed at opposite ends of the brain in distinct expression

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domains that are maintained and restored during brain remodelingand epimorphic brain growth, respectively, and we were not able toidentify cells that coexpress notum and wnt11-6 in any conditions(Fig. 2A,E).

notum and wnt11-6 are required for proper control of brainproportionGiven its regionalized expression within the brain, we next soughtto identify putative functions of notum in the control of brainproportion. Inhibition of notum caused a reduction of cintillo+ andgad+ brain cell proportions in regenerating head fragmentscompared with control animals (Fig. 3A,B). Therefore, notum hasa role in promoting brain cell numbers during conditions of braincell loss through tissue remodeling. notum(RNAi) regenerating headfragments possessed normal regionalized expression of threehomeotic transcription factors [orthopedia (otp) (Umesono et al.,1997), orthodenticle B (otxB) and orthodenticle A (otxA) (Umesonoet al., 1999)] and a brain branch marker [G protein alpha subunit(gpas) (Cebrià et al., 2002b); Fig. S2], suggesting that notumprimarily affects size but not pattern of the brain. The size reductionphenotype appeared to be specific to the brain, because notumRNAi did not change relative pharynx-to-body proportion

(Fig. S3A), length of the pharynx neuropile at the distal end ofthat organ (Fig. S3B) or total body length (Fig. S3C). Finally,inhibition of notum did not significantly alter the density of neuronswithin the brain (Fig. S3D) or average neuronal cell size (Fig. S3E),indicating that notum is likely to regulate brain:body proportionprincipally through the regulation of brain cell number.Furthermore, although previous work identified notum ascontrolling anterior pole formation during head regeneration(Petersen and Reddien, 2011), inhibition of notum in headfragments did not eliminate expression of sFRP-1, a marker of theanterior pole (Fig. S4A). Therefore, the role of notum in brain sizingis likely to be independent of anterior pole formation. Collectively,these results demonstrate a specific function for notum in promotingappropriate brain size in regenerative degrowth.

notum(RNAi) head fragments undergoing brain remodelingadditionally formed an ectopic set of photoreceptors within thehead tip anterior to the original photoreceptors (19 of 21 worms;Fig. S4B). Such animals had anterior chat+ neural tissue associatedwith the ectopic photoreceptors and a more anterior placement ofthe posterior brain boundary (Fig. 3A). We suggest that brain sizereduction through notum RNAi might change the position of an eyefield that could be set up through underlying tissue of the brain.

Fig. 1. Planarian brain:body proportion is restored through regeneration by either increasing or decreasing brain cell number as necessary. (A) FISHdetecting 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 differentlysized uninjured animals plotted against body length. (D) Average numbers of cintillo+ neurons (top, magenta) and gad+ neurons (bottom, cyan) normalizedto 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 brainthrough epimorphosis (G). (F,G) Black dashed lines indicate amputation plane; bottom panels show highermagnification view of top panels. Scale bars: 300 µm inA,B,E and F,G top panels; 150 µm in F,G bottom panels. Anterior, top. d, day.

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Alternatively, notum could have functions in eye placement that alsoaffect brain size. We examined the relationship between the smallbrain and ectopic eye phenotypes by inhibiting ovo, a transcriptionfactor required for production of photoreceptors (Lapan andReddien, 2012). Dual inhibition of notum and ovo caused areduction in brain size and absence of ectopic anteriorphotoreceptors (Fig. S4C,D). Additionally, prep(RNAi)homeostasis animals that form ectopic anterior photoreceptors(Fig. S4E) (Felix and Aboobaker, 2010), similar to notum(RNAi)animals, have normal numbers of cintillo+ cells (Fig. S4F),suggesting a potential separation in requirements for brain sizingand eye placement. We conclude that eye placement functions fornotum are not required for its control of brain size and we did notinvestigate them further.wnt11-6(RNAi) planarians undergo brain expansion (Adell et al.,

2009; Kobayashi et al., 2007) and in the planarianDugesia japonicaform ectopic posterior photoreceptors (Kobayashi et al., 2007),suggestive of opposing functions to notum in brain size control. Wetherefore tested possible functional interactions between notum andwnt11-6 in Schmidtea mediterranea using double RNAi. wnt11-6(RNAi) head fragments had increased brain:body proportionscompared with control animals (Fig. 3A,B) but no defect inphotoreceptor number (Fig. S5A; 49 of 51 animals).wnt11-6(RNAi)animals also had no defects in mediolateral brain organization (Fig.S2) or significant changes in pharynx proportion, pharynx neuropilesize, body size, neuron density or neuron cell size (Fig. S3),

indicating that, like notum, wnt11-6 specifically regulates therelationship between brain cell number and body size. Simultaneousinhibition of notum and wnt11-6 in amputated head fragmentsundergoing brain remodeling completely suppressed the ectopicphotoreceptor and small brain phenotype caused by notuminhibition (Fig. S5A; 65 of 67 animals) and instead resulted in anincreased brain size similar to wnt11-6(RNAi) animals (Fig. 3A,B).qPCR confirmed that the suppressive effects of wnt11-6 dsRNA onthe notum RNAi phenotype were not caused by alteration of notumRNAi efficiency (Fig. S5B). We conclude that wnt11-6 is requiredfor the notum(RNAi) brain size phenotype during remodeling,consistent with a mechanism in which notum inhibits wnt11-6,which in turn normally suppresses brain size.

We next tested whether notum andwnt11-6 also control brain sizeduring formation of a new head through epimorphic regeneration.After decapitation, notum is expressed by 18 h near the anterior-facing wound site, and subsequently by 48-72 h in the new anteriorpole (Petersen and Reddien, 2011). Administration of notumdsRNA prior to injury results in a range of defects, includinghead/tail polarity transformations or defective head regeneration(Petersen and Reddien, 2011). To examine functions for notumspecifically in brain growth, we delivered notum dsRNA to animals24 h after head amputation, reasoning that establishment of poleidentity is likely to precede head and brain formation (Fig. S6A).Such animals succeeded in regenerating a head and forming ananterior pole (Fig. S6B,C) but formed elongated or supernumerary

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 posteriorof 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 commissureexpress 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 detectingexpression of notum (cyan) andwnt11-6 (magenta) in head fragments undergoing brain remodeling (top panels) and trunk fragments forming a new brain throughepimorphosis (bottom panels). Scale bars: 50 µm in A-D; 150 µm in E. Anterior, top. d, day.

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photoreceptors (Fig. S6B; 8 of 12 worms) reminiscent of thephotoreceptor phenotype in notum(RNAi) regenerating headfragments (Fig. S4B). notum(RNAi)-epimorphosis animalsexhibited reduced brain proportions (Fig. 3C,D). Thesephenotypes were also dependent upon wnt11-6, as notum(RNAi);wnt11-6(RNAi)-epimorphosis animals lacked photoreceptor defects(Fig. S6D; 31 of 34 worms) and had increased brain proportions(Fig. 3C,D). The effect of Wnt signaling on brain proportionappears to be specific to wnt11-6, because inhibition of wnt1,another planarian Wnt gene whose activity is known to be affectedby notum (Petersen and Reddien, 2009a, 2011), did not significantlyaffect brain size or suppress the notum(RNAi) small brain phenotype(Fig. S6E). notum and/or wnt11-6 inhibition in trunk fragments didnot alter pharynx proportion or total body size, further indicatingthat the scaling functions of these genes are specific for the brain(Fig. S6F,G). These results support the hypothesis that notum

inhibits wnt11-6 function to control the relationship between brainand body size and suggest that this regulation could occur through aprocess common to both epimorphosis and remodeling.

We then investigated the dynamic emergence of brain proportionphenotypes after notum and wnt11-6 RNAi in epimorphosis andremodeling (Fig. 3E). notum or wnt11-6 inhibition causedprogressive defects in brain:body proportion (Fig. 3E) and inabsolute neuronal cell number (Fig. S7A) that were stronger at latertimes during regeneration (after 4 days). notum/wnt11-6 signaling istherefore unlikely to affect general processes intrinsic to earlywound-induced signaling or all instances of neurogenesis but ratherhas a function specific to brain size attainment. Notably, inhibitionof notum and wnt11-6 each caused an identical change in ultimatebrain:body proportion achieved through both brain epimorphosisand brain remodeling (Fig. 3E, day 21). Consistent with thisinterpretation, prolonged inhibition of notum or wnt11-6 in the

Fig. 3. notum inhibits wnt11-6 to control brain size in regenerative degrowth and growth. (A) Day 21 regenerating head fragments undergoing brainremodeling stained for expression of chat, gad and cintillo after indicated RNAi treatments. (B) Brain:body proportion [cintillo+ (magenta) or gad+ (cyan) cellnumbers 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 withindicated 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 brainmorphology and for cintillo (D; magenta) and gad (D; cyan) to quantify brain size normalized to body length; n≥18 animals per condition. (E) Animals undergoingepimorphosis (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.

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absence of injury caused effects on brain size similar to thoseobserved through regeneration (Fig. S7B,C). Together, these resultssuggest that notum/wnt11-6 signaling might specifically influencetarget brain size. We conclude that notum and wnt11-6 antagonismacts in an essentially constitutive process to control brain cellnumber, potentially through the determination and maintenance of asize set-point for the planarian brain.

wnt11-6 regulates notum expression at the anterior braincommissure to form an inhibitory spatial feedback loopHomeostatic mechanisms, such as set-points, commonly rely onfeedback inhibition to provide output stabilization (Stanger, 2011).Notum acts as a feedback inhibitor of Wnt signaling in multipleanimal species (Flowers et al., 2012; Gerlitz and Basler, 2002;Giráldez et al., 2002; Petersen and Reddien, 2011); therefore, weexamined the potential requirement of wnt11-6 for notumexpression. Inhibition of wnt11-6 reduced numbers of notum-expressing cells in the anterior brain in conditions of homeostasisand remodeling (Fig. 4A,B; Fig. S8A). This function for wnt11-6was specific for the brain, becausewnt11-6RNAi did not noticeablyreduce numbers of notum+ cells at the anterior pole (Fig. 4A,B;Fig. S8A) or induced early after amputation (Fig. S8B), suggesting

that other or redundantly acting Wnts might participate in theseprocesses. Inhibition of beta-catenin-1 decreased notum+ cellnumbers in the anterior commissure and anterior pole and, bycontrast, inhibition of APC (encoding a component of the β-catenindestruction complex) increased the numbers of these notum+ cells(Fig. 4A,B). Therefore, transcriptional activation of notum in theanterior brain is likely to occur through canonical Wnt signaling.We additionally found that the notum+ cells of the brain commissureexpress four of the nine planarian frizzled genes [ frizzled-5/8-2,frizzled-5/8-3, frizzled-5/8-4 and frizzled-1/2/7 (Liu et al., 2013)],suggesting that they should be capable of receiving Wnt signals(Fig. S8C). As notum andwnt11-6were not coexpressed in any cellsduring regeneration (Fig. 2E), we conclude that wnt11-6 acts eitherat a distance or indirectly to activate notum expression. Together,these results indicate that wnt11-6 is required for the expression ofits own secreted inhibitor, notum, within the brain to establish anegative feedback loop across the organ axis.

wnt11-6 is likely to suppress brain growth through non-canonical Wnt signalingWnt family ligands can signal through either canonical, β-catenin-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 fromA (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 theindicated 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 forbrain:body proportion as measured by cintillo+ cell numbers per animal length normalized to control animals (n≥5 animals per condition). All error barsindicate 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.

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pathways (Nusse, 2012). We examined intracellular components ofcanonicalWnt signaling and their relationships to notum andwnt11-6 to clarify the signaling pathways used in control of brain size.RNAi of beta-catenin-1 [at a time of regeneration prior to body-wide formation of ectopic anterior central nervous system structures(Gurley et al., 2008; Iglesias et al., 2008; Petersen and Reddien,2008)] caused a decrease in brain size in heads regenerated fromanterior-facing wounds (Fig. 4C), opposite to the wnt11-6 RNAiphenotype and consistent with previous findings (Owen et al.,2015). wnt11-6(RNAi);beta-catenin-1(RNAi) animals had largebrains similar to wnt11-6 inhibition alone (Fig. 4C), suggestingthat wnt11-6 might act downstream or in parallel to beta-catenin-1to control brain size. We inhibited APC to overactivate canonicalWnt signaling in order to test the hypothesis that beta-catenin-1promotes brain size mainly through activating notum expression.In head fragments undergoing brain remodeling, APC RNAiphenocopied wnt11-6 RNAi to produce an enlarged brain

(Fig. 4D). Furthermore, simultaneous inhibition of APC andnotum resulted in small-brained animals (Fig. 4D), confirmingthat the large brain APC(RNAi) phenotype depends on notum. Thesimplest interpretation of these results is that wnt11-6 activatesnotum through canonical Wnt signaling but wnt11-6 suppressesbrain growth through non-canonical, beta-catenin-1-independentsignaling (Fig. 4E). Inhibition of Dishevelled (Dvl) homologsDvl-1and Dvl-2, genes that function in both canonical and non-canonicalWnt signaling pathways, caused brain enlargement similar townt11-6 RNAi and unlike beta-catenin-1 RNAi (Fig. 4F),consistent with this interpretation.

notum and wnt11-6 regulate neoblast differentiation toinfluence brain sizeWe then sought to identify the cellular mechanisms regulated bynotum and wnt11-6 in control of brain size and hypothesizedthat they are likely to be common to epimorphosis and tissueremodeling. We first tested for candidate functions for notum andwnt11-6 in apoptosis because of their ability to control cell numberand their activation during tissue remodeling (Pellettieri et al.,2010). We measured the role of notum and wnt11-6 in directing celldeath 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 ofTUNEL+ cells throughout the animal at any regenerative time point(Fig. 5B). Therefore, we conclude that notum and wnt11-6 areunlikely to act through injury-induced cell death to direct a changein brain size.

Ultimate organ size is likely to result from a balance between cellloss and cell production. Planarians use neoblasts, a population thatincludes pluripotent stem cells, for regeneration, growth andhomeostatic cell replacement (Reddien et al., 2005b; Wagneret al., 2011). Therefore, we next investigated whether notum orwnt11-6 regulates neoblast-dependent tissue production to controlbrain size. To test this hypothesis, wemeasured cintillo+ cell numberin lethally irradiated animals injectedwith control, notum orwnt11-6dsRNA 2 days prior to head removal (Fig. 6A). As expected,decapitated irradiated trunk fragments did not produce a headblastema or cintillo+ cells (zero cells in three animals examined foreach RNAi condition). Surprisingly, control RNAi irradiated headfragments undergoing brain remodeling lost an excess number ofcintillo+ cells compared with non-irradiated control head fragments,indicating that normal tissue remodeling involves a neoblast-dependent activity that promotes brain cell number. notum(RNAi)non-irradiated or irradiated head fragments all attained a numberof cintillo+ cells similar to irradiated control RNAi head fragments,indicating that notum requires neoblasts to promote brain sizeduring remodeling (Fig. 6A). Additionally, irradiation completelysuppressed the large brain phenotype observed with wnt11-6 RNAi,suggesting thatwnt11-6 also requires neoblasts to affect brain size inall regenerative contexts (Fig. 6A). The simplest interpretation ofthese results is that remodeling involves the production of new braincells in a manner regulated by notum and wnt11-6. To test thishypothesis, we measured the ability of notum(RNAi) or wnt11-6(RNAi) animals undergoing brain remodeling to produce new chat+

cells of the brain after a pulse of bromodeoxyuridine (BrdU) on day 9and before fixation on day 13. notum RNAi caused a reduction inrelative numbers of BrdU+chat+ cells, whereas wnt11-6 RNAiincreased their numbers (Fig. 6B). Together, these results strongly

Fig. 5. notum and wnt11-6 do not control injury-induced cell death.(A,B) Cell death during brain remodeling in regenerating head fragments wasassayed by TUNEL staining over a time series of 21 days. (A) Animals weretreatedwith control, notum orwnt11-6 dsRNA by injection (three injections over3 days), amputated pre- and post-pharyngeally, then TUNEL stained.(B) TUNEL+ cell numbers per fragment area were quantified for all conditionsand time points (n≥5 animals per time point; n.s. P>0.05, two-tailed t-test). Allerror bars indicate s.d. Scale bars: 300 µm. Anterior, top. d, day.

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suggest that new brain cell production contributes to attainment ofappropriate proportion during regenerative tissue remodeling andthat notum andwnt11-6 are likely to control brain size by influencingthe formation of new brain cells.We tested for possible functions of wnt11-6 on global

proliferation, because neoblasts are the only proliferating cells inplanarians (Baguñà, 1976; Newmark and Sánchez Alvarado, 2000;Reddien et al., 2005b).Wemeasured global mitotic index in control,notum(RNAi) andwnt11-6(RNAi) head or tail fragments during brainremodeling or epimorphosis by staining with anti-phophoSer10-histoneH3 (Wenemoser andReddien, 2010). In both remodeling andepimorphosis, notum(RNAi) and wnt11-6(RNAi) animals hadbroadly similar global proliferative activities (Fig. 6C,D; Fig. S9),with differences at selected times (elevated after wnt11-6 RNAi inday 3 tail fragments and day 13 head fragments and reduced afternotumRNAi in day 7 in tail fragments). Thus,wnt11-6 and notum donot appear to control cell division globally throughout regenerationas would be expected for regulation that constitutively maintainsappropriate brain size, although we cannot rule out the possibilitythat they control proliferation of neoblast subpopulations at isolatedwindows in regeneration.

Neoblasts are a heterogeneous population that includes bothpluripotent cells and distinct subpopulations specified forproduction of multiple differentiated tissues (Adler et al., 2014;Cowles et al., 2013; Currie and Pearson, 2013; Forsthoefel et al.,2012; Lapan and Reddien, 2012; Marz et al., 2013; Scimone et al.,2011, 2014a,b; van Wolfswinkel et al., 2014; Vásquez-Doormanand Petersen, 2014; Vogg et al., 2014; Wenemoser et al., 2012). Wehypothesized that notum and wnt11-6 might regulate brain cellproduction by controlling numbers of brain cell progenitors markedby expression of lineage-specific transcription factors. We firstexamined ap2, which is required for production of trpA+ brainneurons and expressed in nearby smedwi-1+ neoblasts (Wenemoseret al., 2012). Like all other neuronal populations tested, trpA+ braincell numbers increased after wnt11-6 RNAi and decreased afternotum RNAi (Fig. S10A,B). As hypothesized, ap2+ smedwi-1+ cellnumbers were elevated afterwnt11-6RNAi and reduced after notumRNAi both in formation of a new brain through epimorphosis(Fig. 7A) and in alteration of a pre-existing brain throughremodeling (Fig. 7B). We examined two additional neoblastsubpopulations that contribute to the brain, lhx1/5-1+smedwi-1+

cells that form serT+ serotonergic neurons of the brain (Currie and

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+ cellsquantified 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 regenerationtime 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 representativeimages). 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.

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Pearson, 2013) (Fig. S10C,D) and coe+smedwi-1+ cells that form avariety of neuronal subtypes [including cpp-1+, chat+, gad+, tph+,th+ and tbh+ neurons (Cowles et al., 2013)]. In all cases, notum(RNAi) animals had fewer brain progenitors and wnt11-6(RNAi)animals had more brain progenitors (Fig. 7A,B). Taken together, wesuggest that notum inhibits wnt11-6 activity, which in turn limitsproduction of differentiated neurons through control of neoblastdifferentiation but not cell death. These results suggest thatappropriate size attainment in regeneration involves regulation ofthe rates or extents of tissue production through organ-specificregionalized signaling.

DISCUSSIONThe mechanisms underlying size attainment in development andregeneration are poorly understood, and planarians offer a powerful

system for dissection of this process. Our studies support a model inwhich negative feedback signaling directs the size of the planarianbrain through reversible regenerative growth (Fig. 8). We proposethat wnt11-6 from the posterior brain inhibits the formation ordivision of brain progenitors to modify the rate of brain cellproduction. Through the canonical Wnt signaling pathway, wnt11-6activates the expression of its own secreted inhibitor, notum, at theopposite end of the organ. In turn, notum promotes brain growththrough its inhibition of non-canonical wnt11-6 activity. Thisprocess operates to maintain brain proportionality in uninjuredanimals and restore appropriate brain:body proportion in animalseither growing a new brain through blastema formation or shrinkinga pre-existing brain through remodeling. The notum/wnt11-6feedback loop does not appear to control the ability to respond toinjury or significantly affect the early rates of brain size increase in

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 orcoe (magenta) in (A) trunk fragments regenerating a new brain or (B) head fragments undergoing brain remodeling 4 days after amputation and treated withindicated dsRNAs. Upper panels, images are representative planes from a confocal stack of the region indicated by the cartoon (white arrows, doublepositive cells; yellow insets show magnified representative double positive cells). Lower panels, graphs show quantifications of progenitor cell numbers (seeMaterials 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.

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epimorphosis or decrease in brain remodeling, but rather controls abrain size set-point. The ability to perform large-scale unbiasedRNAi screens in planarians, the unique ability to achieve organproportions by either increases or decreases in cell number and tomeasure these processes quantitatively in whole animals will enablethis system to be used to identify the genetic architecture underlyingattainment of organ size.Our studies highlight that whole-body regeneration acts primarily

to restore form rather than simply to replace missing tissues. Forexample, decapitation of animals with an average of 45 cintillo+

cells caused regeneration of only 30 new cintillo+ cells fromregenerating trunk fragments (Fig. S1G), but this number perfectlyrestored the proportion of cintillo+ cells to body size (Fig. 1D).Therefore, planarian regeneration does not replace an absolutenumber of cells removed by injury. Likewise, amputated headfragments starting with 45 cintillo+ cells lost 20 of these cellsthrough tissue remodeling (Fig. S1G), and this reduction alsorestored brain proportion (Fig. 1D). Given that uninjured planariansdo not possess a fixed size (Reddien and Sánchez Alvarado, 2004),we argue that restoration of proportion is central to the completionof regeneration and might be a requirement to ensure fidelity insuccessive rounds of asexual reproduction or a property intrinsic towhole-body regeneration.We find unexpected similarities between the mechanisms of

growth control involved in epimorphosis, tissue remodeling andhomeostatic maintenance. After or in parallel to early injury-induced proliferation (Wenemoser and Reddien, 2010), tissuepolarization (Petersen and Reddien, 2009a, 2011) and injury-induced signaling (Gavino et al., 2013; Wenemoser et al., 2012),production of the regeneration blastema might involve an extremeform of tissue turnover shared among aspects of tissue remodelingand normal maintenance, similar to the concept of homeostaticregeneration (Wills et al., 2008a,b). The control of organ size isfrequently described as the result of a balance between cell

proliferation and cell death (Conlon and Raff, 1999). Tissueremodeling activated by injury coincides with waves of earlywound-induced and later systemic cell death dependent on theextent of missing tissue (Pellettieri et al., 2010). We observed norole for notum/wnt11-6 signaling in programmed cell death duringbrain remodeling (Fig. 5) and instead found that notum and wnt11-6controlled the size of the pool of brain progenitors descended frompluripotent neoblasts (Fig. 7). Additionally, depletion of stem cellsby irradiation and notumRNAi caused identical excesses of cell lossto brains undergoing tissue remodeling, and perturbation ofwnt11-6and notum had opposite effects on the rate of neoblast differentiationto brain cells in remodeling (Fig. 6A,B). Together, these resultssuggest that an important component of size regulation throughremodeling is the control of neoblast activity. Likewise, starvation-induced degrowth that reduces size globally involves body-widereduction of differentiation (González-Estévez et al., 2012a), andintestinal remodeling coincides with a significant amount of newcell production in that organ (Forsthoefel et al., 2011). We suggestthat the wnt11-6/notum signaling system can tune the size of thebrain in contexts of regenerative growth and degrowth and in theabsence of injury to allow alteration of the fraction of neoblastsspecified to brain cell fates. Lineage-committed progenitor cellnumber during embryogenesis of several vertebrate model systemshas been shown to be correlated with final organ size (Kicheva et al.,2014; Stanger et al., 2007), suggesting that the regulation ofprogenitor numbers could be an ancient and conserved mechanismfor organ size control.

The use of Wnt signaling for organ size control and anteriorpatterning is also widespread (Petersen and Reddien, 2009b).Production of the Wnt inhibitor Dkk through cell differentiationregulates the size of zebrafish mechanosensory organs bycounteracting Wnt signals required for progenitor proliferation(Wada et al., 2013). Overactivation of β-catenin increases the sizeof the mammalian brain through cell cycle regulation of neuralprogenitors (Chenn and Walsh, 2002, 2003), a phenotype broadlysimilar to planarian APC RNAi (Fig. 4D). Mammalian wnt1,wnt3a and wnt8 participate in brain patterning and proliferation(Erter et al., 2001; Lee et al., 2000; McMahon and Bradley, 1990;Thomas and Capecchi, 1990), but the precise mechanisms thatrelate Wnt ligands and secreted inhibitors for control of brain sizeremain unclear in vertebrates, as do their relationships withadditional regulatory pathways. Perturbation of planarian insulin(Miller and Newmark, 2012) or hippo signaling (Lin and Pearson,2014) causes body-wide defects in neoblast proliferation,suggesting that alternative pathways are used for control of brainsize. Our results reveal a broadly conserved relationship betweenWnt activity and brain size and suggest that the planarian brain isa tractable system for discovery of additional size regulatorypathways.

Growth inhibitors produced as a consequence of differentiationadditionally have broad use in the control of organ size(Gamer et al., 2003; Gomer, 2001; Lander et al., 2009; Stanger,2008). As a negative regulator of brain growth expressed in neuronsof the brain and predicted to encode a secreted protein, wnt11-6shares similarity with the action of molecules such as myostatin(McPherron and Lee, 1997; McPherron et al., 1997) and gdf11 (Wuet al., 2003), proposed chalones that limit cell numbers within thetissue from which they are expressed, the mammalian muscle andolfactory epithelium, respectively. Our studies demonstrate anadditional layer of regulation in regenerative organ size control, inwhich the action of a putative chalone (wnt11-6) is tuned throughthe use of a feedback inhibitor (notum). In principle, restoration and

Fig. 8. notum/wnt11-6 feedback regulation dampens brain celldifferentiation to achieve proper brain:body scaling. (Left) Cartoondepicting planarian brain with neurons (black), wnt11-6-expressing neurons(red with black outline) and other brain-associated cell types (red with whiteoutline) in the posterior brain region (red), notum-expressing neurons at theanterior commissure (green with black outline) and neoblasts that surround thebrain (blue, medial neoblasts shown). (Right) Model for regulatory pathwayinfluencing brain size. wnt11-6 inhibits neoblast production of differentiatedbrain cells (including cintillo+, gad+, trpA+ and tph+ neurons) by suppressingformation or division of neural progenitors (ap2+smedwi-1+, coe+smedwi-1+

and lhx1/5-1+smedwi-1+ cells) from pluripotent neoblasts. wnt11-6 signalsdirectly or indirectly through β-catenin-dependent canonical Wnt signaling toactivate expression of notum in neurons at the anterior brain commissure.wnt11-6 is likely to signal independently of beta-catenin-1 in control ofneoblasts to suppress brain size. notum encodes a secreted protein thatinhibits wnt11-6 function to promote ongoing synthesis of brain cells fromneoblasts. Levels of wnt11-6 and notum signaling control numbers ofneoblasts committing to brain cell fates to influence the size of the brain.

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maintenance of organ proportions could occur by achieving andpreserving maximal activity of growth inhibitors in order to halt celldifferentiation at an appropriate endpoint. However, it is unlikelythat wnt11-6 activity levels are maximally high or low at theendpoint of regeneration, because perturbation of wnt11-6 or notumcan reversibly affect brain size during homeostatic conditions inanimals starting with optimal brain:body proportions (Fig. S7B,C).Instead, maintenance of ongoing growth and regeneration abilitiesmight require constitutively expressed dampening mechanisms(Reddien, 2011), such as wnt11-6 feedback inhibition throughnotum, to prevent cessation of growth regulation. Spatial regulatorymodules that achieve a sustained balance between growth inhibitorsand activators could be essential for regenerative abilities,proportional growth and defining target organ size.

MATERIALS AND METHODSPlanarian culture and irradiation treatmentsAsexual Schmidtea mediterranea (CIW4) were maintained in 1× Montjuicsalts between 18 and 20°C. Gamma irradiation (6000 rad) was performedwith a cesium-137 source irradiator at least 24 h prior to amputation toeliminate all dividing cells.

Whole-mount in situ hybridizationAnimals were fixed and stained as described previously (Pearson et al.,2009). Antibodies were used in MABT containing 10% horse serum forFISH [anti-DIG-POD 1:2000 (Roche), anti-FL-POD 1:1000 (Roche), anti-DNP-POD 1:500 (PerkinElmer)] or NBT/BCIP in situ hybridization [(anti-DIG-AP 1:4000 (Roche)]. For multiplex FISH, peroxidase activity wasquenched between tyramide reactions using 4% formaldehyde (Pearsonet al., 2009) or 100 mM sodium azide (King and Newmark, 2013) for at least1 h at room temperature. Nuclear counterstaining was performed using1:1000 Hoechst 33342 (Invitrogen) in PBSTx (1× phosphate buffered salinewith 0.1% Triton X-100).

RNA interferenceRNA interference by feeding was performed using E. coli HT115 culturesexpressing dsRNA from cDNA cloned into pPR244 (Gurley et al., 2008;Reddien et al., 2005a). For regeneration experiments, animals were fed liver-bacteria mixture four times over 9 days. For long-term homeostasisexperiments, animals were fed RNAi bacterial food every 3 days. dsRNAtargeting C. elegans unc-22 was used as a negative control. For RNAi byinjection, dsRNA was synthesized by in vitro transcription and diluted to2000 ng/μl, then administered to animal fragments by microinjection(Drummond Scientific). For double RNAi, liver-bacteria mixtures or in vitrotranscribed dsRNA were mixed in equal volumes prior to administration,with single gene inhibition controls normalized with control dsRNA. notum,beta-catenin-1, APC, Dvl-1 and Dvl-2 plasmids were described previously(Gurley et al., 2008; Petersen and Reddien, 2011). wnt11-6 was clonedusing primers 5′-TCGCATACAGCTTCAATCACA-3′ and 5′-AATGAT-TTTGTGCCATACGAA-3′.

BrdU labelingDay 9 head fragments from RNAi-fed animals were injected with 5 mg/mlBrdU (Sigma-Aldrich) in water, then 4 days later fixed and stained aspreviously described (Vásquez-Doorman and Petersen, 2014).

Whole-mount immunostainingAnimals were sacrificed in 0.75 M HCl, then fixed with Carnoy’s solution(60% ethanol, 30% chloroform and 10% acetic acid) and bleached overnightwith 6% hydrogen peroxide in methanol. Animals were blocked for 6 h in1× PBSTB (1x phosphate buffered saline, 0.3% Triton X-100, 0.25%bovine serum albumin) and primary and secondary antibody incubationsperformed overnight using rabbit anti-phospho-Histone H3 Ser10 (CellSignaling; 1:3000 in 1× PBSTB) followed by anti-rabbit horseradishperoxidase conjugate (Invitrogen; 1:1000 in 1× PBSTB) and Alexa568-tyramide amplification (1:150; Invitrogen).

Terminal uridine nick-end labeling (TUNEL)TUNEL was performed as described by Pellettieri et al. (2010), withmodifications. Animals were sacrificed in 5% N-acetyl-cysteine in 1× PBS,fixed in 4% formaldehyde in 1× PBSTx, and bleached overnight in 6%hydrogen peroxide in 1× PBSTx. Samples were labeled with DIG-11-dUTP(Roche) by terminal deoxyuridine transferase (TdT) reaction (Fermentas) at37°C for 2 h, then blocked and incubated overnight in anti-DIG-POD(Roche; 1:2000 in 10% horse serum in 1× PBSTx) prior to tyramidedevelopment (Invitrogen).

Image analysisImagingImaging was performed with a Leica M210F dissecting scope with a LeicaDFC295 camera, a Leica DM5500B compound microscope with Optigrid,Leica SP5 or Leica TCS SPE confocal compound microscopes. Fluorescentimages collected by compound microscopy are maximal projections of a z-stack and adjusted for brightness and contrast using Adobe Photoshop.

Cell countingcintillo+ cells and gad+ or trpA+ cells in the medial brain region werecounted manually. chat+ cells from one lobe of each animal were countedfrom a z-series of images using three-dimensional segmentation software inImaris. Animal lengths were measured with ImageJ (National Institutes ofHealth) as visualized with Hoechst. Relative brain length was measuredfrom the most posterior brain branch to the most anterior brain branch asvisualized by chat FISH signal or Hoechst. Samples from similar fragmentsand time points were averaged and significant differences determined bytwo-tailed Student’s t-tests. Cells coexpressingwnt11-6 and chat, notum andchat or notum and collagen were counted manually from z-stack confocalimages (0.5-1 μm thick) using ImageJ. For notum coexpression, imageswere taken of the head region containing both the anterior body pole andanterior brain commissure. For wnt11-6, images were taken of the posteriorof brain. Cells labeled with BrdU and chat or expressing both smedwi1 andeither ap2, coe or lhx1/5 during regeneration were manually blind-countedfrom z-stack images near the posterior cephalic ganglia (BrdU), anterior-facing wound site (epimorphosis) or between the lobes of the cephalicganglia (remodeling) by manual electronic labeling in using ImageJ andchecking for consistency by comparing neighboring planes. H3P+ andTUNEL+ cell numbers were quantified using CellProfiler (Jones et al.,2008; Lamprecht et al., 2007) or ImageJ.

Regression analysisLines of best fit for cintillo+ and gad+ compared with either body (Table S1)or brain length (Table S2) in uninjured animals were determined byregression analysis in Microsoft Excel. Equations and r2 values are shown inTables S1 and S2. Estimates of intact proportions, shown on graphs ofregenerative time courses by a dashed black line, are averages of valuesinterpolated from power regression (the analysis type with highest r2 values)using body or brain length measurements of animals on day 15 ofremodeling or epimorphosis.

AcknowledgementsWe thank the Pearson Laboratory (UToronto) for kindly sharing their TUNEL protocoland Dr Erik Andersen, Adam Hockenberry and all past and present members of thePetersen laboratory for helpful discussions of the manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceived and designed the experiments: E.M.H., C.P.P. Performed theexperiments: 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.

FundingThe authors acknowledge support from a National Institutes of Health institutionalpredoctoral training program [Cellular and Molecular Basis of Disease TrainingProgram 2T32GM008061-31 to E.M.H.] and an Ellison Medical Foundation NewScholar in Aging Research Award [AG-NS-0835-11 to C.P.P.], a National Institutes

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of Health Director’s New Innovator Award [1DP2DE024365-01 to C.P.P.] and anAmerican Cancer Society institutional research grant [ACS-IRG 93-037-15 toC.P.P.]. The funders had no role in study design, data collection and analysis,decision to publish or preparation of the manuscript. Deposited in PMC for releaseafter 12 months.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.123612/-/DC1

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