A comparative analysis of planarian regeneration specificity reveals tissue polarity contributions of the axial cWnt signalling gradient

Reviewer #1 (Public review):

Summary:

In the manuscript entitled 'A comparative analysis of planarian regeneration specificity reveals tissue polarity contributions of the axial cWnt signalling gradient.' Cleland et al. study the robustness of regenerating a head or a tail in the proper position in two different planarian species (S. mediterranea and G. sinensis). The authors find that the expression of notum, a Wnt inhibitor that is triggered after any cut, shows different dynamics of expression in both planarian species, being more symmetrical in the species that display a higher number of double-headed or Janus heads (G. sinenesis), which they refer to a less robust regeneration. The authors claim that the reduced robustness of G. sinensis regeneration is partially explained by this anterior-posterior symmetric expression of notum, since in S. mediterranea, which shows a 'robust regeneration' it appears asymmetric. So, the first claim of the manuscript is that the symmetry in notum expression could underlie the poor robustness of regenerating a head/tail in small bipolar regenerating planarian fragments.

Then, they analyse the role of a proposed tail-to-head cWnt signalling gradient during the regeneration of heads and tails in the same planarian species. To do so they develop an antibody that allows the quantification of b-catenin activity along the AP axis, together with a pharmacological approach that reduces the pre-existent cWnt gradient without affecting the wound-induced. Through this strategy the authors can demonstrate the slope of the b-catenin activity, which is a very nice result, and that it changes according to the size of the animal. Furthermore, they are able to demonstrate that by reducing the cWnt signalling in the pre-existent tissue, there is an increase in the number of double-headed regenerates (Janus heads) and that it depends on the body size and on the decreasing steepness of the cWnt gradient. This result relies on G. sinensis species since the drug is not so effective in S. mediterranea. Thus, the authors' second claim is that the slope of the cWnt gradient may contribute to head-tail regeneration specificity in planarians.

To conclude, it is proposed that regeneration of the correct identity in each wound depends on multiple cues acting in parallel and that their species-specificity provides variations in the regenerative capability of the different planarian species.

The study has great potential to have a high impact on the regeneration community, since the opportunity to compare mechanisms between close species provides the framework for understanding the essential mechanism of regeneration.

Strengths:

The project has several strengths. The authors are able to reproduce the Janus heads phenotypes described by Morgan TH by analysing different planarian species. This is of great importance in the planarian field, because with the current model species, S. mediterranea, this could not be reproduced. So, these results demonstrate that small planarian fragments do make errors during regeneration, giving rise to double-headed animals, which supports the well-known hypothesis that it exists an anteroposterior gradient underlying anteroposterior identity during regeneration. However, and importantly, it does not occur in all planarian species. So, there are differences between planarian species in the robustness of regeneration and may be in the mechanisms that drive this regeneration. The finding of different behaviours and gene expressions in different planarian species is very interesting and promising in the field of regeneration.

A second strength of the study is the demonstration of the b-catenin1 slope in planarians and how it changes with the animal size, and also the establishment of a method to decrease it in the pre-existent tissue but not in the wound. This strategy allows us to examine specifically the role of the pre-existent cWnt signal, demonstrating that it does have a role in the decision of making head or tail during regeneration, which was an essential question in the field of planarians and animal regeneration.

Weaknesses:

(1) The finding that notum, which is the main head determinant identified in planarians, has a different dynamic in both planarian species is very suggestive. However, the different dynamics of notum expression during regeneration, which is the basis of the subsequent rationale, is not properly demonstrated, nor is its correlation with the robustness in regenerating a proper head/tail identity. Main concerns regarding this point:

a) The authors observe that 'In regenerating S. mediterranea 2 mm trunk pieces cut from 6 mm animals, notum expression was induced predominantly at anterior-facing wounds as early as 6 h post-amputation (Figure 2A), as previously reported (Petersen and Reddien 2011)'. However, in the graphics in Figures 2B and C, the expression of notum at 6h is shown as symmetric. It definitely does not agree with the in situ, with the text, or with the published data. How was it measured? It should be corrected and explained since it is the basis of the subsequent rationale.

b) Then, when measuring notum in G. sinensis the authors conclude: 'Strikingly and in sharp contrast to S. mediterranea, the number of notum expressing cells was nearly identical between anterior and posterior wounds without any discernible A/P asymmetry at any of the examined time points (Figures 2E-F)'. However, in the in situ results of 12 h regenerating G. sinensis, there is a clear difference in notum expression between anterior and posterior wounds. Is it not representative of the image? Again, how exactly the measurements were performed? Are dots or pixels quantified? It is not explained in the text. This is a crucial result that has to be consistent.

c) A more general weakness of this part of the manuscript is that even if the authors demonstrate that in G. sinensis the expression of notum is symmetrical in contrast to S. mediterranea, this is just an observation of 1 species that has symmetrical notum and regenerates less robustly than 1 species that has asymmetrical expression and regenerates more robustly. If they for instance look at the expression of wnt1, maybe they also see differences between both species that could be linked to their different regeneration properties (related to this, see below the comment on wnt1 expression). That is to say, comparing 1 to 1 species cannot give any cause-effect evidence.
Furthermore, the authors rely on the fact that notum inhibition rescues the wild-type phenotype to conclude that is the symmetric expression of notum that underlies the appearance of Janus heads. This is what can be read in the results: 'Significantly, the rescue of wild-type regenerates by notum(RNAi) suggests that the symmetric G. sinensis notum expression contributes to the formation of double-heads and thus to reduced regeneration specificity'; and in the Summary: We found that the reduced regeneration robustness of G. sinensis was partially explained by wound site-symmetric expression of the head determinant notum, which is highly anterior-specific in S. mediterranea.' However, notum RNAi decreases notum in both wounds, so it does not produce an asymmetric expression (at least this is not shown). So, it does not link the symmetry or asymmetry of notum with the appearance of Janus heads.

d) If the authors want to maintain the claim that the symmetry of notum is one of the reasons that explain the increase in Janus head phenotype in G. sinensis, there are several possibilities to test it. For instance:

i) Analyse notum expression in different planarian species and relate its symmetry or asymmetry with the appearance of Janus heads. If the claim is true, the species that are more robust should show more asymmetric expression of notum. This would sustain strongly the first claim, and would really be a breakthrough in the field of regeneration.

ii) Another possibility is a more in-depth analysis of notum expression in the species of the study. If the authors show that larger fragments show fewer Janus heads, and also that it depends on the anteroposterior level of the fragments, they could try to relate the rate of Janus heads with the degree of asymmetry in notum expression in both wounds. For instance, they could analyze notum expression in bipolar regenerating fragments along the anteroposterior axis in both species; it should be more symmetric in G sinenesis, in all fragments, according to Figure 2 L. Or they could analyze notum expression in bipolar regenerating fragments of different sizes, mainly in 1 or 2 mm fragments of big planarians, since they are the fragments analyzed that form or not the Janus heads. In G sinensis the expression of notum should be more symmetrical than in S. mediterranea in these fragments.

iii) The authors could design an experiment to demonstrate that the symmetry in the expression of notum affects the rate of Janus heads. The experiment that the authors show is the rescue of the Janus heads in G. sinensis after notum RNAi. However, notum RNAi suppresses notum in both wounds, thus not making them asymmetric. Furthermore, the rescue could be explained by the posteriorizing effect that notum RNAi has in planarians, as reported by several authors. A possibility could be to inhibit APC, which increases notum expression in S. mediterranea (Petersen and Reddien 2011). If APC RNAi in G. sinenesis produces an increase in notum in both wounds and the rate of Janus heads is not rescued, then it would support the hypothesis that notum symmetry is the cause of the Janus heads. However, if it produces an increase of notum in an asymmetric manner, then the Janus phenotype should be rescued.

(2) The second weakness of the study is related to the methodology used to support the second claim, that the slope of bcatenin1 activity has a role in the decision of regeneration - a head and a tail in the correct tip. The main concerns relate to the specificity of the anti-bcatenin1 antibody and to the broad effect of C59 in the secretion of all Wnts.

a) Raising an antibody against beta-catenin1 that allows the quantification by western blot is a strength of the study, since beta-catenin1 is the key element of the cWnt pathway, and their levels are directly associated with the activation of the pathway. Since this is one of the tools that support the second claim of the study, a characterization of the antibody and additional tests to prove its specificity are required. The authors show a Western blot in which the band intensity decreases after beta-catenin1 inhibition in both species. Further analysis should be shown:
i) Demonstration that the intensity of the band increases after APC or Axin inhibition.
ii) Does the antibody work in immunohistochemistry? It would provide further evidence of the specificity of a nuclear signal could be demonstrated.
iii) Explanation and discussion of the protocol used to analyse the levels of b-catenin1 activity along the anteroposterior axis is required. It has been reported that beta-catenin1 is highly expressed and required in the brain in planarians, and also in the pharynx, and in the sexual organs (Hill and Petersen 2015, Sureda-Gomez et al 2016). How is it then explained the anterior-to-posterior gradient of expression of beta-catenin1 seen in this study in both species? Has the pharynx been removed before the protein extraction? What about the beta-catenin1 activity demonstrated in the brain? Why is it not reflected in the western blot analysis using the antibody? This point should be clarified.

b) The second tool used in the second part of the manuscript is the drug C59, which inhibits Porcupine, a protein required for palmitoylation and secretion of Wnts. Because Porcupine could be required for the secretion of all Wnts, the phenotype obtained with the drug could be the sum of the inhibition of cWNT signal (wnt1 for instances) and non-canonical WNT (as wnt5). This is in fact the phenotype resulting after the inhibition of Wntless in planarians (Adell et al. 2009), which is also required for the secretion of Wnts. Thus, in the phenotypes resulting from C59 treatment the analysis of the nervous system and posterior/anterior markers is required. Looking at the in vivo phenotype it appears that in fact the drug is affecting both canonical and no canonical pathways since the animal with protrusions in the lateral part (Figure 4B-double head, or Supplementary Figure 3A) is very similar to the one reported after Wntless inhibition. In case the phenotypes observed also show non-canonical Wnt inhibition, this should be clearly shown and discussed.

The above-mentioned weaknesses are the most important concerns about the present manuscript. However, there are other concerns related to a further analysis of the phenotypes and the analysis of additional Wnt elements as wnt1, which are essential to complete the study and are directly discussed with the authors.

Reviewer #2 (Public review):

Summary:

This study identifies a key role for bodywide canonical Wnt gradients in controlling the outcome of regeneration within planarians, likely acting in parallel to injury-induced cues that also use tissue asymmetry to control this process. In S. Mediterranea a central part of this decision process is the asymmetric expression of the Wnt inhibitor notum specifically at injury sites facing in the anterior direction to promote head formation and inhibit tail formation through regulation of canonical Wnt signaling pathways. Leveraging classic studies by T.H. Morgan over a century ago, which found that amputated thin transverse fragments occasionally incorrectly regenerate 2 heads rather than a head and a tail in a species of Girardia planarians, this study identifies a closely related species G. Sinensis which undergoes errors to regeneration specificity under similar challenges. Morgan had proposed that his results might arise from the use of a "gradient of materials" providing axis information across the body axis such that small tissue fragments are too narrow to interpret gradient differences, leading to head/tail polarity defects in regeneration. The authors show very convincingly that this species of planaria undergoes notum expression after injury, but unlike in S. Mediterranea, this occurs symmetrically at the onset of regeneration. Using RNAi, they show notum participates in the regeneration of mispolarized heads (though interestingly apparently not in normal head regeneration unlike in Smeds, at least under these conditions). G. Sinensis planarians, like many organisms, have abundant expression of Wnt genes posteriorly. To test whether this gradient of Wnts may participate in the regeneration distinct from any Wnt signals activated after injury, the authors use chemical inhibition to reduce Wnt signaling prior to injury and then alleviate inhibition following injury by removal of the drug and confirming successful washout of the drug using mass spec. They also raise a new antibody that can detect beta-catenin-1 in this species in order to monitor the body-wide cWnt gradient after these treatments, and correlate this with outcomes on the head/tail regeneration decision. Using this approach, they find that homeostatic inhibition of porcupine (required for Wnt secretion) could dampen the cWnt/beta-catenin gradient and increase the incidence of inappropriate head regeneration at posterior-facing wounds. In addition, they find that the cWnt gradient is less steep in larger animals that also concurrently have a higher incidence of mistakes in regeneration specificity. Together, the paper presents compelling experiments and analysis to support the conclusion that cWnt gradients are an important determinant of head/tail identity determination decisions in G. Sinensis, and thereby proposes a plausible model that the notum asymmetry present in S. Mediterranea could act in parallel to support the higher regeneration robustness observed in that species.

Strengths:

This is a great paper, an instant classic. It addresses an enduring problem that Morgan and others initiated more than a century ago and brings a new synthesis of ideas to clarify an important mechanism. I also like the term "regeneration specificity" which can provide a nice unification and generalization of ideas that other authors have variously described as regeneration patterning or regeneration polarity. The work is a tour de force that creatively builds new tools and observations to leverage a new model of planarian species for unraveling general mechanisms of regeneration decision-making. The experiments are rigorously conducted and I find the overall data to be quite compelling. I have some comments for the authors to consider below for drawing out the interpretation and also clarifying the underlying mechanism.

Comments:

(1) The G. Sinesis species showed accurate head/tail specificity in 2mm thick fragments but was strongly impaired at 1 mm thick. I am assuming that outcomes of pieces greater than 2mm would make similarly robust head/tail choices, implying a rather sharp transition occurring between 1 and 2 mm. In that case, in the gradient model, are there theoretical reasons to predict that polarity outcomes would decline sharply rather than gradually as size thickness decreases? I think the muscle fibers themselves are thought to have lengths on the order of 200 microns, so I wonder what could account for the characteristic length of less than 1mm here? From the lab's prior analysis of beta-cat gradient, is this perhaps the minimal length where a difference in bcat protein levels can be detected? This is not essential to resolve in this draft (in my view), just a very interesting question arising from the present study. Relatedly, it seems that the slope of cWnt at the wound site itself might not be enough information for polarity because at a highly granular level, this should be identical at posterior-facing wounds from trunk fragments versus thin transverse fragments obtained at the same AP position, yet trunk fragments succeed at regeneration specificity whereas thin transverse fragments fail.

(2) The paper nicely shows strong evidence that notum expression is definitely symmetric at the first occurrence of its expression by 6 hours in D. Sinensis, and this is a really important result of the paper. At 12 hours, it does look to me in the FISH experiments that there is more persistence of expression at the anterior-facing wound versus the posterior-facing wounds (Fig 2D), although the methods for quantification in Fig2E/F do not show a difference in expression at the two wound sites at this time point. Could this difference arise from differences in the perdurance or timing of early wound-induced signaling at the two wound sites that was perhaps too subtle to detect in the quantification methods used? Or perhaps these images do not represent the population? On a related note, the quantification method seems to fail to show that in 6h Smeds, notum expression is indeed asymmetric. Probably the issue here is not the data in the FISH images themselves which strongly support the author's interpretations, but rather a deficiency or limitation of the quantification method used, which should be resolved so that the conclusions from the single FISH images can be interpreted robustly. For example, some authors have used a method of counting notum+ cells and I wonder if this could provide better quantitative information here.

(3) Given that the double-headed phenotype is observed from thin transverse fragments, ideally, the symmetry of notum could be established to occur in those types of fragments as well. This experiment would clarify that notum is expressed at posterior-facing wounds in the very same types of fragments that undergo the highest levels of mistakes in regeneration specificity.

(4) Is wnt1 expressed symmetrically at wound sites in this species? It seems there are cases like acoels where wound-induced Wnt activation can occur asymmetrically but through preferential expression of Wnts at posterior-facing wounds, rather than notum. It would be interesting to know although I also think the work the authors already have done in this study itself already constitutes a very comprehensive advance and could be the subject of future work.

(5) I agree that notum is relatively much more strongly expressed at the far posterior region in D. Senesis than in Smeds, but it does seem from the RNAseq data it also has some locally enriched expression at the anterior pole. Because the RNAseq analysis involves scaling expression across the regions for each gene, it is difficult to know if the anterior expression is relatively lower or perhaps even about the same level of expression as the anterior pole expression of this gene in Smeds. Though not essential to make the desired arguments, in situs on notum in the intact animals would be helpful to clarify this. Relatedly it would be fascinating to know whether D. Senesis notum undergoes anterior-pole expression around the 72 hour or similar timepoint as in Smeds.

(6) The assessment of beta-catenin gradients was done through protein extractions from whole tissue fragments. However, it has been shown in other planarian species that beta-catenin can have strong tissue-specific expression in, for example, the pharynx, brain, and reproductive systems. Some supporting evidence or argument should be presented to clarify the interpretation that the graded expression observed by western blotting cannot be fully explained by this kind of tissue-specific expression of beta-catenin rather than representing a true signaling gradient as interpreted by the authors. For example, if this antibody could be used in immunostaining, this could support the beta-catenin signaling gradient. Alternatively, information about the location of the pharynx or any other posterior reproductive tissues in D. Sinensis could be calibrated with respect to the fragment bins used for the gradient--perhaps a portion of the C59-dependent body-wide gradient measured here occurs fully within tail tissue that lacks other regionalized tissue that could be a potential additional source of beta-catenin. Further discussion and interpretation, or additional experiments, should be included to rule out alternative confounding sources of beta-catenin in order to clarify the interpretation of the western blot as representing a beta-catenin signaling gradient.

(7) I find the analysis in Figure 5 to be quite compelling for showing the importance of cWnt/Bcat gradients in contributing to head/tail determination, and I also think that the author's discussion of the limitations of the approach are well articulated and considered. Based on prior literature, it also seems very likely that there is a third redundantly acting component to regeneration specificity, which is the amplification of small differences in cWnt in a directional-dependent manner early in the regeneration process (24-72 hours in Smeds). This would explain why post-amputation with porcupine inhibitor in D. Sinensis caused 100% penetrant defects in regeneration specificity while the pre-treatment paradigm caused a weaker effect (25-40% for larger animals). In Smeds, it is known already that delivery of dsRNAs against beta-catenin-1, wnt1, and notum only after injury caused polarity defects, and thus all three genes certainly have a function relevant for head/tail after injury (Petersen and Reddien 2008, 2009, 2011- please note these experiments were reported in the text of these studies and not in individual figures). This evidence, combined with extensive FISH and complementary RNAi studies in the field, strongly suggests that some combination of the 6-18h injury-induced phase but also very likely the subsequent "pole-specific phase" of wnt1 expression is likely to be important for driving or enacting the tail fate program and is therefore a component of the regeneration specificity mechanism described here.

(8) Prior work has also demonstrated roles for Wnt genes expressed in gradients to participate in regeneration specificity. In particular, inhibition of the wntP-2/wnt11-5 gene, which is expressed in an animal-wide gradient, strongly enhanced the effects of inhibition of wnt1, which is the earliest wound-activated Wnt gene, to cause 100% penetrant posterior head regeneration phenotypes in S. mediterranea (Petersen and Reddien 2009). These observations are complementary to the present study by implicating Wnts expressed in bodywide gradients in the process of regeneration decision-making. Given that this study also showed that wnt1 is necessary for new wntP-2 expression during the wound-induced early phase and that wnt1 activation does not require beta-catenin for its expression, collectively suggest a more complex process involved in gradient detection and the involvement of wound signals likely beyond only autoregulation of the cWnt gradient or notum asymmetry mechanisms. Although this paper is cited already, framing the present study more fully in context with this and other relevant prior work would be helpful to contextualize the advance for the field.

Reviewer #3 (Public review):

Summary:

In this study, the authors revisit the hypothesis of gradient-based polarity specification during planarian regeneration proposed over a century ago, but here they apply molecular techniques and a valuable comparative approach. By using a comparative analysis with classic and modern planarian model organisms, the authors have identified variable molecular mechanisms that different planarian species utilize to ensure that the proper tissues are regenerated following wounding.

Strengths:

The comparative approach of using 2 different planarian species allowed the study to elucidate different molecular mechanisms that planarians utilize in re-establishing anterior-posterior axis polarity during regeneration. Without this comparative approach, the mystery of T.H. Morgan's data classic studies that demonstrate mistakes in this axis re-polarization would remain unanswered. Furthermore, the use of both a modern molecular model species and another more classical planarian species, which the authors have fully developed with molecular tools and techniques, sheds light on the diversity of genetic processes that closely related species seem to utilize in regeneration. To dissect the role of a long-hypothesized canonical cWnt signaling gradient, the authors developed a novel strategy using chemical genetics to titer this gradient, which led to phenotypes with enhanced aberrant axis polarity re-establishment. Together these experimental approaches establish a well-supported initial model for explaining the molecular mechanisms that different planarian species utilize to allow for proper regeneration of lost tissues.

Weaknesses:

While pharmacological perturbation of signaling pathways could produce off-target effects, the authors provide well-documented evidence that canonical Wnt signaling is altered with drug treatment. The correlation between altered cWnt signaling gradients and the incidence of double-headed regeneration is strong, but it is not clear that the axial cWnt signaling gradient is the ultimate cause of the modified regeneration polarity. However, the model established here and supported by considerable data provides a useful alternative to the mechanism of notum upregulation that has been well-documented in the Schmidtea mediterranea, the workhouse model in planarian research. Throughout the manuscript, the authors suggest that Girardia sinensis lost the ability to upregulate notum at anterior-facing wounds, but until additional planarian species are evaluated, it remains plausible (and equally parsimonious) that S. mediterranea could have innovated a novel strategy to re-establish axis-polarity through asymmetric notum expression.

The study is very well-designed with considerable confirmation of results, especially in the novel use of the pharmacological inhibitor C59. This study is invaluable in its comparative approach, finding that well-established molecular processes may not explain similar developmental outcomes for different species; this corroborates the need to study additional model organisms and how an evolutionary approach to the study of development is imperative.