Some animals can regrow tissues that have been amputated. A group of flatworms called planarians are often used as a model to study the regeneration process because they are able to restore any lost tissue or even an entire animal from even tiny pieces of the body. For regeneration to be successful, it is critical to identify which tissues or regions of the body need to be replaced.

The planarian body is divided into three main parts: head, trunk and tail. Several genes involved in specifying what tissues regenerate are very active (or “highly expressed”) in muscle cells in different regions of the planarian body. Some of the genes are involved in mechanisms that allow cells to communicate with each other, such as the Wnt and FGF signaling pathways. These genes could form a coordinated system to control regeneration, but their precise roles remain poorly understood.

Two groups of researchers have now independently identified genes that provide cells with information about their location in the flatworm body. Lander and Petersen identified three genes that are expressed in an overlapping manner along the body of uninjured animals. One of the genes – known as ptk7 – is mainly produced in the trunk region, while the second gene (wntP-2) is produced from the tail and the third (ndl-3) is produced from the head region. The wntP-2 and ptk7 encode components of the Wnt signaling pathway, while ndl-3 encodes a protein involved in FGF signaling.

Lander and Petersen used a technique called RNAi to lower the activity of the three genes individually or in pairs, and then examined whether this affected the ability of the worms to regenerate. Inhibition of any of the three genes resulted in an expansion of the trunk tissues into the tail region, indicating that the normal role for these genes is to stop cells adopting the trunk “identity”.

Another study by Scimone, Cote et al. found that two separate sets of genes – including wntP-2 and ndl-3 – are needed to correctly position tissues in the head and trunk of planarians. Together these findings suggest that the Wnt and FGFRL pathways act in a body-wide system that co-ordinates where and which new tissues form during regeneration. A future challenge will be to decipher the complete network of genes that provides the positional information needed for regeneration.

Robust pattern control is a central but poorly understood feature of regenerative abilities (Wolpert, 1969; French et al., 1976). Animals cannot anticipate how a given injury will alter tissue composition, so regeneration likely depends critically on the re-establishment of missing tissue identity. Planarians use pluripotent stem cells to regenerate from nearly any amputation to restore a complete set of regionalized tissues, including cephalic ganglia in the anterior and a pharynx and mouth in the trunk, and are a model of positional restoration after amputation (Reddien, 2011; Adler and Sánchez Alvarado, 2015). Canonical Wnt signaling controls anterior-versus-posterior pole identity in planarian regeneration, with principal upstream determinants wnt1 expressed at the posterior pole (Petersen and Reddien, 2009; Gurley et al., 2010) and the secreted Wnt inhibitor notum expressed at the anterior pole (Petersen and Reddien, 2011), both activated transcriptionally early after wounding. Inhibition of Wnt signaling components β-catenin-1, wnt1, Evi/wntless, Dvl-1/2 and teashirt causes regeneration of ectopic heads (Gurley et al., 2008; Iglesias et al., 2008; Petersen and Reddien, 2008; Petersen and Reddien, 2009; Owen et al., 2015; Reuter et al., 2015); conversely, inhibition of APC or notum can cause regeneration of ectopic tails (Gurley et al., 2008; Petersen and Reddien, 2011). Other pathways participate in head or tail regeneration, with Hedgehog signaling influencing injury-induced wnt1 expression (Rink et al., 2009), several transcription factors required for head formation (prep, foxD, zic-1/zicA, pbx, egr-4) (Felix and Aboobaker, 2010; Blassberg et al., 2013; Chen et al., 2013; Fraguas et al., 2014; Scimone et al., 2014; Vásquez-Doorman and Petersen, 2014; Vogg et al., 2014) and/or tail formation (junli-1, pitx, pbx) (Blassberg et al., 2013; Chen et al., 2013; Currie and Pearson, 2013; Marz et al., 2013; Tejada-Romero et al., 2015), and influenced by gap junction and calcium signaling (Oviedo et al., 2010; Zhang et al., 2011). However, comparatively little is known about the restoration of positional information along the head-to-tail body axis through regeneration. Expression profiling and homology searching have identified a cohort of factors related to Wnt, Hox, and FGF signaling expressed regionally in domains along the anteroposterior (A-P) axis in planarians (Cebrià et al., 2002; Petersen and Reddien, 2008; Reddien, 2011; Owen et al., 2015; Reuter et al., 2015). These factors could in principle form a molecular coordinate system that re-specifies axis identity in regeneration and have been termed 'positional control genes' (PCGs) (Witchley et al., 2013). However, few phenotypes of positional displacement have been reported from perturbation of PCGs (Cebrià et al., 2002; Kobayashi et al., 2007), so it remains unclear how the majority of these genes participate in regeneration.

To uncover programs responsible for patterning along the body axis, we examined PCGs as defined by prior homology and expression profiling studies (Petersen and Reddien, 2008; Reddien, 2011; Witchley et al., 2013; Owen et al., 2015; Reuter et al., 2015) and found a unique expression pattern for a planarian homolog of ptk7, expressed in an animal-wide graded fashion maximally in the trunk, and also the CNS and pharynx (Figure 1A, Figure 1—figure supplement 1). Ptk7 proteins encode cell-surface Wnt co-receptors with a predicted intracellular pseudokinase domain that participate in noncanonical, β-catenin-independent Wnt signaling (Lu et al., 2004), and can either weakly suppress or activate canonical β-catenin-dependent Wnt signaling in a context-dependent manner (Peradziryi et al., 2011; Puppo et al., 2011; Hayes et al., 2013; Bin-Nun et al., 2014). Like other described PCGs, planarian ptk7 was expressed in collagen+ cells of the body-wall musculature (Witchley et al., 2013) and aspects of its expression domain could become re-established after amputation even in irradiated animals lacking neoblasts and the ability to form new tissues (Figure 1A–C). In ptk7(RNAi) animals amputated to remove both head and tail, regeneration produced a normal head and tail (35/35) but caused formation of an ectopic posterior mouth at a high penetrance (83%, n=35) and, more rarely, formation of an ectopic posterior pharynx with broadly normal orientation with respect to the primary body axis (14%, n=35) (Figure 1D–E). Thus, ptk7 limits trunk identity in planarian regeneration.

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We next used RNAi and a phenotypic enhancement assay to identify other PCGs that participate with ptk7 in trunk identity regulation (Figure 1—figure supplement 2). Co-inhibition of ptk7 with either wntP-2/wnt11-5/wnt4b (hereafter referred to as wntP-2), ndl-3, Dvl-2 (Almuedo-Castillo et al., 2011), or fzd-1/2/7 caused the strongest enhancement of the ectopic pharynx phenotype, resulting in 100% of animals affected, whereas co-inhibition with other PCGs affected this phenotype more weakly and below statistical significance under these conditions. wntP-2 encodes a Wnt gene expressed in a graded fashion from the posterior (Figure 1—figure supplement 2) and based on phylogenetic analyses has been proposed to be either a Wnt11 (Gurley et al., 2010) or Wnt4 (Riddiford and Olson, 2011) family member. Planarian ndl-3 is expressed in a graded fashion from the anterior (Rink et al., 2009) (Figure 1—figure supplement 2), and encodes a member of the conserved FGFRL/nou-darake class of cell-surface molecules that possess an FGF-receptor-like extracellular domain but lacks a tyrosine kinase intracellular domain and are thus proposed to function as FGF signaling inhibitors (Cebrià et al., 2002; Gerber et al., 2009). fzd-1/2/7 encodes a predicted Wnt receptor expressed broadly (Figure 1—figure supplement 2). Individual inhibition of ptk7, wntP-2, and ndl-3 caused formation of ectopic posterior mouths (30/35 ptk7(RNAi) animals, 22/33 wntP-2(RNAi) animals, 14/23 ndl-3(RNAi) animals), and ectopic posterior pharynges (5/35 ptk7(RNAi) animals, 8/33 wntP-2(RNAi) animals, 2/23 ndl-3(RNAi) animals) in amputated animals regenerating both their heads and tails compared to controls (0/46 animals) (Figure 2A). We verified knockdown of ptk7, wntP-2, and ndl-3 using in situ hybridizations and qPCR (Figure 2—figure supplement 1A–B). Inhibition of any pairwise combinations of the three genes enhanced the trunk expansion phenotypes (Figure 2A–B) (animals with ectopic pharynges: 45/55 wntP-2(RNAi);ptk7(RNAi), 29/32 ptk7(RNAi);ndl-3(RNAi), 24/40 wntP-2(RNAi);ndl-3(RNAi)), with double-RNAi animals also frequently forming two ectopic pharynges (Figure 2A) and co-inhibition of ptk7 and wntP-2 producing highest penetrance and expressivity of trunk duplication phenotypes. The orientation of ectopic pharynges was generally oblique, and sometimes inverted, but the majority in all conditions pointed toward the posterior rather than anterior direction (Figure 2C). Live animals with ectopic pharynges were observed during feeding, and the duplicated pharynx could obtain food (Figure 2D, Video 1), suggesting normal functionality of this organ. Taken together, ptk7, wntP-2 and ndl-3 form a core group of regionally expressed PCGs that jointly suppress trunk identity during posterior regeneration.

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wntP-2 and ptk7 were expressed regionally in the absence of injury, so we examined whether they act only in regeneration or constitutively to regulate trunk regionalization. Prolonged co-inhibition of wntP-2 and ptk7 produced animals with an ectopic pharynx, indicating that these genes together restrict trunk identity homeostatically in the absence of injury (Figure 2E). Furthermore, the fact that such animals ultimately formed multiple ectopic mouths extending toward the posterior suggests that graded activities of wntP-2 and/or ptk7, rather than their control of a binary switch in developmental outcomes, could pattern the tail and trunk regions.

We next examined whether trunk suppression mediated by wntP-2, ptk7, and ndl-3 was operational in all regeneration contexts, as is the case for several characterized patterning genes in planarians. By contrast, under RNAi conditions that produced an 80–100% penetrant pharynx duplication in regenerating trunk fragments, head and tail fragments from wntP-2(RNAi);ptk7(RNAi) animals or ndl-3(RNAi);ptk7(RNAi) animals formed only a single laminin+ pharynx as did control animals (Figure 2F). Eventually, after prolonged dsRNA feeding after regeneration, such animals could form ectopic pharynges (76 days of RNAi, n=3 of 12 animals examined), consistent with homeostatic functioning of the three genes. However, these results indicate that ptk7, wntP-2 and ndl-3 suppress trunk expansion in a context-dependent manner and suggest they may provide information about trunk absence or presence during regeneration.

We investigated the anatomy of ptk7(RNAi);wntP-2(RNAi) and ptk7(RNAi);ndl-3(RNAi) regenerating trunk fragments to determine the extent of the axis under control of the three genes. We first examined the influence of ptk7, wntP-2, or ndl-3 inhibition on expression of PCGs and tissue-specific genes marking A-P axial identity (Figure 3A–B). Such ptk7(RNAi);wntP-2(RNAi) or ptk7(RNAi);ndl-3(RNAi) regenerating animals had normal anterior pole and brain regions (marked by notum, ndk) and a normal pre-pharyngeal region anterior to the original pharynx (marked by wnt2 and novel gene SMU15014980), expansion of trunk-related peripharyngeal cells (expressing mmp1, FoxA, and SMU15007112), a reduced domain of PCGs expressed in the posterior (wnt11-1, fzd-4-1 and Abd-Ba), and normal expression of wnt1 at the posterior pole. We performed additional examinations of the brain (marked by chat and cintillo) and far posterior (marked by wnt11-2) in ptk7(RNAi);wntP-2(RNAi) animals and found no apparent differences compared to control animals (Figure 3—figure supplement 1A–B). Thus, ptk7, wntP-2, and ndl-3 normally promote anterior tail identity at the expense of the trunk and do not strongly affect head or tail formation. Both the pre-existing and ectopic pharynx were capable of regeneration after amputation by sodium azide treatment (Adler et al., 2014), suggesting that wntP-2/ptk7 signaling acts in part to limit the size of the trunk region within the posterior rather than only functioning to position the anterior extent of newly made trunk tissue (Figure 3C). We next inhibited ptk7 and wntP-2 in a regenerating sexual strain of S. mediterranea that forms reproductive organs posterior to the pharynx upon attainment of appropriate size. Such animals formed both an ectopic pharynx and ectopic reproductive organs marked by laminin and dmd-1 expression respectively (Chong et al., 2013), indicating ptk7 and wntP-2 regulate trunk regionalization beyond only control of pharynx and mouth formation (Figure 3—figure supplement 1C).

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The pharynx is formed from FoxA+ precursors derived from smedwi-1+ neoblasts. Because inhibition of trunk identity genes produced an ectopic pharynx, we reasoned this structure likely arose from FoxA+ progenitor cells. Indeed, ptk7(RNAi);wntP-2(RNAi) animals regenerating an ectopic pharynx produced an ectopic domain of FoxA+ cells at a time (7 days of regeneration) prior to appearance of a fully formed ectopic pharynx (14 days of regeneration) (Figure 3C). Expression domains of ptk7 (Figure 1B) and wntP-2 (Petersen and Reddien, 2009; Gurley et al., 2010) can be altered by amputation independently of neoblasts, so we suggest these genes likely function to regulate axis organization upstream of controlling neoblast fates.

The unidirectional nature of these axis patterning phenotypes as expansion of trunk at the expense of tail identity prompted us to determine more precisely the nature of the regionalized expression of ptk7, wntP-2 and ndl-3 mRNAs. Visual inspection of colorimetric in situ hybridizations suggested that ndl-3, ptk7, and wntP-2 are expressed in overlapping domains along the anteroposterior axis (Figure 4A). We verified this interpretation by quantifying the staining intensity of colorimetric in situ hybridizations along a lateral region running from head to tail (Figure 4B). ndl-3 staining intensity was maximal in the anterior in a region of graded ptk7 staining. ptk7 staining was maximal in the trunk region in which wntP-2 and ndl-3 staining form opposing gradients. wntP-2 staining was maximal in the posterior tail in a region of relatively less ptk7 expression. The graded expression detected in this manner could arise from regional differences in the abundance of cells that uniquely express each factor or in regulation of cells that express combinations of the three genes. To test these models, we performed triple FISH to simultaneously detect expression of all three genes in eight domains along the head-tail axis, which broadly confirmed the regionalized expression behavior of anterior/pre-pharyngeal maximal ndl-3 expression, trunk maximal ptk7 expression, and tail maximal wntP-2 expression (Figure 4C). We sought to verify this trend quantitatively and segmented the images to examine cells expressing any of the three genes (see Materials and Methods) then determined the mean FISH intensity for each gene per cell. This demonstrated that regions of maximal expression of ndl-3, ptk7, and wntP-2 are comprised of cells with high expression of these genes (Figure 4D). We next examined pairwise comparisons of expression of each gene across the body axis (Figure 4E). This approach identified cells that co-express wntP-2 and ndl-3 (in particular in head-to trunk proximal regions R3-R5, Figure 4E), ptk7 and ndl-3 (head-to-trunk regions R2-R4), and ptk7 and wntP-2 (trunk-to-tail regions R5-R7). We explored whether this approach could identify the spatial distribution of discrete states of cells expressing all combinations of the three genes. We pooled the cell-based expression data across all regions to determine an approximate threshold to define higher versus lower expression for each gene, then assigned each measured cell into one of eight expression classes representing each combination of high versus low expression of each ptk7, wntP-2, and ndl-3 and determined their distribution across the head-tail axis (Figure 4—figure supplement1A–C). This approach identified domains enriched for each expression state, finding a cohort with high wntP-2 expression and low ptk7 and ndl-3 expression in the far posterior, a cohort co-expressing ptk7 and wntP-2 in the anterior tail and trunk regions, a cohort only expressing ptk7 and not wntP-2 or ndl-3 centered in the trunk, cohorts of triple-positive cells and ptk7+ndl-3+ cells in the pre-pharyngeal region, and cohort of ndl-3+ cells in the anterior. These results suggest that a complexity of PCG cell expression states populate regions of the body axis and that a region of high wntP-2 and ptk7 expression exists in the anterior tail at a location where these two genes act together to suppress trunk identity.

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We examined the involvement of canonical Wnt signaling on expression of these factors, as this pathway has multiple functions in patterning the primary body axis (Petersen and Reddien, 2009). β-catenin-1 inhibition in uninjured animals severely reduced the expression of ptk7, wntP-2 and ndl-3 (Figure 5A), consistent with prior analyses of their expression requirements in regeneration (Owen et al., 2015; Reuter et al., 2015). We additionally inhibited APC, encoding an intracellular negative regulator of beta-catenin stability and examined regenerating animals for expression of the three trunk regulatory genes (Figure 5—figure supplement 1). Such animals regenerated anterior tails that expressed wntP-2 throughout, that expressed ptk7 strongly in a region near the amputation plane and away from the terminus, and that lacked ndl-3 expression. These results suggest that beta-catenin upregulation can be sufficient for tail axis formation in conjunction with wntP-2 and ptk7 expression. Taken together, normal levels of beta-catenin signaling are important for the normal expression of pkt7, wntP-2 and ndl-3.

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We next examined candidates for signaling that could occur downstream of wntP-2, ptk7, and ndl-3. Ptk7 proteins can signal through several pathways, including as a coreceptor for Wnt/Frizzled signaling (Lhoumeau et al., 2011). The results of our ptk7 RNAi enhancement screen suggested a candidate molecular pathway in which wntP-2 signals through ptk7 and fzd1/2/7 to suppress trunk identity. We verified that RNAi of fzd1/2/7 alone resulted in ectopic mouth and pharynx formation similar to ptk7 and/or wntP-2 inhibition (Figure 5—figure supplement 2A). Similarly, we verified that Dvl-2 but not Dvl-1 could interact with ptk7 genetically, suggesting the involvement of Dvl-2 in trunk suppression (Figure 5—figure supplement 2B). Ptk7 proteins can act in planar cell polarization so we tested for possible functional interactions between ptk7 and components of the Planar Cell Polarity pathway vangl1, vangl2, DAAM1 and ROCK but inhibition of these genes did not increase or decrease the occurrence of ectopic mouth or pharynx phenotypes (Figure 5—figure supplement 3). We additionally tested whether a downstream step in trunk suppression could be promotion or inhibition of beta-catenin activity. However, co-inhibition of ptk7 and wntP-2 or ptk7 and ndl-3 had no detectable effect on expression of beta-catenin-dependent transcripts axin-B and teashirt (Figure 5B, Figure 5—figure supplement 4A–B) (Owen et al., 2015; Reuter et al., 2015). β-catenin-1(RNAi) animals lose trunk regional identity and their pharynx while becoming completely anteriorized (Iglesias et al., 2008), so we tested for possible functional interactions between beta-catenin signaling and trunk control genes by inhibiting ptk7/wntP-2 along with APC. We could not detect any enhancement or suppression of ectopic pharynx formation in that assay, further suggesting independence between ptk7/wntP-2 and APC/β-catenin-1 signaling (Figure 5—figure supplement 4C). These observations are consistent with the clear distinction between the β-catenin-1 RNAi phenotype of ectopic head production (Gurley et al., 2008; Iglesias et al., 2008; Petersen and Reddien, 2008) as compared to the ptk7(RNAi);wntP-2(RNAi) and ptk7(RNAi);ndl-3(RNAi) phenotypes of ectopic trunk formation without affecting the identity of the anterior and posterior poles. Together, these results strongly suggest that trunk regionalization can be separable from pole identity and that ptk7 and wntP-2 likely do not operate exclusively through β-catenin-1 to pattern the trunk and tail.

Regeneration can involve the re-definition of positional identity within pre-existing tissues, but the mechanisms controlling this process are unclear. We examined the expression and activities of ptk7, wntP-2, and ndl-3 in forming a mouth and pharynx (expressing laminin) within the pre-existing tail tissue (Figure 6B). In amputated tail fragments, the mouth and pharynx were formed during the first 5 days of regeneration, with expression of laminin evident as early as 3 days. wntP-2 was initially expressed throughout the amputated tail, but its expression restricted posteriorly starting around day 2, reaching a minimum around day 4, and re-establishing to intact proportions around day 7 (Petersen and Reddien, 2009; Gurley et al., 2010). ndl-3 expression was initially absent in the tail fragments, emerged at day 1 in the far anterior then spread posterior to occupy the anterior half of the regenerating fragments by 4 days. ptk7 expression remained broad in tail fragments through early times in regeneration and re-established a trunk-proximal domain evident by 7 days. Notably, the position of the newly regenerated pharynx correlated with a domain in which wntP-2 expression was reduced and ndl-3 was elevated.

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We next examined the functions of ptk7, wntP-2 and ndl-3 in positional information control within regenerating tail fragments. In tail fragments, inhibition of ptk7 caused a posterior shift to the location of the mouth and pharynx (Figure 6A, Figure 6—figure supplement 1A), an effect enhanced by co-inhibition of wntP-2 or ndl-3 though not caused by wntP-2 or ndl-3 inhibition alone (Figure 6—figure supplement 1B). Reduced doses of wntP-2 and ptk7 dsRNA resulted in intermediate placement phenotypes, and the distributions of placement phenotypes were not biphasic (Figure 6—figure supplement 1C), suggesting the activity of these genes could regulate pharynx position in a graded fashion rather than controlling a switch between two alternate organ locations. ptk7(RNAi);wntP-2(RNAi) tail fragments regenerated with posteriorly restricted expression of fzd4-1 and wnt11-1 and posteriorly expanded expression of sFRP-2, without strongly affecting the positioning of the brain (ndk) or pre-pharyngeal regions (SMU15014980) (Figure 6—figure supplement 2D). Together, these experiments suggest that ptk7, along with wntP-2 and ndl-3, has a primary activity in controlling the positioning of trunk/tail tissues in regeneration.

To examine the relationship between the expression and function of ptk7 and wntP-2 signaling in regional identity re-establishment, we analyzed the time of emergence and critical time for pharynx placement phenotypes in regenerating tail fragments. RNAi of ptk7 for three days prior to tail amputation resulted in a posterior shift in the location of the newly formed laminin expression domain detected as early as 3 days of regeneration (Figure 6—figure supplement 2A). These results indicate that ptk7 acts before day 3 of regeneration. To determine a lower bound for the latest time of action for trunk control genes, we performed timed delivery of dsRNA via injections performed at successive two-day intervals during regeneration then examined the position of the laminin expression domain 7 days after amputation. Injections of wntP-2 and ptk7 dsRNA only prior to amputation, or as late as day 1 and day 2, were capable of posteriorly shifting the laminin expression domain (Figure 6—figure supplement 2B). Taken together, these results suggest that ptk7/wntP-2 signaling has functions in pharynx positioning after day 1 and before day 3, coinciding with the timing of wntP-2’s posterior restriction and ndl-3’s anterior expression. This occurs prior to the ultimate reestablishment of ptk7, wntP-2 and ndl-3 expression into finalized trunk, posterior, and anterior domains by 7–14 days, suggesting a separation between processes that control initial organ placement and ultimate proportion restoration (Figure 6A).

The coordinate regulation of wntP-2 and ndl-3 in regeneration led us to examine candidate transcriptional interactions among wntP-2, ndl-3, and ptk7. We did not detect strongly apparent changes to these expression domains after single inhibition of the other two genes by WISH, FISH or qPCR (Figure 6—figure supplement 3A–C). We tested for possible mutual expression requirements in tail fragments, reasoning this might provide a sensitized context in which the expression domains normally undergo regulation. wntP-2 expression was reduced in ndl-3(RNAi) tail fragments, particularly along the lateral edges (Figure 6C), and posteriorly restricted in ptk7(RNAi) regenerating tail fragments (Figure 6—figure supplement 4A), suggesting that these genes can normally promote expression of wntP-2 in regenerating tail fragments. By contrast, ndl-3 expression was expanded posteriorly in ptk7(RNAi) and ptk7(RNAi);wntP-2(RNAi) tail fragments (Figure 6D, Figure 6—figure supplement 4B). We inhibited ptk7 and wntP-2 in uninjured animals to determine whether this effect was specific for regenerating tail fragments. Such animals expressed ndl-3 ectopically posterior to the pharynx at a time (by 12 days of homeostatic RNAi) prior to significant pharynx formation (Figure 6E–F, Figure 6—figure supplement 3A–B). Analysis of these ectopic ndl-3+ cells by double-FISH revealed that the majority (41/63) co-expressed collagen and were located within the plane of the body-wall musculature (Figure 6F). These observations argue for a specific regulatory relationship in which ptk7 and wntP-2 together suppress expression of ndl-3 in the posterior. Alternatively, the nascent pharynx could exert influence over the expression of ndl-3 in a manner indirectly controlled by ptk7 and wntP-2’s suppression of pharynx identity. Together these experiments indicate ptk7, wntP-2 and ndl-3 can directly or indirectly engage in regulatory interactions in homeostatic maintenance of tissue pattern and also in re-establishment of pattern in regeneration.

These experiments suggest a molecular model in which trunk identity and axis position are determined by low wntP-2 activity signaling through the co-receptor ptk7 and receptor fzd1/2/7, which together could provide competence for beta-catenin-independent outputs necessary for trunk and anterior tail patterning (Figure 7A). ndl-3 is expressed in the trunk region yet acts with the same sign as wntP-2 and ptk7 to suppress posterior trunk expansion, either through a parallel or downstream process engaged in trunk suppression (Figure 7A) or perhaps due to its ability under some circumstances to promote robust expression of wntP-2 (Figure 6C). In the tail region of uninjured animals and regenerating trunk fragments, high levels of wntP-2/ptk7 activity prevent the acquisition of trunk identity and enable the formation of anterior tail tissue (Figure 7B). By contrast, head fragments initially have lower expression of wntP-2 and ptk7, which could facilitate their formation of trunk tissue through regeneration. Amputated tail fragments would be expected to initially possess high levels of wntP-2 and ptk7 activity, but a regeneration expression regulatory program reduces wntP-2 mRNA from the anterior to allow trunk regionalization to occur at an appropriate location. These models suggest that the expression status of wntP-2 and ptk7 could provide information about the presence or absence of pre-existing tissues used in determining regeneration outcomes. A previously proposed animal-wide gradient of β-catenin-1 activity (Reuter et al., 2015) could set up the axis into distinct ptk7, wntP-2 and ndl-3 expression domains refined by wntP-2 and ptk7 repression of ndl-3 expression. However, our experiments argue that a β-catenin-1-independent signaling output downstream of wntP-2 and ptk7 likely act to suppress trunk regional identity and thereby control placement of a trunk/tail boundary along the axis according to a gradation of their activities within the posterior. The identification of trunk expansion phenotypes independent of head/tail identity transformations suggests that whole-body regeneration involves a regulatory hierarchy of anterior/posterior pole formation followed by subsequent regional subdivision.

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Our results thus establish a link between Wnt, Ptk7 and FGFRL proteins in regeneration patterning and axis formation. In mice and zebrafish, Ptk7 deletion causes defects in axis formation including a lack of convergent extension within the trunk and tail and a mispolarized auditory epithelium in mice, similar to disruption of core planar cell polarity components that signal independently of beta-catenin (Lu et al., 2004; Hayes et al., 2013). However, studies in Drosophila, zebrafish, Xenopus and mammalian tissue culture have found conflicting evidence that Ptk7 can also either promote (Puppo et al., 2011; Bin-Nun et al., 2014) or inhibit (Peradziryi et al., 2011; Hayes et al., 2013) beta-catenin-dependent signaling in a context-dependent manner. The regional identity defects we observe after ptk7 RNAi in planarians are not obviously the result of defective planar cell orientations, are phenocopied by inhibition of a Wnt gene, and do not globally affect beta-catenin transcriptional targets or beta-catenin-dependent processes. We suggest that Ptk7 proteins can control tissue fate through an alternate mechanism, perhaps by coordinating the activities of cell cohorts within a field. Planarians and most other animals have expression of multiple Wnts in posterior domains, pointing to their ancient use in organizing the primary body axis (Petersen and Reddien, 2009). The use of Ptk7 proteins for trunk/tail regionalization could therefore have an ancient origin and allow posterior Wnts to produce distinct signaling outcomes for combinatorial pattern control.

We also find with wntP-2 and ndl-3 a second example in planarians of shared patterning regulation between Wnt and FGFRL factors. Whereas wntP-2/wnt11-5/wnt4b and ndl-3 restrict the domain of the trunk, wntA/wnt11-6/wnt4a (Kobayashi et al., 2007) and nou darake (Cebrià et al., 2002) restrict the domain of the head and brain. Intriguingly, in both cases, the FGFRL genes have most prominent expression in the anterior but act with the same sign as the Wnt genes to promote more posterior identity outside of the domain of their own expression. In mammals, FGFRL1 and Wnt4 are each required for formation of the metanephric kidney from intermediate mesoderm, suggesting this positive regulatory relationship is conserved (Stark et al., 1994; Gerber et al., 2009).

Pattern restoration in regeneration has been proposed to involve intercalation (French et al., 1976) or progressive specification of adjacent regions (Roensch et al., 2013) to restore positional values across a field disrupted by amputation. In planarians, asymmetric wound-induced expression of notum provides information about wound site directionality used to program anterior-versus-posterior pole fates (Petersen and Reddien, 2011). By contrast, we find genes encoding signaling molecules that are constitutively expressed in body-wide mRNA gradients and are used for control of positional information in regeneration. Graded expression of paracrine factors across fields of cells could enable patterning over the large length-scales necessary for adult regeneration. The interactions between this tissue-wide positional information present at the time of injury, combined with wound-induced directional cues, could account for robust pattern control in regeneration.

Note added in proof: while this manuscript was under review, Sureda-Gómez et al. reported that wnt11-5 RNAi causes a pharynx duplication phenotype (Sureda-Gómez et al., 2015).

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Author details

1. Rachel Lander

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

   Contribution

   RL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

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

   CPP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   christian-p-petersen@northwestern.edu

   Competing interests

   The authors declare that no competing interests exist.

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

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