Regenerative ability in adulthood is widespread but unevenly distributed across the animal kingdom, with some species displaying high regenerative capacity while other representatives of the same phyla display a more limited capability. By contrast, the ability to maintain tissue integrity and functionality via homeostatic maintenance throughout adulthood is more common (Poss, 2010). Regeneration is initiated by injury, and so it involves unique inputs beyond those needed for tissue maintenance and growth, such as wound healing, injury-induced activation of proliferation, tissue re-patterning, and the integration of new and old tissues. However, beyond initial responses to injury, the processes to produce new adult tissue through homeostatic maintenance or regeneration appear to occur through substantially similar mechanisms involving the shared use of tissue progenitors and stem cells for the formation of new differentiated cells. Organisms with strong regenerative ability in many cases also undergo abundant homeostatic maintenance in the absence of injury, making them ideal systems to interrogate the requirements for these processes (Newmark and Sánchez Alvarado, 2000; Elliott and Sánchez Alvarado, 2013; Maden et al., 2013; Srivastava et al., 2014; Rodrigo Albors et al., 2015; Schaible et al., 2015; Bodnar and Coffman, 2016). Indeed, functional studies of gene function in highly regenerative organisms, including planarians and zebrafish, indicate that a large majority of factors required for regeneration are also required for tissue maintenance in uninjured animals (Reddien et al., 2005; Whitehead et al., 2005; Wills et al., 2008).

Despite the similarity between regeneration and growth programs, most animals exhibit an age-dependent reduction in the ability for de novo tissue formation without a coinciding loss of proliferative growth or tissue maintenance. Examples of this phenomenon can be found across most metazoan phyla, including Xenopus limbs (Dent, 1962; Slack et al., 2004), the distal tips of mammalian digits (Borgens, 1982; Reginelli et al., 1995; Lehoczky et al., 2011), Drosophila imaginal discs (Harris et al., 2016), and mouse myocardial tissue (Drenckhahn et al., 2008; Porrello et al., 2011). Therefore, the mechanisms accounting for age-associated loss of regenerative capacity are unlikely to derive from generic reductions in cell proliferation or differentiation. An alternative cause of regeneration attenuation could be developmental loss of embryonic axis patterning systems, which can provide robust positional and scaling information early in embryogenesis (Reversade and De Robertis, 2005) but are generally not sustained into adulthood in organisms with low regenerative ability in maturity.

Adult freshwater planarians, which have a nearly unlimited ability to undergo regeneration and tissue replacement through homeostasis, use constitutive positional information as an essential upstream regulator of regeneration (Elliott and Sánchez Alvarado, 2013). These animals continually express patterning molecules that demarcate the main body axes and are used for regional identity determination through regeneration: Wnt and FGFRL signaling for the anteroposterior (AP) axis (Gurley et al., 2008; Petersen and Reddien, 2008; Petersen and Reddien, 2009a; Gurley et al., 2010; Petersen and Reddien, 2011; Hill and Petersen, 2015; Lander and Petersen, 2016; Scimone et al., 2016), BMP signaling for the dorsoventral (DV) axis (Molina et al., 2007; Reddien et al., 2007; Gaviño and Reddien, 2011; Molina et al., 2011), and Slit/Wnt5 signaling for the mediolateral (ML) axis (Cebrià et al., 2007; Gurley et al., 2010). Genes from these pathways are expressed mainly within cells of the body-wall musculature (Witchley et al., 2013) and are regionally restricted (Lander and Petersen, 2016; Scimone et al., 2016) to mark territories across each axis. Although some patterning factors are induced by injury and function early in regeneration (Petersen and Reddien, 2009a; Gurley et al., 2010; Petersen and Reddien, 2011; Wenemoser et al., 2012; Roberts-Galbraith and Newmark, 2013; Wurtzel et al., 2015), the majority are expressed in specific axial territories in uninjured animals and shift their expression domain during the regeneration process to restore missing body regions (Petersen and Reddien, 2009a; Gurley et al., 2010; Lander and Petersen, 2016; Scimone et al., 2016). Perturbations to these factors can result in tissue duplications or alterations to regional proportionality, either in regenerating animals (Bartscherer et al., 2006; Owen et al., 2015; Lander and Petersen, 2016; Scimone et al., 2016) or in animals undergoing RNAi inhibition over a period of prolonged tissue homeostasis in the absence of injury (Hill and Petersen, 2015; Reuter et al., 2015; Lander and Petersen, 2016; Stückemann et al., 2017). For example, RNAi of the Wnt inhibitor notum produces ectopic eyes anteriorly in the head (Hill and Petersen, 2015), RNAi of the Wnt gene wnt11-6/wntA and the Wnt receptor fzd5/8-4 produces ectopic eyes posteriorly in the head (Scimone et al., 2016), and RNAi of the Wnt gene wntP-2/wnt11-5 and the Wnt co-receptor ptk7 produces ectopic pharynges within the tail (Lander and Petersen, 2016; Scimone et al., 2016). Some dynamic expression changes of positional control genes can occur in animals depleted of stem cells, for example, expression of wntP-2 and ptk7 in regenerating head fragments, suggesting that at least some patterning information is not dependent on the ability to produce of missing tissues (Petersen and Reddien, 2009a; Gurley et al., 2010; Lander and Petersen, 2016). Therefore, patterning factors are influential in regulating axis composition both in regeneration and in homeostatic maintenance in the absence of injury.

All mature tissues in planarians derive from a body-wide pool of adult pluripotent stem cells of the neoblast population (Wagner et al., 2011; Guedelhoefer and Sánchez Alvarado, 2012). Therefore, a compelling model to account for the robustness of both pattern restoration through regeneration and pattern maintenance through perpetual homeostasis is the use of positional cues to precisely control the differentiation and targeting of planarian neoblast stem cells for tissue production at correct locations (Reddien, 2011). Indeed, for the D/V axis, BMP signaling can either directly or indirectly influence the specification of neoblasts into dorsal or ventral epidermal progenitors (Wurtzel et al., 2017), and BMP signaling is necessary to maintain D/V axis asymmetry both in regeneration and through homeostasis (Molina et al., 2007; Reddien et al., 2007; Gaviño and Reddien, 2011; Molina et al., 2011). Likewise, Wnt signaling along the A/P axis can regulate neoblast specification in both contexts as well (Hill and Petersen, 2015; Reuter et al., 2015; Lander and Petersen, 2016). One expectation of this model is that the sites of organ regeneration and organ homeostasis should be identical along the body axis even if patterning information is experimentally modified.

We investigated this model by examining the regenerative and homeostatic properties of tissue duplication phenotypes generated by pattern disruption through RNAi treatment. We focused our analysis on regeneration and maintenance of the planarian eye, a simple, well characterized, and regionally restricted organ that can be specifically removed and easily studied. Using RNAi of Wnt signaling components and surgical strategies to shift head patterning information either to the anterior or posterior, our analyses indicate that sites of organ homeostasis do not always coincide with sites of organ regeneration. These results suggest that patterning molecules have a primary function to control the location of regeneration and that mature tissue can undergo growth and tissue homeostasis through progenitor acquisition independent of more precise positional cues. Collectively, these properties could account for the integration of new and pre-existing tissues during regeneration and suggest potential mechanistic differences between tissue regeneration and tissue homeostasis.

To investigate the regenerative competency and homeostatic stability of duplicated tissue structures, we first sought to establish a reliable method for the production of duplicated organs in planarians. NOTUM is a evolutionarily conserved secreted Wnt inhibitor that deacylates Wnt ligands to prohibit binding Frizzled receptors (Kakugawa et al., 2015; Zhang et al., 2015). In planarians, notum is an integral regulator of anterior identity and pattern (Petersen and Reddien, 2011; Hill and Petersen, 2015). notum(RNAi) head fragments and uninjured animals undergo anterior shifts to axial identity to produce a set of anterior eyes located anterior to the original, pre-existing eyes (Figure 1A). Both the ectopic and pre-existing eyes contain a normal distribution of cell types (photoreceptor neurons expressing opsin, and pigment cups expressing tyrosinase) and enervate the brain, as seen by detecting their neuronal processes with anti-ARRESTIN staining (Figure 1B–C). Additionally, we tested the functionality of both sets of eyes in light avoidance assays that measure travel time away from a light source through an illuminated arena. Negative phototaxis still occurred in animals with only pre-existing eyes or only ectopic eyes. Light avoidance behavior was eliminated only when all eyes were removed, indicating the functionality of both the ectopic and pre-existing eyes to detect light (Figure 1—figure supplement 1A–E).

Figure 1 with 3 supplements see all

Download asset Open asset

We next examined the regenerative properties of pre-existing versus supernumerary eyes generated by notum RNAi. Resection of normal planarian eyes results in eye regeneration over approximately 2 weeks (Deochand et al., 2016; LoCascio et al., 2017). In notum(RNAi) animals, resection of newly formed supernumerary eyes consistently resulted in regeneration of new eyes in the same position. However, in nearly all cases, no regeneration occurred following removal of a pre-existing eye (Figure 1D, Figure 1—figure supplement 2A–B). We were able to generate notum(RNAi) animals with three sets of eyes either at a low frequency from either prolonged homeostatic inhibition of notum or by tail removal of 4-eyed notum(RNAi) animals (Figure 1—figure supplement 3A–B). In all cases, only the most anterior eyes of such animals could regenerate after removal (Figure 1—figure supplement 3C). Additionally, removal of all three eyes from one side of the animal similarly resulted in regeneration of only the most anterior eye, suggesting that failure of posterior eye regeneration is not due to the presence of an anterior eye. Together these results indicate that pattern alteration by notum inhibition likely shifts a zone of competence for eye regeneration toward the anterior of the animal.

Based on current models of positional control in planarian regeneration, we anticipated that non-regenerative, pre-existing eyes in notum(RNAi) animals would eventually disappear through failed homeostasis. To examine this possibility, we monitored individual 4-eyed notum(RNAi) animals over an extended time (over 200 days), representing more than three times the approximate length of complete eye turnover (~60 days; [Lapan and Reddien, 2012]). Both the regenerative ectopic eyes and the non-regenerative pre-existing eyes persisted throughout the entire 200 day experiment (Figure 2A). The longevity of non-regenerative eyes suggested these organs could be homeostatically maintained despite their loss of regenerative ability.

Figure 2 with 3 supplements see all

Download asset Open asset

To test whether non-regenerative eyes are actively maintained through stem cell activity or are instead retained as static tissue devoid of both cell gains and losses, we examined the functional requirement of eye cell differentiation for their persistence. Four-eyed notum(RNAi) animals were subjected to 60 days of RNAi inhibition of ovo, a transcription factor that serves as a master regulator of planarian eye differentiation from neoblasts (Lapan and Reddien, 2012). Both the notum(RNAi) regenerative eyes and non-regenerative eyes disappeared with similar kinetics during ovo RNAi treatment, suggesting that pre-existing eyes are actively maintained through homeostasis (Figure 2B). To confirm these predictions, we used BrdU labeling to detect the differentiation of new eye cells. Within the eye lineage, proliferative neoblasts give rise to non-dividing eye progenitors that then terminally differentiate into mature eye cells (Lapan and Reddien, 2011; Lapan and Reddien, 2012). Therefore, the incorporation of BrdU into eye tissues allows detection of recently differentiated cells in the growing eye. We found similar numbers of BrdU +mature eye cells (opsin+) within both the regenerative and non-regenerative notum(RNAi) eyes 7 and 14 days after BrdU pulsing (Figure 2C, Figure 2—figure supplement 1), indicating that both regenerative and non-regenerative eyes are maintained by stem cell activity. BrdU incorporation was lower in each of the notum(RNAi) eyes compared to control eyes, but the total number of BrdU +eye cells was not altered by notum RNAi, suggesting that in such animals differentiating eye cells are partitioned across multiple eyes. Furthermore, we observed that both regenerative and non-regenerative eyes in notum(RNAi) animals undergo significant size increases in response to animal feeding (Figure 2—figure supplement 2). Together, these results indicate that pattern alteration through inhibition of notum shifted the location of regeneration but not the location of eye tissue maintenance.

We next tested whether the region of the pre-existing eye might be deficient in expression of wound-induced genes, which would provide a candidate explanation for why these organs cannot regenerate. Expression of the early wound-induced factors jun-1 and fos-1 as well as the late factor gpc-1 appeared normal after resection of either anterior or posterior notum(RNAi) eyes (Figure 2—figure supplement 3). Therefore, the inability of pre-existing posterior eyes to regenerate following resection is not likely due to a failure in injury responsiveness.

An alternative explanation for the inability of pre-existing notum(RNAi) eye to regenerate could be positional discrepancies with respect to the rest of the body. We next examined the position of the regenerative and non-regenerative notum(RNAi) eyes with respect to anteriorly expressed positional control genes (PCGs) and the brain. In 4-eyed notum(RNAi) animals, pre-existing eyes were located far from the anteriorly expressed sFRP-1 and within the pre-pharyngeal region of ndl-3 expression, distinct from the location of normal eyes (Figure 3A). Consistent with these findings, non-regenerative notum(RNAi) eyes were located much more posterior than control eyes with respect to the primary body axis (Figure 3B). However, notum(RNAi) regenerative eyes were located somewhat more anteriorly with respect to the body axis compared to control eyes. notum RNAi can affect multiple aspects of patterning within the animal anterior (Petersen and Reddien, 2011; Hill and Petersen, 2015), so we hypothesized that if notum RNAi shifted the site of eye regeneration more anterior, then this site may be well positioned with respect to other anterior tissue such as the brain. Consistent with this hypothesis, non-regenerative eyes were considerably displaced with respect to cintillo+ chemosensory neurons of the head (Figure 3C). Using Hoechst staining to demarcate the cephalic ganglia, we found that eyes typically formed at a particular location along the anterior-posterior axis of the brain (Figure 3D), consistent with previous reports in other planarian species (Agata et al., 1998). Intriguingly, while notum(RNAi) non-regenerative eyes were located at a more posterior position with respect to the brain, regenerative eyes in noutum(RNAi) animals were located at the same relative position as control eyes. Therefore, the site of regeneration correlates with a particular relative location with respect to other anterior tissues, either because of a role for the brain in eye positioning or because the eye and brain are both subject to independent control by an upstream process. notum itself is expressed within an anterior domain of the brain in chat+ neurons and also at the anterior pole within the body-wall musculature and both brain size and ectopic eye phenotypes from notum RNAi are suppressed by RNAi of wnt11-6 (Hill and Petersen, 2015), which is consistent with either possibility. These observations suggest that notum inhibition shifted the locations of multiple tissues within the anterior, including the target site for eye regeneration, leaving behind mispositioned pre-existing eyes at a location outside of this region.

Figure 3

Download asset Open asset

If positional control genes such as notum regulate the proportionality of many regional tissues, what mechanism explains the ability of non-regenerative eyes to undergo homeostatic maintenance? We considered two possible explanations for this phenomenon, either that mature eyes have an ability to induce their own progenitors in order to sustain themselves through homeostasis, or that eyes can acquire nearby eye progenitors regardless of the site of eye regeneration. Normal eye homeostasis involves migration of eye progenitors that specify from neoblasts within the anterior of the animal at a distance from the differentiated eye (Lapan and Reddien, 2011, 2012). In principle, these progenitors could migrate to incorporate into either the anterior or posterior eyes of notum(RNAi) animals. To test this, we first examined the numbers and distribution of ovo+ eye progenitors in 4-eyed notum(RNAi) animals. notum inhibition did not increase the number of eye progenitors per animal, and progenitors could be detected in the vicinity of both the regenerative and non-regenerative eyes (Figure 4A). We scored the position of eye progenitors across several animals and examined their distribution by normalizing their position to the axis defined by the head tip to the pharynx. notum RNAi appeared to cause a slight anterior shift to the domain of eye cell specification but that did not substantially change the abundance of eye progenitors near either the anterior or posterior eyes (Figure 4A). Therefore, both anterior and posterior eyes would likely have similar access to eye progenitors, consistent with the observation that the rate of BrdU incorporation into each notum(RNAi) eye is similar (Figure 2C). Furthermore, we found that nearby tissue removal, which is known to induce additional eye progenitors (LoCascio et al., 2017), did not enable regeneration of posterior notum(RNAi) eyes (Figure 4—figure supplement 1). Together these observations suggest that inability to regenerate is not due to a lack of access to nearby eye progenitor cells and that homeostatic maintenance of nonregenerative eyes can likely be homeostatically maintained by passively acquiring migratory eye progenitors.

Figure 4 with 2 supplements see all

Download asset Open asset

The lack of increased numbers of eye progenitors or increased total BrdU eye cell labeling in 4-eyed notum(RNAi) animals argues against a mechanism in which differentiated eye tissue can induce eye progenitor cells. By contrast, a mechanism in which mature eyes incorporate progenitors without affecting specification predicts that non-regenerative eyes should compete with regenerative eyes for acquisition of a limited pool of eye progenitors. Consistent with this model, we found that despite generating additional eyes, notum inhibition did not alter total numbers of eye cells, but rather resulted reduced numbers of cells per eye (Figure 4B), likely due to a reallocation of the eye progenitor cell pool across an increased number of organs. To further test this model, we resected a posterior eye from 4-eyed notum(RNAi) animals, then counted numbers of eye cells from the ipsilateral anterior eye, using the contralateral anterior eye as an internal control. After 16 days of recovery, the posterior eye did not regenerate, as seen previously, but the anterior eye on the side of injury grew substantially larger than its contralateral counterpart (Figure 4C, top). Likewise, when we resected an anterior eye, the ipsilateral posterior eye enlarged compared to its contralateral counterpart (Figure 4C, bottom), through the size of this effect was smaller, likely due to the ability for the anterior eye to regenerate. We confirmed prior observations that removal of an eye does not substantially alter the number of ovo+ eye progenitors on injured versus uninjured sides of the body (LoCascio et al., 2017), both in control and notum(RNAi) animals (Figure 4—figure supplement 2). We interpret these experiments to mean that eyes can compete with each other for acquisition of a limited pool of migratory eye progenitor cells. Together, these observations suggest that mature eyes can incorporate migratory progenitors independent of the site of eye regeneration.

The ability for patterning alteration to uncouple the sites of regeneration and homeostasis could be a phenomenon either specific to notum inhibition, a property specific to eyes, or alternatively reflect a fundamental difference in the mechanisms of organ regeneration and homeostatic maintenance. To examine the generality of these observations, we performed similar experiments in animals after inhibition of wnt11-6/wntA and fzd5/8-4 (Figure 5A), which act oppositely to notum to restrict head identity. wnt11-6(RNAi);fzd5/8-4(RNAi) animals form supernumerary eyes posterior to their set of pre-existing anterior eyes (Figure 5—figure supplement 1A–B). In these animals, ectopic posterior eyes regenerated after resection (7/10 animals), whereas pre-existing anterior eyes did not (11/11 animals), indicating that the treatment shifted the site of regeneration posteriorly (Figure 5A). Like notum(RNAi) animals, both the pre-existing and supernumerary eyes of wnt11-6(RNAi);fzd5/8-4(RNAi) animals persisted for extended periods of time (Figure 5—figure supplement 2A) and were able to incorporate BrdU +cells through new differentiation (Figure 5—figure supplement 2B). These experiments verify that homeostasis can occur independent of the site of regeneration in a context other than notum inhibition.

Figure 5 with 3 supplements see all

Download asset Open asset

To examine whether the phenomenon of shifting the site of regeneration is specific only to eyes, we focused on the pharynx, a regionalized tissue of the trunk that can be specifically removed and regenerate (Adler et al., 2014). wntP-2, ndl-3, or ptk7 RNAi causes a posterior duplication of the pharynx, leaving behind a pre-existing anterior pharynx (Sureda-Gómez et al., 2015; Lander and Petersen, 2016; Scimone et al., 2016). In previous studies, it has been shown that the use of sodium azide to cause specific removal of both pharynges from wntP-2(RNAi);ptk7(RNAi) animals allowed for the regeneration of both organs (Lander and Petersen, 2016). However, this amputation method likely leaves behind pharynx-associated tissue such as the pharyngeal cavity and the surrounding bifurcated intestine (Adler et al., 2014), which could play a role in the determination of the site of pharynx regeneration. We reasoned that a broader amputation that removes this surrounding tissue would be a stronger test of the regenerative competence of these duplicated tissues. We generated animals with two pharynges after dual inhibition of wntP-2 and ptk7, then performed amputations that removed either the anterior or posterior pharynx and their surrounding tissues. The ectopic posterior pharynx had almost normal capacity for regeneration while the anterior pre-existing pharynx displayed strongly diminished regenerative ability, suggesting that wntP-2(RNAi);ptk7(RNAi) animals undergo a posterior shift to the site of trunk tissue regeneration (Figure 5B). Despite this alteration, uninjured wntP-2(RNAi);ptk7(RNAi) animals acquired BrdU within both pharynges after a 14 day pulse, indicating that pharynges with high or low regenerative ability both incorporate new cells homeostatically (Figure 5—figure supplement 2C). We conclude that modification of trunk patterning can alter the target site for pharynx regeneration away from the pre-existing pharynx without eliminating its ability to undergo homeostatic maintenance.

We additionally tested whether nou darake (ndk) RNAi, which produces ectopic brain tissue and ectopic eyes posteriorly into the prepharyngeal region, would similarly modify the site of eye regeneration (Figure 5—figure supplement 3) (Cebrià et al., 2002). Intriguingly, these animals displayed an unaltered site of eye regeneration, with pre-existing anterior eyes succeeding at regeneration while ectopic eyes failed to regenerate. These observations point to a distinction between the activities of Wnt11-6/WntA (Kobayashi et al., 2007) and nou darake, an FGFRL factor, and indicate that modification of the planarian AP axis content by RNAi does not necessarily alter the site of organ regeneration. Furthermore, these results suggest a specificity of Wnt factors in controlling the target location of organ regeneration along the primary body axis.

Finally, we tested whether the uncoupling of the site of eye regeneration and maintenance only occurs artificially after experimental gene perturbation or could occur as part of the normal regenerative process. Planarians undergo a natural process of patterning alteration after amputation, in which positional control gene expression domains become altered in order to replace regional identities lost to injury as well as accommodate new reduced body proportions (Petersen and Reddien, 2009a; Gurley et al., 2010). Notably, the tissue remodeling process typically does not appear to produce intermediate states in which new well-positioned tissues are formed prior to the elimination of improperly positioned ones. However, spontaneous appearance of ectopic eyes has been reported at low frequencies, indicating errors can occur in this process (Sakai et al., 2000). To specifically test robustness of pattern control through remodeling, we performed a series of amputations along the primary body axis of the animal that would require an increasing amount of tissue remodeling. Our results confirmed that regenerating head fragments typically undergo remodeling through regeneration without producing a second set of eyes (Figure 6A). However, animals that underwent particularly severe truncations to the body axis occasionally produced supernumerary eyes during regeneration (Figure 6A). These results suggest that severe axis rescaling can naturally shift the putative site of eye regeneration to a location distinct from the pre-existing organ.

Figure 6 with 1 supplement see all

Download asset Open asset

This observation suggested that tissue remodeling might normally involve the ability for pre-existing eyes to absorb progenitors while they are mispositioned. We hypothesized that this model would suggest that the site of eye regeneration might become distinct from the position of the pre-existing eyes during this type of regeneration. To test this, we examined the consequences of axis rescaling on the site of eye regeneration by resecting eyes from amputated head fragments in a timeseries after amputation. We fixed and stained these animals after 12 days of eye regeneration, and determined the relative location of the newly regenerated eye (opsin+ and tyrosinase+ cells), using the location of the midline (marked by slit expression) and the uninjured contralateral eye as a reference. Surprisingly, resection at early times in remodeling (days 2 and 4) resulted in eye regeneration at an anteriorly displaced position (Figure 6B), and these times correlate approximately with a time of dynamic alterations to patterning gene expression of zic-1, wnt2-1, ndl-2, ndl-3 and wnP-2 (Figure 6—figure supplement 1) (Petersen and Reddien, 2008, 2009b; Gurley et al., 2010; Vásquez-Doorman and Petersen, 2014; Vogg et al., 2014; Lander and Petersen, 2016; Scimone et al., 2016). Regeneration at an anterior position was dependent on complete eye removal rather than injury itself because partial resection of an eye during remodeling did not result in eye regeneration at a displaced location (Figure 6C).

This displacement to the site of eye regeneration eventually decayed as head fragment regeneration proceeded (Figure 6B), so we hypothesized that tissue remodeling might eventually realign these tissues with the target location of regeneration. To test this hypothesis, we measured the position of uninjured, pre-existing eyes versus resected, regenerating eyes with respect to the A/P axis of the brain in head fragments undergoing tissue remodeling through whole-body regeneration. Pre-existing eyes indeed gradually regained their proper position at a more anterior location with respect to the brain over several weeks of tissue remodeling (Figure 6D, gray). By contrast, eye removal during this process caused regeneration of a new eye located at the appropriate final position with respect to the brain (Figure 6D, red). Collectively, these results suggest that pre-existing tissue exerts an effect on the location of stem cell differentiation during normal tissue remodeling and can actually slow the processes by which regeneration restores proportionality, thus ensuring the maintenance of form during this transformation.

Our observations indicate that the location of regeneration can be altered by experimental perturbation of patterning factors or during the normal process of positional information rescaling after severe amputation (Figure 7A–B). Both the eyes and the pharynx use progenitors that must migrate distantly from the position where they are specified to their final differentiated location (Lapan and Reddien, 2011; Lapan and Reddien, 2012; Adler et al., 2014), indicating that these adult organs either can absorb progenitors that happen to encounter them or, more likely, use active trophic mechanisms for acquiring them. Our data argue that once an organ is formed, it can acquire progenitors to homeostatically maintain itself for long periods of time, perhaps indefinitely, even if it is not correctly placed with respect to patterning gene expression domains. These observations help to reconcile the fact that planarians generally regenerate perfectly, but that it is possible to recover rare variants with ‘mistakes’ in the process of asexual reproduction, including disorganized supernumerary eyes that are incapable of regeneration (Sakai et al., 2000). Given the requirement for progenitors in organ maintenance and the inability for mature eyes to produce their own progenitors, we suggest that homeostatic eye maintenance is likely only possible within the domain occupied by ovo+ progenitors. These interpretations suggest that patterning factors have an important role in precisely specifying the site for initiation of organ formation through regeneration but do not necessarily specify the sites of growth. We suggest that the maintenance of form in the absence of injury therefore likely involves both the use of positional control genes and the ability of existing tissues to acquire nearby progenitors for maintenance.

Figure 7

Download asset Open asset

Given the ability for positional control genes to shift their domains according to the size of the new axis, a hypothetical mechanism for the restoration of form through tissue remodeling could have been new production of tissues in proper locations, followed by slow decay of old tissues in incorrect positions. However, this has generally not been observed for tissue remodeling in planarians (Reddien and Sánchez Alvarado, 2004). Instead, severe truncations that require extensive remodeling generally cause a slow transformation toward normal form without intermediates involving duplicated tissue (Morgan, 1898). The discovery that mispositioned organs can be homeostatically maintained provides a candidate model to help explain the process of tissue remodeling (sometimes called morphallaxis in planarians) (Morgan, 1898). Early after severe axis truncations, patterning genes dynamically shift in order to restore positional information across the body axis. New domains of progenitor specification may be defined and perhaps be mostly restored in short timescales, but pre-existing progenitors and mature tissues remain. Waves of injury-induced cell death likely accelerate the turnover of pre-existing tissues (Pellettieri et al., 2010), but do not appear to fully eliminate them. The ability for mispositioned organs such as eyes and pharynx to acquire progenitors, combined with the relatively slow turnover of adult organs, would then result in a gradual realignment of the positional system with respect to mature tissue. A similar process could occur in remodeling of other tissues, such as the planarian brain, or through alternate mechanisms that await discovery. The maintenance of mature tissues even when in potential conflict with the positional system could play a vital role in proper integration of new and old tissue.

Adult regenerative abilities are widespread but unevenly distributed across animal species, so an enduring question has been how these capabilities are lost or gained through evolution and organismal development. While many strongly regenerative species also maintain their tissues through ongoing tissue homeostasis, many other species maintain their tissues homeostatically without possessing strong regenerative ability as adults (Poss, 2010). The alteration of patterning information can be sufficient to trigger the formation of a new axis or to enhance regenerative ability in flatworm species that are refractory at head regeneration (Liu et al., 2013; Sikes and Newmark, 2013; Umesono et al., 2013), suggesting constitutive patterning information is vital for regeneration. Our discovery that regeneration and homeostasis can be uncoupled in a highly regenerative organism suggests a potential model for how adult regenerative ability could be lost in development or evolution. Growth and homeostatic maintenance of tissues derived from nearby progenitors would not necessarily require ongoing patterning information after axis regionalization is defined in early development. After the completion of patterning, the signaling states that enable positional gene expression could therefore be lost without sacrificing the ability for tissues to grow, be maintained, or perhaps even heal simple wounds. Whole body regeneration could have been an ancient property, as it is shared among representatives of Radiata, planarians, and acoel flatworms, with increasing evidence for common and conserved specific regulatory programs within these groups (Srivastava et al., 2014; Raz et al., 2017). Notably, these species retain patterning information constitutively during adulthood (Reddien et al., 2007; Gurley et al., 2008; Petersen and Reddien, 2008; Lengfeld et al., 2009; Srivastava et al., 2014; Raz et al., 2017), while other non-regenerative species still capable of substantial post-embryonic growth and tissue maintenance are not thought to maintain axis organization programs after embryogenesis. The loss of patterning information in adulthood therefore could account for losses of regenerative ability without the elimination of proliferation and growth.

1. 1. Adler CE
   2. Seidel CW
   3. McKinney SA
   4. Sánchez Alvarado A

   (2014) Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria

   eLife 3:e02238.

   https://doi.org/10.7554/eLife.02238

   • PubMed
   • Google Scholar
2. 1. Agata K
   2. Soejima Y
   3. Kato K
   4. Kobayashi C
   5. Umesono Y
   6. Watanabe K

   (1998) Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers

   Zoological Science 15:433–440.

   https://doi.org/10.2108/zsj.15.433

   • PubMed
   • Google Scholar
3. 1. Bartscherer K
   2. Pelte N
   3. Ingelfinger D
   4. Boutros M

   (2006) Secretion of Wnt ligands requires Evi, a conserved transmembrane protein

   Cell 125:523–533.

   https://doi.org/10.1016/j.cell.2006.04.009

   • PubMed
   • Google Scholar
4. 1. Bodnar AG
   2. Coffman JA

   (2016) Maintenance of somatic tissue regeneration with age in short- and long-lived species of sea urchins

   Aging Cell 15:778–787.

   https://doi.org/10.1111/acel.12487

   • PubMed
   • Google Scholar
5. 1. Borgens RB

   (1982) Mice regrow the tips of their foretoes

   Science 217:747–750.

   https://doi.org/10.1126/science.7100922

   • PubMed
   • Google Scholar
6. 1. Cebrià F
   2. Guo T
   3. Jopek J
   4. Newmark PA

   (2007) Regeneration and maintenance of the planarian midline is regulated by a slit orthologue

   Developmental Biology 307:394–406.

   https://doi.org/10.1016/j.ydbio.2007.05.006

   • PubMed
   • Google Scholar
7. 1. Cebrià F
   2. Kobayashi C
   3. Umesono Y
   4. Nakazawa M
   5. Mineta K
   6. Ikeo K
   7. Gojobori T
   8. Itoh M
   9. Taira M
   10. Sánchez Alvarado A
   11. Agata K

   (2002) FGFR-related gene nou-darake restricts brain tissues to the head region of planarians

   Nature 419:620–624.

   https://doi.org/10.1038/nature01042

   • PubMed
   • Google Scholar
8. 1. Dent JN

   (1962) Limb regeneration in larvae and metamorphosing individuals of the South African clawed toad

   Journal of Morphology 110:61–77.

   https://doi.org/10.1002/jmor.1051100105

   • PubMed
   • Google Scholar
9. 1. Deochand ME
   2. Birkholz TR
   3. Beane WS

   (2016) Temporal regulation of planarian eye regeneration

   Regeneration 3:209–221.

   https://doi.org/10.1002/reg2.61

   • PubMed
   • Google Scholar
10. 1. Drenckhahn JD
    2. Schwarz QP
    3. Gray S
    4. Laskowski A
    5. Kiriazis H
    6. Ming Z
    7. Harvey RP
    8. Du XJ
    9. Thorburn DR
    10. Cox TC

    (2008) Compensatory growth of healthy cardiac cells in the presence of diseased cells restores tissue homeostasis during heart development

    Developmental Cell 15:521–533.

    https://doi.org/10.1016/j.devcel.2008.09.005

    • PubMed
    • Google Scholar
11. 1. Elliott SA
    2. Sánchez Alvarado A

    (2013) The history and enduring contributions of planarians to the study of animal regeneration

    Wiley Interdisciplinary Reviews: Developmental Biology 2:301–326.

    https://doi.org/10.1002/wdev.82

    • PubMed
    • Google Scholar
12. 1. Gaviño MA
    2. Reddien PW

    (2011) A Bmp/Admp regulatory circuit controls maintenance and regeneration of dorsal-ventral polarity in planarians

    Current Biology 21:294–299.

    https://doi.org/10.1016/j.cub.2011.01.017

    • PubMed
    • Google Scholar
13. 1. Guedelhoefer OC
    2. Sánchez Alvarado A

    (2012) Amputation induces stem cell mobilization to sites of injury during planarian regeneration

    Development 139:3510–3520.

    https://doi.org/10.1242/dev.082099

    • PubMed
    • Google Scholar
14. 1. Gurley KA
    2. Elliott SA
    3. Simakov O
    4. Schmidt HA
    5. Holstein TW
    6. Sánchez Alvarado A

    (2010) Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response

    Developmental Biology 347:24–39.

    https://doi.org/10.1016/j.ydbio.2010.08.007

    • PubMed
    • Google Scholar
15. 1. Gurley KA
    2. Rink JC
    3. Sánchez Alvarado A

    (2008) Beta-catenin defines head versus tail identity during planarian regeneration and homeostasis

    Science 319:323–327.

    https://doi.org/10.1126/science.1150029

    • PubMed
    • Google Scholar
16. 1. Harris RE
    2. Setiawan L
    3. Saul J
    4. Hariharan IK

    (2016) Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs

    eLife 5:e11588.

    https://doi.org/10.7554/eLife.11588

    • PubMed
    • Google Scholar
17. 1. Hill EM
    2. Petersen CP

    (2015) Wnt/Notum spatial feedback inhibition controls neoblast differentiation to regulate reversible growth of the planarian brain

    Development 142:4217–4229.

    https://doi.org/10.1242/dev.123612

    • PubMed
    • Google Scholar
18. 1. Kakugawa S
    2. Langton PF
    3. Zebisch M
    4. Howell S
    5. Chang TH
    6. Liu Y
    7. Feizi T
    8. Bineva G
    9. O'Reilly N
    10. Snijders AP
    11. Jones EY
    12. Vincent JP

    (2015) Notum deacylates Wnt proteins to suppress signalling activity

    Nature 519:187–192.

    https://doi.org/10.1038/nature14259

    • PubMed
    • Google Scholar
19. 1. King RS
    2. Newmark PA

    (2013) In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea

    BMC Developmental Biology 13:8.

    https://doi.org/10.1186/1471-213X-13-8

    • PubMed
    • Google Scholar
20. 1. Kobayashi C
    2. Saito Y
    3. Ogawa K
    4. Agata K

    (2007) Wnt signaling is required for antero-posterior patterning of the planarian brain

    Developmental Biology 306:714–724.

    https://doi.org/10.1016/j.ydbio.2007.04.010

    • PubMed
    • Google Scholar
21. 1. Lander R
    2. Petersen CP

    (2016) Wnt, Ptk7, and FGFRL expression gradients control trunk positional identity in planarian regeneration

    eLife 5:e12850.

    https://doi.org/10.7554/eLife.12850

    • PubMed
    • Google Scholar
22. 1. Lapan SW
    2. Reddien PW

    (2011) dlx and sp6-9 Control optic cup regeneration in a prototypic eye

    PLoS Genetics 7:e1002226.

    https://doi.org/10.1371/journal.pgen.1002226

    • PubMed
    • Google Scholar
23. 1. Lapan SW
    2. Reddien PW

    (2012) Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration

    Cell Reports 2:294–307.

    https://doi.org/10.1016/j.celrep.2012.06.018

    • PubMed
    • Google Scholar
24. 1. Lehoczky JA
    2. Robert B
    3. Tabin CJ

    (2011) Mouse digit tip regeneration is mediated by fate-restricted progenitor cells

    PNAS 108:20609–20614.

    https://doi.org/10.1073/pnas.1118017108

    • PubMed
    • Google Scholar
25. 1. Lengfeld T
    2. Watanabe H
    3. Simakov O
    4. Lindgens D
    5. Gee L
    6. Law L
    7. Schmidt HA
    8. Ozbek S
    9. Bode H
    10. Holstein TW

    (2009) Multiple Wnts are involved in Hydra organizer formation and regeneration

    Developmental Biology 330:186–199.

    https://doi.org/10.1016/j.ydbio.2009.02.004

    • PubMed
    • Google Scholar
26. 1. Liu SY
    2. Selck C
    3. Friedrich B
    4. Lutz R
    5. Vila-Farré M
    6. Dahl A
    7. Brandl H
    8. Lakshmanaperumal N
    9. Henry I
    10. Rink JC

    (2013) Reactivating head regrowth in a regeneration-deficient planarian species

    Nature 500:81–84.

    https://doi.org/10.1038/nature12414

    • PubMed
    • Google Scholar
27. 1. LoCascio SA
    2. Lapan SW
    3. Reddien PW

    (2017) Eye absence does not regulate planarian stem cells during eye regeneration

    Developmental Cell 40:381–391.

    https://doi.org/10.1016/j.devcel.2017.02.002

    • PubMed
    • Google Scholar
28. 1. Maden M
    2. Manwell LA
    3. Ormerod BK

    (2013) Proliferation zones in the axolotl brain and regeneration of the telencephalon

    Neural Development 8:1.

    https://doi.org/10.1186/1749-8104-8-1

    • PubMed
    • Google Scholar
29. 1. Molina MD
    2. Neto A
    3. Maeso I
    4. Gómez-Skarmeta JL
    5. Saló E
    6. Cebrià F

    (2011) Noggin and noggin-like genes control dorsoventral axis regeneration in planarians

    Current Biology 21:300–305.

    https://doi.org/10.1016/j.cub.2011.01.016

    • PubMed
    • Google Scholar
30. 1. Molina MD
    2. Saló E
    3. Cebrià F

    (2007) The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians

    Developmental Biology 311:79–94.

    https://doi.org/10.1016/j.ydbio.2007.08.019

    • PubMed
    • Google Scholar
31. 1. Morgan TH

    (1898) Experimental studies of the regeneration of Planaria maculata

    Archiv für Entwickelungsmechanik der Organismen 7:364–397.

    https://doi.org/10.1007/BF02161491

    • Google Scholar
32. 1. Newmark PA
    2. Sánchez Alvarado A

    (2000) Bromodeoxyuridine specifically labels the regenerative stem cells of planarians

    Developmental Biology 220:142–153.

    https://doi.org/10.1006/dbio.2000.9645

    • PubMed
    • Google Scholar
33. 1. Owen JH
    2. Wagner DE
    3. Chen CC
    4. Petersen CP
    5. Reddien PW

    (2015) teashirt is required for head-versus-tail regeneration polarity in planarians

    Development 142:1062–1072.

    https://doi.org/10.1242/dev.119685

    • PubMed
    • Google Scholar
34. 1. Pearson BJ
    2. Eisenhoffer GT
    3. Gurley KA
    4. Rink JC
    5. Miller DE
    6. Sánchez Alvarado A

    (2009) Formaldehyde-based whole-mount in situ hybridization method for planarians

    Developmental Dynamics 238:443–450.

    https://doi.org/10.1002/dvdy.21849

    • PubMed
    • Google Scholar
35. 1. Pellettieri J
    2. Fitzgerald P
    3. Watanabe S
    4. Mancuso J
    5. Green DR
    6. Sánchez Alvarado A

    (2010) Cell death and tissue remodeling in planarian regeneration

    Developmental Biology 338:76–85.

    https://doi.org/10.1016/j.ydbio.2009.09.015

    • PubMed
    • Google Scholar
36. 1. Petersen CP
    2. Reddien PW

    (2008) Smed-betacatenin-1 is required for anteroposterior blastema polarity in planarian regeneration

    Science 319:327–330.

    https://doi.org/10.1126/science.1149943

    • PubMed
    • Google Scholar
37. 1. Petersen CP
    2. Reddien PW

    (2009a) Wnt signaling and the polarity of the primary body axis

    Cell 139:1056–1068.

    https://doi.org/10.1016/j.cell.2009.11.035

    • PubMed
    • Google Scholar
38. 1. Petersen CP
    2. Reddien PW

    (2009b) A wound-induced Wnt expression program controls planarian regeneration polarity

    PNAS 106:17061–17066.

    https://doi.org/10.1073/pnas.0906823106

    • PubMed
    • Google Scholar
39. 1. Petersen CP
    2. Reddien PW

    (2011) Polarized notum activation at wounds inhibits Wnt function to promote planarian head regeneration

    Science 332:852–855.

    https://doi.org/10.1126/science.1202143

    • PubMed
    • Google Scholar
40. 1. Porrello ER
    2. Mahmoud AI
    3. Simpson E
    4. Hill JA
    5. Richardson JA
    6. Olson EN
    7. Sadek HA

    (2011) Transient regenerative potential of the neonatal mouse heart

    Science 331:1078–1080.

    https://doi.org/10.1126/science.1200708

    • PubMed
    • Google Scholar
41. 1. Poss KD

    (2010) Advances in understanding tissue regenerative capacity and mechanisms in animals

    Nature Reviews Genetics 11:710–722.

    https://doi.org/10.1038/nrg2879

    • PubMed
    • Google Scholar
42. 1. Raz AA
    2. Srivastava M
    3. Salvamoser R
    4. Reddien PW

    (2017) Acoel regeneration mechanisms indicate an ancient role for muscle in regenerative patterning

    Nature Communications 8:1260.

    https://doi.org/10.1038/s41467-017-01148-5

    • PubMed
    • Google Scholar
43. 1. Reddien PW
    2. Bermange AL
    3. Kicza AM
    4. Sánchez Alvarado A

    (2007) BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration

    Development 134:4043–4051.

    https://doi.org/10.1242/dev.007138

    • PubMed
    • Google Scholar
44. 1. Reddien PW
    2. Bermange AL
    3. Murfitt KJ
    4. Jennings JR
    5. Sánchez Alvarado A

    (2005) Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria

    Developmental Cell 8:635–649.

    https://doi.org/10.1016/j.devcel.2005.02.014

    • PubMed
    • Google Scholar
45. 1. Reddien PW
    2. Sánchez Alvarado A

    (2004) Fundamentals of planarian regeneration

    Annual Review of Cell and Developmental Biology 20:725–757.

    https://doi.org/10.1146/annurev.cellbio.20.010403.095114

    • PubMed
    • Google Scholar
46. 1. Reddien PW

    (2011) Constitutive gene expression and the specification of tissue identity in adult planarian biology

    Trends in Genetics 27:277–285.

    https://doi.org/10.1016/j.tig.2011.04.004

    • PubMed
    • Google Scholar
47. 1. Reginelli AD
    2. Wang YQ
    3. Sassoon D
    4. Muneoka K

    (1995)

    Digit tip regeneration correlates with regions of Msx1 (Hox 7) expression in fetal and newborn mice

    Development 121:1065–1076.

    • PubMed
    • Google Scholar
48. 1. Reuter H
    2. März M
    3. Vogg MC
    4. Eccles D
    5. Grífol-Boldú L
    6. Wehner D
    7. Owlarn S
    8. Adell T
    9. Weidinger G
    10. Bartscherer K

    (2015) Β-catenin-dependent control of positional information along the AP body axis in planarians involves a teashirt family member

    Cell Reports 10:253–265.

    https://doi.org/10.1016/j.celrep.2014.12.018

    • PubMed
    • Google Scholar
49. 1. Reversade B
    2. De Robertis EM

    (2005) Regulation of ADMP and BMP2/4/7 at opposite embryonic poles generates a self-regulating morphogenetic field

    Cell 123:1147–1160.

    https://doi.org/10.1016/j.cell.2005.08.047

    • PubMed
    • Google Scholar
50. 1. Roberts-Galbraith RH
    2. Newmark PA

    (2013) Follistatin antagonizes activin signaling and acts with notum to direct planarian head regeneration

    PNAS 110:1363–1368.

    https://doi.org/10.1073/pnas.1214053110

    • PubMed
    • Google Scholar
51. 1. Rodrigo Albors A
    2. Tazaki A
    3. Rost F
    4. Nowoshilow S
    5. Chara O
    6. Tanaka EM

    (2015) Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration

    eLife 4:e10230.

    https://doi.org/10.7554/eLife.10230

    • PubMed
    • Google Scholar
52. 1. Rouhana L
    2. Weiss JA
    3. Forsthoefel DJ
    4. Lee H
    5. King RS
    6. Inoue T
    7. Shibata N
    8. Agata K
    9. Newmark PA

    (2013) RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics

    Developmental Dynamics 242:718–730.

    https://doi.org/10.1002/dvdy.23950

    • PubMed
    • Google Scholar
53. 1. Sakai F
    2. Agata K
    3. Orii H
    4. Watanabe K

    (2000) Organization and regeneration ability of spontaneous supernumerary eyes in planarians -eye regeneration field and pathway selection by optic nerves-

    Zoological Science 17:375–381.

    https://doi.org/10.2108/jsz.17.375

    • PubMed
    • Google Scholar
54. 1. Schaible R
    2. Scheuerlein A
    3. Dańko MJ
    4. Gampe J
    5. Martínez DE
    6. Vaupel JW

    (2015) Constant mortality and fertility over age in Hydra

    PNAS 112:15701–15706.

    https://doi.org/10.1073/pnas.1521002112

    • PubMed
    • Google Scholar
55. 1. Scimone ML
    2. Cote LE
    3. Rogers T
    4. Reddien PW

    (2016) Two FGFRL-Wnt circuits organize the planarian anteroposterior axis

    eLife 5:e12845.

    https://doi.org/10.7554/eLife.12845

    • PubMed
    • Google Scholar
56. 1. Sikes JM
    2. Newmark PA

    (2013) Restoration of anterior regeneration in a planarian with limited regenerative ability

    Nature 500:77–80.

    https://doi.org/10.1038/nature12403

    • PubMed
    • Google Scholar
57. 1. Slack JM
    2. Beck CW
    3. Gargioli C
    4. Christen B

    (2004) Cellular and molecular mechanisms of regeneration in Xenopus

    Philosophical Transactions of the Royal Society B: Biological Sciences 359:745–751.

    https://doi.org/10.1098/rstb.2004.1463

    • PubMed
    • Google Scholar
58. 1. Srivastava M
    2. Mazza-Curll KL
    3. van Wolfswinkel JC
    4. Reddien PW

    (2014) Whole-body acoel regeneration is controlled by Wnt and Bmp-Admp signaling

    Current Biology 24:1107–1113.

    https://doi.org/10.1016/j.cub.2014.03.042

    • PubMed
    • Google Scholar
59. 1. Stückemann T
    2. Cleland JP
    3. Werner S
    4. Thi-Kim Vu H
    5. Bayersdorf R
    6. Liu SY
    7. Friedrich B
    8. Jülicher F
    9. Rink JC

    (2017) Antagonistic self-organizing patterning systems control maintenance and regeneration of the anteroposterior axis in planarians

    Developmental Cell 40:248–263.

    https://doi.org/10.1016/j.devcel.2016.12.024

    • PubMed
    • Google Scholar
60. 1. Sureda-Gómez M
    2. Pascual-Carreras E
    3. Adell T

    (2015) Posterior wnts have distinct roles in specification and patterning of the planarian posterior region

    International Journal of Molecular Sciences 16:26543–26554.

    https://doi.org/10.3390/ijms161125970

    • PubMed
    • Google Scholar
61. 1. Thi-Kim Vu H
    2. Rink JC
    3. McKinney SA
    4. McClain M
    5. Lakshmanaperumal N
    6. Alexander R
    7. Sánchez Alvarado A

    (2015) Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ

    eLife 4:e07405.

    https://doi.org/10.7554/eLife.07405

    • Google Scholar
62. 1. Umesono Y
    2. Tasaki J
    3. Nishimura Y
    4. Hrouda M
    5. Kawaguchi E
    6. Yazawa S
    7. Nishimura O
    8. Hosoda K
    9. Inoue T
    10. Agata K

    (2013) The molecular logic for planarian regeneration along the anterior-posterior axis

    Nature 500:73–76.

    https://doi.org/10.1038/nature12359

    • PubMed
    • Google Scholar
63. 1. Vogg MC
    2. Owlarn S
    3. Pérez Rico YA
    4. Xie J
    5. Suzuki Y
    6. Gentile L
    7. Wu W
    8. Bartscherer K

    (2014) Stem cell-dependent formation of a functional anterior regeneration pole in planarians requires Zic and Forkhead transcription factors

    Developmental Biology 390:136–148.

    https://doi.org/10.1016/j.ydbio.2014.03.016

    • PubMed
    • Google Scholar
64. 1. Vásquez-Doorman C
    2. Petersen CP

    (2014) zic-1 Expression in Planarian neoblasts after injury controls anterior pole regeneration

    PLoS Genetics 10:e1004452.

    https://doi.org/10.1371/journal.pgen.1004452

    • PubMed
    • Google Scholar
65. 1. Wagner DE
    2. Wang IE
    3. Reddien PW

    (2011) Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration

    Science 332:811–816.

    https://doi.org/10.1126/science.1203983

    • PubMed
    • Google Scholar
66. 1. Wenemoser D
    2. Lapan SW
    3. Wilkinson AW
    4. Bell GW
    5. Reddien PW

    (2012) A molecular wound response program associated with regeneration initiation in planarians

    Genes & Development 26:988–1002.

    https://doi.org/10.1101/gad.187377.112

    • PubMed
    • Google Scholar
67. 1. Whitehead GG
    2. Makino S
    3. Lien CL
    4. Keating MT

    (2005) fgf20 is essential for initiating zebrafish fin regeneration

    Science 310:1957–1960.

    https://doi.org/10.1126/science.1117637

    • PubMed
    • Google Scholar
68. 1. Wills AA
    2. Kidd AR
    3. Lepilina A
    4. Poss KD

    (2008) Fgfs control homeostatic regeneration in adult zebrafish fins

    Development 135:3063–3070.

    https://doi.org/10.1242/dev.024588

    • PubMed
    • Google Scholar
69. 1. Witchley JN
    2. Mayer M
    3. Wagner DE
    4. Owen JH
    5. Reddien PW

    (2013) Muscle cells provide instructions for planarian regeneration

    Cell Reports 4:633–641.

    https://doi.org/10.1016/j.celrep.2013.07.022

    • PubMed
    • Google Scholar
70. 1. Wurtzel O
    2. Cote LE
    3. Poirier A
    4. Satija R
    5. Regev A
    6. Reddien PW

    (2015) A generic and cell-type-specific wound response precedes regeneration in planarians

    Developmental Cell 35:632–645.

    https://doi.org/10.1016/j.devcel.2015.11.004

    • PubMed
    • Google Scholar
71. 1. Wurtzel O
    2. Oderberg IM
    3. Reddien PW

    (2017) Planarian epidermal stem cells respond to positional cues to promote cell-type diversity

    Developmental Cell 40:491–504.

    https://doi.org/10.1016/j.devcel.2017.02.008

    • PubMed
    • Google Scholar
72. 1. Zhang X
    2. Cheong SM
    3. Amado NG
    4. Reis AH
    5. MacDonald BT
    6. Zebisch M
    7. Jones EY
    8. Abreu JG
    9. He X

    (2015) Notum is required for neural and head induction via Wnt deacylation, oxidation, and inactivation

    Developmental Cell 32:719–730.

    https://doi.org/10.1016/j.devcel.2015.02.014

    • PubMed
    • Google Scholar

Author details

1. Eric M Hill

   Department of Molecular Biosciences, Northwestern University, Evanston, United States

   Contribution

   Conceptualization, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1426-2573

2. Christian P Petersen

   1. Department of Molecular Biosciences, Northwestern University, Evanston, United States
   2. Robert Lurie Comprehensive Cancer Center, Northwestern University, Evanston, United States

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing

   For correspondence

   christian-p-petersen@northwestern.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7552-6865

• 3,244

  views

• 501

  downloads

• 19

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.