In order to regenerate tissues successfully, stem cells must detect injuries and restore missing cell types through largely unknown mechanisms. Planarian flatworms have an extensive stem cell population responsible for regenerating any organ after amputation. Here, we compare planarian stem cell responses to different injuries by either amputation of a single organ, the pharynx, or removal of tissues from other organs by decapitation. We find that planarian stem cells adopt distinct behaviors depending on what tissue is missing to target progenitor and tissue production towards missing tissues. Loss of non-pharyngeal tissues only increases non-pharyngeal progenitors, while pharynx removal selectively triggers division and expansion of pharynx progenitors. By pharmacologically inhibiting either mitosis or activation of the MAP kinase ERK, we identify a narrow window of time during which stem cell division and ERK signaling produces pharynx progenitors necessary for regeneration. These results indicate that planarian stem cells can tailor their output to match the regenerative needs of the animal. Many animals can repair and regrow body parts through a process called regeneration. Tiny flatworms called planaria have some of the greatest regenerative abilities and can regrow their whole bodies from just a small part. They can do this because around a fifth of their body is made of stem cells, which are cells that continuously produce new cells and turn into other cell types through a process called differentiation.Measuring the gene activity in stem cells from planaria shows that these cells are not all the same. Different groups of stem cells have specific genes turned on which are needed to regrow certain body parts. It is unclear whether all stem cells respond to injuries in the same way, or whether the stem cells that respond are specific to the type of injury. For example, stem cells needed to repair the gut may respond more specifically to gut injuries than to other damage.Bohr et al. studied how stem cells in planaria respond to different injuries, by comparing an injury to a specific organ to a more serious injury involving several organs. The specific injury was the loss of the pharynx, the feeding organ of the flatworm, while the more serious injury was the loss of the entire head. Within hours of removing the pharynx, stem cells that were poised to develop into pharyngeal cells became much more active than other stem cell types. When the head was removed, however, a wide range of stem cells became active to make the different cell types required to build a head. This suggests that stem cells monitor all body parts and respond rapidly and specifically to injuries.These findings add to the understanding of regeneration in animal species, which is of great interest for medicine given humans’ limited ability to heal. Many of the genetic systems that control regeneration in planaria also exist in humans, but are only active before birth. In the long-term, understanding the key genes in these processes and how they are controlled could allow regeneration to be used to treat human injuries. When faced with injury or disease, many animals can repair or even replace damaged tissue. This process of regeneration is observed across animal species, and is often fueled by tissue-resident stem cells . In respSchmidtea mediterranea is an ideal model organism to study the interaction between injury and tissue repair due to their virtually endless ability to regenerate  for piwi-1 and the pharynx-specific progenitor marker FoxA, 3 days after pharynx or head amputation. We then quantified pharynx progenitors in the same region, anterior to the pharynx  caused a significant increase in pharynx progenitors compared to intact controls. We also performed flank resections, which removed tissue from regions adjacent to the pharynx but did not damage the pharynx itself. Despite being previously shown to increase new cells into the uninjured pharynx with BrdU labeling , intestine (+gata-4/5/6), the excretory system (+six-1/2) and the nervous system (+pax6a)   after ei(pax6a+) . Analysioutcomes . No chanoutcomes . Togethepax6a, are required for regeneration of their cognate organ (FoxA(RNAi), knockdown of other organ-specific progenitor markers did not impact the recovery of feeding ability , nervous system (+pax6a), and muscle (+myoD) progenitor division increased 2 days after head amputation, while intestinal (+gata-4/5/6) progenitors did not. Additionally, division of nervous system and muscle progenitors increased only after head but not pharynx amputation   , unless putation . Therefoputation . This fiIn planarians, pulse-chase experiments using thymidine analogs have shown that cell division contributes to the production of regenerated tissues . Our res+) stem cells  kinase pathway drives proliferation and differentiation during development and regeneration in many organisms . In planExposing animals to PD0325901 (PD), an inhibitor of the upstream ERK-activating MEK kinase, blocks ERK phosphorylation and permanently inhibits regeneration following substantial anterior tissue removal . To detelaminin. Animals exposed to PD for 5 days, beginning 0 or 1 day after amputation, lacked a pharynx, while pharynges in animals exposed beginning 2 days after amputation were comparable to controls  (activin-1 and -2 (fst(RNAi) prevents regeneration following substantial anterior tissue removal  animals regained the ability to feed at a normal rate  , which a1 and -2 . Like ER removal . Howeverminishes . Likewisminishes , on a siminishes . Therefoputation , but notputation , suggestputation  may not mal rate , indicat+ piwi-1+FoxA cells typically observed after pharynx amputation   animals to regenerate is entirely dependent on their failure to reset positional information, as inhibition of wnt signals that restrict head formation rescue these regeneration defects  , which pin (fst) . Interes defects . Therefo removal , but notpoptosis . Therefoegfr-3 is required to activate ERK during regeneration  and the fibroblast growth factor receptor (FGFR) have been shown to play critical roles in signaling upstream of ERK in many organisms , making neration  and is aneration . Other sectively . Furtherpression . Whetherpression .FoxA expression in stem cells after pharynx or head removal, we have uncovered shifts in stem cell heterogeneity that depend on the presence or absence of a particular organ. Intriguingly, we find that both the overall number of +FoxA pharynx progenitors, as well as those that are actively dividing, increase only after pharynx removal. Therefore, stem cells sense the absence of the pharynx and channel their proliferative output toward the population of stem cells required to regenerate it. Combined with our analysis of non-pharyngeal progenitor dynamics after different amputations, our results suggest that the heterogeneity of the stem cell population can be differentially deployed depending on the severity of the injury and the particular tissues that need repair.By studying the dynamics of + cells within the pharynx, raising a conundrum regarding the source of these cells. One possibility is that other, non-+FoxA progenitors which have yet to be identified, may contribute to pharynx regeneration. However, no other tissue-specific transcription factors or proposed pharynx progenitor markers  appear to be required for pharynx regeneration  in planaria water containing 3% DMSO for 4 hr either immediately or 24 hr after amputation and fixed 7 days after amputation.Animals were fixed as previously described  with min2O2 overnight. Animals were then treated with proteinase K (10 µg/mL proteinase K and 0.1% SDS in PBSTx) for 15 min, and post-fixed in 4% formaldehyde in PBSTx for 10 min. A F-ara-EdU development solution was made containing PBS + 1 mM CuS04 and 100 µM Oregon Green 488 azide (Thermo Fisher O10180). Freshly made 100 mM ascorbic acid was added to this solution immediately before administering it to samples, which were then incubated for 30 min in the dark. Following a few rinses with PBSTx, animals were post-fixed, rinsed 2x in PBSTx, and put through in situ (see below). Following in situ, animals were placed in K block  at room temperature for 4 hr or 4°C overnight. To detect F-ara-EdU, animals were incubated with 1:1000 anti-Oregon Green-HRP (Thermo Fisher A21253) and counterstained with DAPI in K block at 4°C overnight. Antibodies were washed off in PBSTx, pre-incubated with tyramide (1:2000 FAM) for 10 min and developed for 15 min.For F-ara-EdU detection, following fixation, animals were rehydrated and bleached in 6% Hin situ hybridizations were performed as described in in situ hybridizations were performed as in 2O2 in 0.5x SSC) for 2 hr, then treated with proteinase K . Following overnight hybridizations at 56°C, samples were washed 2x each in wash hybe (5 min), 1:1 wash hyb:2X SSC-0.1% Tween 20 (10 min), and 2X SSC-0.1% Tween 20 (30 min), 0.2X SSC-0.1% Tween 20 (30 min) at 56°C followed by 3 × 10 min PBSTx washes at room temperature. Subsequently, animals were placed in blocking solution (0.5% Roche Western Blocking Reagent and 5% inactivated horse serum diluted in PBSTx). Animals were then incubated with an appropriate antibody: 1:1000 anti-DIG-POD (Roche 11207733910) or 1:1000 anti-FITC-POD (Roche 11426346910) in blocking solution at 4°C overnight followed by several washes with PBSTx. For development with FAM (1:2000) or Cy3 (1:7500), animals were preincubated with tyramide in borate buffer for 30 min and then developed with 0.005% H2O2 in borate buffer for 45 min. For development with rhodamine, animals were pre-incubated with tyramide (1:5000) for 10 min and developed for 15 min. To inactivate peroxidases, animals were treated with 200 mM sodium azide or 4% H2O2 in PBSTx for 1 hr, then rinsed with PBSTx >6 times before application of the next antibody.Colorimetric in situ, animals were incubated with anti-phosphohistone H3 (Ser10) antibody  diluted 1:1000 in blocking solution (0.5% Roche Western Blocking Reagent and 5% inactivated horse serum in PBSTx) for 2 days at 4°C. Primary was washed off with PBSTx followed by incubation with goat anti-rabbit-HRP (Thermo Fisher 31460) diluted 1:2000 in PBSTx overnight at 4°C. Antibody was washed off with PBSTx and samples were pre-incubated and developed with rhodamine tyramide as described above.For H3P detection, following in situ and immunostaining experiments, DAPI [5 µg/mL]  was added along with the last antibody (except for colorimetric in situ). After the final development, animals were soaked in ScaleA2  . To maintain consistent sample thickness, animals were mounted in wells cut from a double layer of double stick tape .For all % DABCO)  for at l2, 100 mM KCl, 10% glycerol, 0.05% NP40, and 0.5 mM DTT) (C (Thermo Fisher). After quantification, Bolt LDS sample buffer (Life Technologies B0007) was added to the extracts and 100 µg of each sample was run on a polyacrylamide gel . The gel was transferred onto a PVDF Immobilon membrane (Merck Millipore IPFL00010) using the Pierce Power Blot Cassette system (Thermo Scientific), then treated with Odyssey blocking buffer (LI-COR 927–40000) for 1 hr at RT. Membranes were incubated overnight at 4°C with mouse anti-tubulin (Sigma/Millipore T5168) and rabbit anti-Phospho-p44/42 MAPK (Erk1/2) , diluted 1:1000 in blocking buffer. The blot was washed 3 × 10 min in TBST (TBS +10% Tween 20) and incubated for 1 hr at RT with goat anti-mouse Alexa Flour 488 (Thermo Fisher A11029) and goat anti-rabbit IRDye 800CW (LI-COR 926–32211) secondary antibodies . Membranes were then washed as before and imaged using a Bio-Rad ChemiDoc MP. Western blots were repeated at least twice with comparable results.Ten animals per condition were snap frozen in lysis buffer   containiNocodazole (Sigma M1404) was administered in 24 or 48 hr increments at 50 ng/mL. PD0325901  and UO126  were administered at 10 µM and 25 µM, respectively. Drugs were diluted in planaria water containing 0.05% DMSO. Animals were rinsed three times after treatment and either fixed immediately, or transferred to a new dish and rinsed daily until further experimentation.Animals were fed 20 µL of colored food (4:1 liver:milliQ water with 2% red food coloring) in a petri dish. After approximately 30 min, the number of animals with red intestines were scored. For time courses, feeding assays started at 4 days post-amputation and any animals that ate were removed from the dish. Feeding assay time courses were repeated at least three times with ~20 animals assayed per experiment.in vitro-synthesized double-stranded RNA (dsRNA). dsRNA was diluted to a final concentration of 400 ng/µL in colored food. RNAi food was administered every 3 days, six times in total, except for gata-4/5/6 and six-1/2, which caused phenotypes after 1–2 feeds. C. elegans unc22 dsRNA was used as a control. Amputations were carried out 5–7 days after the last feed. All RNAi experiments were repeated at least twice with ~10 animals per experimental group.RNAi was performed as previously described , with inin situ hybridizations and live worms were imaged on a Leica M165F. Fluorescent in situ hybridizations were imaged on a Zeiss 710 confocal microscope using a 25x objective with 2.28 µm z-sections. ImageJ software was used for processing and quantification  beginning at the first +piwi-1 cell. For H3P analysis, the entire pre-pharyngeal region was imaged, captured at 0.6x zoom. Quantification included 30 z-sections (68.4 µm) beginning at the first H3P+ cell and was normalized to area. Representative confocal images are partial projections of ~5 z-sections from regions that were used for quantification, re-imaged at 4x zoom. For F-ara-EdU quantification, images were captured at 1x zoom. In most cases, the pharynges and brains of each experimental group were imaged from the same animal. All visible F-ara-EdU+ cells in the pharynx and all F-ara-EdU++ChAT cells in the brain were quantified throughout the entire organ. Representative F-ara-EdU images are projections of the entire analyzed region. In bar graphs, symbols represent individual animals, and shapes distinguish biological replicates. Statistical analysis was performed using PRISM-Graphpad version nine or GraphPad Quick Calcs to perform one-way ANOVA with Tukey test, unpaired t-test or Fisher’s Exact test as indicated in figure legends. *p≤0.05; **p≤0.01; ***p≤0.001; and ****p≤0.0001.For Sequences for all transcripts used in this study were cloned using the following primers: In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:How stem cells respond to missing tissue is an important question for understanding regenerative processes. Here, Bohr et al. address this question by comparing how stem cells respond to loss of a single organ (the pharynx) versus loss of many tissues (after head amputation) in the planarian, an organism that can regenerate its entire body from a tiny piece of tissue. The authors find that the stem cells respond to loss of the pharynx by producing more pharynx progenitors; this increase is not observed after removal of non-pharyngeal tissues. Thus, the planarian's stem cells are able to "sense" when certain tissues are missing and target their fates accordingly.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "Planarian stem cells sense the identity of missing tissues to launch targeted regeneration" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in All three reviewers recognized the potential significance of this work, but also shared the same concerns about sample sizes, lack of biological replicates, and insufficient details about cell quantification. Given the interest in the question being pursued, if these issues can be addressed satisfactorily, a revised paper could be considered here as a new submission. We have included the reviews below and hope you will find the comments helpful.Reviewer #1:This manuscript by Bohr et al. explores how planarian stem cells respond to the loss of a specific organ: the pharynx. The previously proposed "target-blind" model of planarian regeneration  posited that stem cells do not respond directly to missing tissues, but rather replace missing cell types based on their normal rates of homeostatic turnover. In contrast, Bohr et al. suggest that planarian stem cells can sense and respond to the loss of specific missing tissues, using the pharynx as a case study. The authors conclude that planarians may use more than one mode of regeneration, depending upon the target being regenerated (eye vs. pharynx).The question explored in this paper is of fundamental importance, and providing an alternative model by which planarian stem cells regenerate missing tissues should be of interest to a broad readership. Unfortunately, in its current form, the manuscript presents enticing preliminary findings, rather than robust experimental observations. Currently, the manuscript has limited samples sizes and experimental replicates, which is unfortunate. Because this paper is attempting to refute a previously published model it is critical that the data are clear and convincing. If not, these findings could be summarily dismissed without appropriate debate. If the authors can show the robustness and rigor of their results, and address the major issues listed below, this manuscript would represent a significant contribution to our understanding of planarian regeneration.1. Throughout the manuscript, experiments were either not repeated, or the number of biological replicates was not reported. In most cases, it appears that experiments were done only once (with the exception of the drug treatments). Numbers of biological replicates and sample sizes should be explicitly stated and the data from different replicates reported for Figures 1D-G, 2B-D, 3C, 3E-F, 4B, 4D-H, 5C-D, and 6A-E.2. The authors do not sufficiently describe their methods for imaging and quantifying cells . The size of the area covered to collect these data is unclear. High-magnification images are shown: are these the areas that were imaged? If so, their results could be biased by choosing small regions of interest. Ideally, the authors should quantify more than one region per animal. Also, they do not describe the depth of the z-stacks collected or how these stacks were normalized/standardized across conditions. All their conclusions hinge on the quantification of progenitor populations in response to different amputation paradigms or chemical treatments, so the standards for imaging and quantification must be clearly reported.3. Inappropriate statistical tests were used throughout. The use of multiple t-tests amplifies the chance of a Type I error and is especially problematic when up to 7 comparisons were made! The authors should use one-way ANOVA with multiple comparison corrections for all experiments with more than two groups.4. Figures 1D-E show that upon pharynx amputation but not head amputation, FoxA+ piwi+ pharynx progenitors increase. These data suffer from the quantification issues highlighted above: how the data were quantified is not sufficiently described, only 3 data points were taken , the experiment appears to have been performed only one time, and the wrong statistical test was used. Rather than reporting the number of FoxA+ piwi-1+ cells counted, the authors should quantify the total number of doubly positive cells as a percentage of piwi-1+ cells, as was previously published . The authors also fail to specify whether the change observed between "3 dpa phx" and "3 dpa head" is significant, which is a material point.5. Figure 2D also suffers from the inadequate quantification practices described above. Ideally, FoxA+ cells should be quantified as a percentage of the H3P+ cells observed.6. The authors use "stem cell", "progenitor", "stem cell progenitor", and "progenitor stem cells" in a mixed and confusing way throughout the paper. For example, in lines 174-175 the authors state that "proliferation of FoxA+ stem cells precedes the increase in pharynx progenitors." This refers to FoxA+ H3P+ cells vs. FoxA+ piwi-1+ cells, but the only difference is that the former are stem cells in the act of mitosis. Is a distinction being made? Elsewhere in the paper, FoxA+ piwi+ cells are referred to as stem cells. The terminology used needs more clarity and consistency.-1 has a more expansive expression pattern and is co-expressed with progenitor markers? If double FISH was performed with the progenitor markers reported , would they all overlap with tgs-1? These experiments need to be performed to make any claims about FoxA expression in the context of pluripotency.Along these lines, what is a FoxA+ PSC (Line 168)? Are the authors suggesting that FoxA is a pluripotency marker? Or are the authors saying that tgs7. In Figure 3C, how does pharynx regeneration occur over such a long period of time after nocodazole treatment in the 1-2 day window? Does target-blind regeneration occur once this window is missed? The authors should repeat analysis of FoxA+ progenitors at later time points in this condition, and/or show rates of BrdU incorporation into the pharynx with and without nocodazole treatment in this window.8. The use of the inhibitor PD in Figure 4 is problematic. No data are shown to verify that phosphorylation of ERK was inhibited in these experiments. A citation of previous use is not sufficient. The effect of PD on WT uncut animals with regards to FoxA+ cells is not shown and is a necessary control. To address questions of drug specificity, the authors should corroborate their findings with a second inhibitor of ERK signaling like U0126, which has already been shown to work in planarians .9. The conclusion that ERK signaling functions in regulating differentiation but not proliferation is premature (line 249-264). Figures 4E-H should be quantified as percentages of piwi-1+ and H3P+ cells, especially since there is decreased proliferation overall with PD treatment.10. The use of head fragments to compare eye regeneration vs. pharynx regeneration is inappropriate. Previous studies have already shown that the absence of eyes is not required to induce ovo+ progenitor amplification . Thus, this is not a surprising result (line 343-345) and the authors are mis-citing previous observations . The region where ovo+ cells was quantified in Figure 6A is not justified or explained. The yellow box is placed in a medial region where ovo+ cells do not normally reside. The authors should image within the laterally positioned ovo+ streams that have been previously described .11. Rather than using head fragments, the authors should repeat the flank resection experiments shown previously . This previous study showed that increased BrdU incorporation into the pharynx occurred following flank resection even though the pharynx was present. That result may have been 1) an artifact of increased BrdU staining due to stimulation of proliferation upon injury, 2) caused by unintentional damage to cells associated with the pharynx, or 3) a response to the loss of FoxA+ progenitor populations that surround the pharynx rather than the loss of the differentiated organ. The authors have the opportunity to revisit this published observation by quantifying the FoxA+ progenitor response during flank resection +/- pharynx. Without these data, this story is incomplete and therefore the conclusion of a targeted regeneration response is not yet convincing.12. The negative results that proliferation and ERK signaling are dispensable for eye regeneration in Figure 6 are weak and unconvincing. The regenerated eyes appear smaller; this should be quantified (number of PRNs per eye). If the small pharynges that form in Figures 3D and 4C are considered a deleterious phenotype, why is the same standard not applied to the eye? Also, existing eye progenitors could have been sufficient for eye regeneration in these drug treatments. Furthermore, eyes did not regenerate after nocodazole treatment 50% of the time. Is it not more likely that the observations reported are dosage and timing artifacts? How has proliferation been affected? These observations do not live up to the claims made.13. The authors claim that ovo+ cells are not proliferative (H3P+) even in cases where there is eye progenitor amplification (head amputation), but the data are not shown (line 321). They should be. Indeed, previous publications have never shown that ovo+ cells proliferate. This might mean that there are proliferating eye progenitors that precede expression of ovo. The authors should discuss this alternative.+ cells into the regenerating pharynx should be higher than in uninjured controls. Conversely, the rate of BrdU incorporation into the regenerating eye should remain unchanged between injured (i.e. eye-resected) and control animals . Once the authors have established the prediction above, they have the opportunity to show the effects of nocodazole, PD, and U0126 on BrdU incorporation in the regenerating eye vs pharynx following eye resection and pharynx amputation in the same animal. This way, the authors can directly test the requirement for proliferation and/or ERK signaling in both tissues.14. The authors claim that eye regeneration does not require proliferation or ERK signaling but pharynx regeneration does. This conclusion hinges on the gross observation that eyes can regenerate in the presence of nocodazole and PD (see point 12 above). These data are coarse and the interpretations are unconvincing. Instead, their model can be directly tested using BrdU pulse-chase experiments. According to the authors' model, one would predict that following pharynx amputation, the rate of incorporation of BrdUReviewer #2:Bohr, Shiroor, and Adler investigate how stem cells respond to the loss of specific tissues in planarians. The planarian stem cell population (neoblasts) are distributed throughout the planarian body and include pluripotent stem cells and a wide range of lineage-committed progenitor cells. How this heterogenous pool of cells behave post-injury or amputation is incompletely understood. The discovery of markers to label stem cell progeny have opened the door to investigate stem cells respond to tissue loss. However, the anatomy of planarians makes it difficult to surgically remove or damage specific organs. The PI of this study developed an assay to remove the pharynx by "chemical amputation" to study the mechanisms underlying regeneration of this organ without drastically perturbing or injuring other tissues. Using this approach, this paper investigates how a well-defined population of FoxA+ progenitors respond to pharynx removal at early time-points during the regeneration. Their data suggest that stem cells are able to detect loss of the pharynx and respond by generating significantly more cells fated to become pharynx, whereas amputation of non-pharyngeal tissues does not have an obvious effect on pharynx progenitor specification dynamics. In addition, using pharmacological treatments, the authors show that cell proliferation and ERK signaling are required for the expansion of pharynx progenitors and cell differentiation. In contrast, other cell types in the planarian eye do not appear to require proliferation or ERK signaling, suggesting that stem cell responses "target blind" as suggested in a previous study, but are rather tuned to specific missing tissues.This work has the potential to make a significant contribution to the field by advancing our understanding of how the planarian heterogenous stem cell population responds to the loss of a specific organ. However, the report is preliminary as presented. It appears that the authors performed many experiments a single time. In addition, description of the methods is insufficient. Consequently, before this work can be considered for publication, the authors need to chiefly demonstrate the reproducibility of the data and robustness of the observations.1. The authors need to replicate experiments to increase the sample size for most experiments.2. Details for imaging and quantification should be explicitly stated in the methods, and the reported cell count numbers should be normalized as appropriate for each set of experiments.-1 (which was only examined at 1 dpa). Could the authors clarify what they think FoxA+ stem cells represent? Are these self-renewing pluripotent stem cells or lineage-committed progenitors? Can the authors get some insight by scanning their images of PH3+ cells expressing FoxA visibly undergoing metaphase? Are daughter cells uniformly FoxA+ as reflected in the model? At least in one of the cells shown in the nocodazole treated controls it appears that both daughter cells express FoxA . I suggest showing some higher magnification images to support the interpretations/conclusions. Others have posited , that not every dividing cell may be a long-term self-renewing stem cell and whether a transient amplifying cells exist or contribute to regeneration in planarians is unknown. Adler and Sánchez Alvarado (2015) discuss the role of transient states and how the transcriptional profiles change in response to regeneration. It wasn't clear to me how the authors think about these cells based on the limited number of experiments and analysis, and there are a few places where the terminology is inconsistent, especially in reference to proliferating ovo+ progenitors (P. 14). The authors need to be clear, and it might be helpful to illustrate their model in one of the early figures or to include it in the final model, which omits tgs-1 due to the limited number of experiments performed with this marker gene .3. Although the authors mention "lineage-tracing" experiments, they do not perform DNA analog pulse-chase experiments to analyze a temporal progression and spatial localization of stem cells to FoxA+ progenitors after pharynx removal. The authors rely on PH3-staining in conjunction with FoxA, and supplementary experiments using the pluripotent stem cell marker tgs4. The pharynx is complex and there is no data to assess what the contribution of other progenitor populations might be. I don't think or think it is unlikely that FoxA+ progenitors are solely responsible for reconstructing the pharynx. The authors should examine how other progenitor populations behave during the process of pharynx regeneration by extending the timeline of progenitor cell analysis. This would reveal if there is fluctuation in progenitor dynamics as animals regenerate the pharynx or re-scale proportions after pharynx regeneration. For example, can the authors test if they are able to detect a contribution of neural progenitors to regeneration of the pharyngeal nervous system? And if so, when during the regeneration process does it take place in the context of their study?Reviewer #3:In this manuscript, Bohr et al. examine how the pluripotent stem cell system of planarians responds to organ-specific damage. If and how the differentiation of specific cell types is dynamically regulated is a conceptually fascinating problem in planarians and in general stem cell research. The authors address this problem by comparing the stem cell response between a single-organ amputation (the pharynx) versus broad tissue loss (decapitation). Their findings indicate that only the removal of the pharynx triggers the increased differentiation of pharyngeal cell types, while the loss of non-pharyngeal tissues upregulates the differentiation of progenitors of multiple organs, but not the pharynx. Further, the authors implicate temporally restricted ERK signaling as a regulatory component in the differentiation of pharyngeal cell types. These observations are also important because they contrast with the previously proposed "target blind" model  that posits the differentiation of different cell types at constant relative proportions, with the rate of stem cell divisions as global production rate regulator. In contrast, the observations by Bohr et al. provide further evidence for more flexibility and specificity within the planarian stem cell system ("target consciousness") in the sense of lineage-specific adjustments in the production rates of specific cell types.eLife.That said, the manuscript generally suffers from an overly narrow focus. Important questions remain regarding the specificity of the stem cell response to pharynx amputation and multiple experiments lack important controls (see below). Moreover, the authors have overlooked that a "target conscious" progenitor response has already been demonstrated by the selective proliferation of protonephridial marker expressing neoblasts in response to protonephridial damage by RNAi . As a result, the current manuscript would require substantial additional experimentation to consolidate its findings sufficiently for publication in 1. Specificity of the stem cell responseThe central premise of the paper is the selective amplification of pharyngeal progenitors in response to pharynx amputation. This the authors conclude on basis of i) an increase in the absolute number of foxA+/piwi-1+ cells in a specific area, while ii) de-capitation has no effect on the absolute number of foxA+/piwi-1+ cells in the same area. This approach is an insufficient demonstration of specificity, as the known phenomenon of wound-induced stem cell activation might also change the absolute number of specific neoblast subclasses and might do so in an injury-dependent manner.To account for this important caveat, the authors need to i) quantify RELATIVE proportions of foxA+/piwi-1+ cells out of total piwi cells  and ii) they need to include other organ progenitors in the initial analysis. The latter is also critical because the pharynx is a complex organ comprising descendants of multiple lineages  and it is not clear whether the foxA+/piwi-1+ cells indeed serve as a singular origin of all constituting lineages (as assumed by the authors), or if they only provide a subset of pharyngeal cells with a rate-limiting role in pharynx assembly . In face of such uncertainty, iii) the quantification of new cell incorporation into the pharynx versus other tissues via BrdU labeling would be necessary to address this caveat and to provide a global perspective on the specificity of the response independent of incompletely characterized marker genes.In addition, the following experimental design problems need to be addressed or better documented, including:• The authors provide insufficient methodological detail on progenitor quantifications, even though the entire manuscript rests on this assay. What are their criteria for scoring a piwi-1+ cell as double-positive for the often weak and noisy lineage labels? If done "by eye", was double-blind scoring used? Were all cells in a given Z-stack counted or only specific planes? If the latter, by which criteria were image planes selected for quantification? How were the specific specimens out of an experimental cohort selected for imaging/quantification? Though not necessarily a unique shortcoming of this particular study, these points simply need to be adequately addressed in order to rigorously support quantitative differences between experimental conditions .• The authors appear not to distinguish at all between technical replicates  versus biological replicates . This is significant, because (i), the use of the standard error of the mean (SEM) that the authors employ throughout is not really an appropriate measure for a single biological replicate with 3 animals – the standard deviation (SD) would seem a more appropriate measure in this context (SD). (ii), the number of worms quantified for each experiment is generally low  given the observed variability in the data . Similarly, for kinetic experiments as in Figure 1H or 2C, it is simply crucial to ensure that the error bars include the variation in response dynamics between multiple replicates due to the drift in the baseline fraction of H3P+ cells or varying staining efficiencies , rather than the technical variation in a single experimental cohort only. Please address these concerns by adding more specimens and a thorough description of the experimental design.2. Timeline of pharynx regenerationThe pharynx regeneration timeline and associated events that the authors present are insufficiently supported by experimental data. The conclusion in line 215 "that proliferation in a critical window of 1 to 2 days after pharynx amputation produces a population of progenitors that are likely essential for pharynx regeneration" rests (i) on the diagnosis of a "proliferative peak of FoxA+ stem cells that occurs after pharynx amputation " (line 202). However, rather than a "proliferative peak", Figure 2C shows a broad "proliferative plateau" of FoxA+ stem cells between 6h and 3 days after amputation. Similarly, the foxA+/H3P+ quantification after pharynx amputation in Figure 4G also displays a lack of a peak of foxA+/H3P+ from 1d to 2d after amputation. (Ii), the associated nocodazole experiments suffer from the fact that the authors did not quantify the impact of the drug on the abundance of foxA+/piwi-1+ cells during treatment intervals from 0-1d and 2-3d after pharynx amputation. Therefore, the authors cannot rule out that nocodazole treatment might have similar effects on the abundance of foxA+/piwi-1+ cells throughout the 1-3 d post-amputation time interval, with the more severe organ-level phenotype of the 1-2d treatment window being caused by some other effect of the drug . Similar concern apply regarding the statement in line 242, "… a window 1-2 days after amputation in which activation of ERK signaling is important for pharynx regeneration." Here, (i) the quantification of the end-stage phenotype of drug treatment during the 1-2d time interval (regain of feeding ability) is missing. (ii) Similarly, the examination of the consequences of PD treatment on foxA+ expression in piwi-1+ cells in panel 4D-H employs drug soaking for 3 days, yet the corresponding end-stage phenotype of 3-day drug treatment is not shown. (iii) the implications of ERK in pharynx regeneration are tentative. Even though the PD compound is initially correctly introduced as "MEK inhibitor", the authors subsequently switch to the factually wrong "ERK inhibitor" designation . Further, additional experimental evidence for the assumed Erk inhibition as the cause of the observed phenotypes would be desirable to rigorously support the conclusion.These caveats need to be addressed if a cell biological timeline is to remain part of this manuscript.3. Integration with the existing literatureThe authors need to better integrate their findings with the literature. First, they need to cite the findings of Vu et al., which explicitly demonstrated a specific increase in the fractional abundance of piwi-1+/protonephridial marker+ cells in response to RNAi-mediated damage to protonephridia. As such, this study already demonstrates the main point of Bohr et al., namely that the planarian stem cell system is capable of "target conscious" progenitor provision. In the very least, the authors should credit these results as additional evidence for their model. A further finding that they should discuss is the demonstration by LoCascio et al.  that flank region cut-outs cause a significant increase in pharynx cell incorporation over baseline, despite the absence of injury to the pharynx. How do the authors reconcile the discrepancy between these data and their own? In general, the discussion would benefit greatly from a more explicit comparison between the "target blind" model versus their data, as well as a broader perspective on the regulation of stem cell homeostasis. All three reviewers recognized the potential significance of this work, but also shared the same concerns about sample sizes, lack of biological replicates, and insufficient details about cell quantification. Given the interest in the question being pursued, if these issues can be addressed satisfactorily, a revised paper could be considered here as a new submission. We have included the reviews below and hope you will find the comments helpful.Reviewer #1:This manuscript by Bohr et al. explores how planarian stem cells respond to the loss of a specific organ: the pharynx. The previously proposed "target-blind" model of planarian regeneration  posited that stem cells do not respond directly to missing tissues, but rather replace missing cell types based on their normal rates of homeostatic turnover. In contrast, Bohr et al. suggest that planarian stem cells can sense and respond to the loss of specific missing tissues, using the pharynx as a case study. The authors conclude that planarians may use more than one mode of regeneration, depending upon the target being regenerated (eye vs. pharynx).The question explored in this paper is of fundamental importance, and providing an alternative model by which planarian stem cells regenerate missing tissues should be of interest to a broad readership. Unfortunately, in its current form, the manuscript presents enticing preliminary findings, rather than robust experimental observations. Currently, the manuscript has limited samples sizes and experimental replicates, which is unfortunate. Because this paper is attempting to refute a previously published model it is critical that the data are clear and convincing. If not, these findings could be summarily dismissed without appropriate debate. If the authors can show the robustness and rigor of their results, and address the major issues listed below, this manuscript would represent a significant contribution to our understanding of planarian regeneration.We thank the reviewer for recognizing the importance of the questions we have pursued here. However, we would like to clarify that our intention was not to refute the previously published model. This confusion likely arose because of a lack of clarity in our writing. In the current version, we explicitly test the ‘non-targeted’ model with EdU incorporation  and flank resection experiments . Our data shows that the ‘targeted’ model is the primary mode by which the pharynx regenerates, but the ‘non-targeted’ model also operates immediately after wounding. We have added an entire section in the discussion to make it plainly apparent to readers that both mechanisms likely operate during regeneration.1. Throughout the manuscript, experiments were either not repeated, or the number of biological replicates was not reported. In most cases, it appears that experiments were done only once (with the exception of the drug treatments). Numbers of biological replicates and sample sizes should be explicitly stated and the data from different replicates reported for Figures 1D-G, 2B-D, 3C, 3E-F, 4B, 4D-H, 5C-D, and 6A-E.We agree that the manuscript suffered from a general lack of experimental replicates and small sample sizes, as addressed in the overview above. Taking into account these essential criticisms, we have repeated each experiment 2-3 times, increased overall sample sizes, and explicitly marked distinct biological and technical replicates on graphs, where possible. We have also included a table detailing biological and technical replicates for each relevant figure.2. The authors do not sufficiently describe their methods for imaging and quantifying cells . The size of the area covered to collect these data is unclear. High-magnification images are shown: are these the areas that were imaged? If so, their results could be biased by choosing small regions of interest. Ideally, the authors should quantify more than one region per animal. Also, they do not describe the depth of the z-stacks collected or how these stacks were normalized/standardized across conditions. All their conclusions hinge on the quantification of progenitor populations in response to different amputation paradigms or chemical treatments, so the standards for imaging and quantification must be clearly reported.We agree that the previous version of the manuscript lacked clarity with regard to imaging and quantification. We have now addressed these important criticisms by clearly describing them in the figure legends and the Image acquisition, quantification and statistical analysis section of the methods (beginning on line 759). We now explicitly state the following standardized parameters for a given experiment: 1) the region analyzed within the animal, 2) the x-y area measured, 3) thickness (z-sections) for a given experiment, and 4) the z-section where quantification began.3. Inappropriate statistical tests were used throughout. The use of multiple t-tests amplifies the chance of a Type I error and is especially problematic when up to 7 comparisons were made! The authors should use one-way ANOVA with multiple comparison corrections for all experiments with more than two groups.As suggested, all figures in which multiple comparisons are made now use one-way ANOVA to determine statistical significance.4. Figures 1D-E show that upon pharynx amputation but not head amputation, FoxA+ piwi+ pharynx progenitors increase. These data suffer from the quantification issues highlighted above: how the data were quantified is not sufficiently described, only 3 data points were taken , the experiment appears to have been performed only one time, and the wrong statistical test was used. Rather than reporting the number of FoxA+ piwi-1+ cells counted, the authors should quantify the total number of doubly positive cells as a percentage of piwi-1+ cells, as was previously published . The authors also fail to specify whether the change observed between "3 dpa phx" and "3 dpa head" is significant, which is a material point.We have addressed the concerns with biological replicates, sample sizes and statistical analysis as described in our responses to points 1 and 3. We have also attempted to indicate when relevant differences are not statistically significant, as is the case for the comparison in question above.FoxA+piwi-1+ cells were represented as a relative percentage . However, similar quantification of ovo+ eye progenitors is represented as absolute numbers rather than percentages . Without clear quantification standards in the literature, we initially evaluated both strategies, but found the results to be very similar. We have included two figures of side-by-side comparisons of these two quantification strategies for key panels from our manuscript (Author response images 1 and 2). Given the complex dynamics of the stem cell population, we feel that representing the data as relative percentages oversimplifies our analysis. Because our progenitor analysis initially compares multiple injury scenarios, we are confident that the changes we see in progenitors occur independently of broad injury responses. Further, representing the data as means of absolute numbers of cells in a standardized area/thickness allows us to indicate individual animals and biological replicates on graphs, providing a more transparent view of the data to readers. Based on these criteria, we have maintained our strategy of representing the data as absolute values, along with extensive additions to the manuscript clarifying where and how the data were obtained.As for representing our data as the absolute number of cells rather than as a proportion, the reviewer is correct in noting that this strategy is a departure from our previous paper, where 5. Figure 2D also suffers from the inadequate quantification practices described above. Ideally, FoxA+ cells should be quantified as a percentage of the H3P+ cells observed.We have addressed this concern as described to points 1-4 above.6. The authors use "stem cell", "progenitor", "stem cell progenitor", and "progenitor stem cells" in a mixed and confusing way throughout the paper. For example, in lines 174-175 the authors state that "proliferation of FoxA+ stem cells precedes the increase in pharynx progenitors." This refers to FoxA+ H3P+ cells vs. FoxA+ piwi-1+ cells, but the only difference is that the former are stem cells in the act of mitosis. Is a distinction being made? Elsewhere in the paper, FoxA+ piwi+ cells are referred to as stem cells. The terminology used needs more clarity and consistency.-1 has a more expansive expression pattern and is co-expressed with progenitor markers? If double FISH was performed with the progenitor markers reported , would they all overlap with tgs-1? These experiments need to be performed to make any claims about FoxA expression in the context of pluripotency.Along these lines, what is a FoxA+ PSC (Line 168)? Are the authors suggesting that FoxA is a pluripotency marker? Or are the authors saying that tgspiwi-1+ or H3P+ cells, 2) we use the term “organ-specific progenitors” to refer to cells double-positive for progenitor markers and either H3P or piwi-1 as labeled in Figure 2 and 3, and 3) we removed all instances of the confusing terms “stem cell progenitor” and “progenitor stem cells” throughout. In addition, we removed the tgs-1 data and any claims about pluripotent stem cells, which we agree were preliminary and inconclusive.We agree that our language, particularly with regard to terminology surrounding stem cells, progenitors, and organ progenitors lacked consistency. We have now modified the language in the following ways: 1) we refer to “stem cells” as 7. In Figure 3C, how does pharynx regeneration occur over such a long period of time after nocodazole treatment in the 1-2 day window? Does target-blind regeneration occur once this window is missed? The authors should repeat analysis of FoxA+ progenitors at later time points in this condition, and/or show rates of BrdU incorporation into the pharynx with and without nocodazole treatment in this window.We agree that it would be valuable to know how animals eventually regenerate after perturbation of pharynx progenitors and whether non-targeted mechanisms take over to slowly regenerate this organ. However, we have struggled to devise an experiment to clearly define these parameters. Nocodazole treatment during the 1-2 day window likely results in culling of pharynx progenitors by apoptosis. Because progenitors are not undergoing mitosis at equivalently high rates in intact animals, nocodazole treatment would not perturb them similarly, making comparisons difficult. Therefore, administration of BrdU during this window could potentially label completely distinct stem cell populations.8. The use of the inhibitor PD in Figure 4 is problematic. No data are shown to verify that phosphorylation of ERK was inhibited in these experiments. A citation of previous use is not sufficient. The effect of PD on WT uncut animals with regards to FoxA+ cells is not shown and is a necessary control. To address questions of drug specificity, the authors should corroborate their findings with a second inhibitor of ERK signaling like U0126, which has already been shown to work in planarians .FoxA+piwi-1+ in intact animals after treatment with PD and UO .We have addressed all the concerns raised above. 1) All MEK inhibitor experiments have been repeated with UO126, with comparable results . 2) A western blot verifies that MEK inhibitor treatment for as little as 24 hours prevents ERK phosphorylation . 3) We now include data showing that exposure to either inhibitor blocks regeneration in tail fragments, indicating that the pharynx regeneration defect is not due to inefficient inhibition . 4) We now include quantification of 9. The conclusion that ERK signaling functions in regulating differentiation but not proliferation is premature (line 249-264). Figures 4E-H should be quantified as percentages of piwi-1+ and H3P+ cells, especially since there is decreased proliferation overall with PD treatment.FoxA+ H3P+ cells and total H3P+ cells are done in the same animals, one can extrapolate that the proportional data would look similar. In fact, a decrease in overall H3P would inflate the relative numbers of FoxA+ H3P+ cells. We have added text to clarify this in the Results section (line 426-429).In the current version of the manuscript we have made every effort to dissect the role of ERK signaling in stem cell division versus differentiation. We have now extensively tested the timeline for ERK requirement in pharynx regeneration. Based on either PD or UO exposure , our data now clearly shows that ERK activity is not required prior to the stem cell division that occurs 1-2 days after pharynx amputation. Further, even though it is true that PD and UO exposure do reduce overall stem cell division, these drug treatments do not impact division of pharynx progenitors . Because analysis of 10. The use of head fragments to compare eye regeneration vs. pharynx regeneration is inappropriate. Previous studies have already shown that the absence of eyes is not required to induce ovo+ progenitor amplification . Thus, this is not a surprising result (line 343-345) and the authors are mis-citing previous observations . The region where ovo+ cells was quantified in Figure 6A is not justified or explained. The yellow box is placed in a medial region where ovo+ cells do not normally reside. The authors should image within the laterally positioned ovo+ streams that have been previously described .We agree that this experiment did not add much to the overall findings, nor did we adequately explain our rationale for including it. It has been removed from this version.11. Rather than using head fragments, the authors should repeat the flank resection experiments shown previously . This previous study showed that increased BrdU incorporation into the pharynx occurred following flank resection even though the pharynx was present. That result may have been 1) an artifact of increased BrdU staining due to stimulation of proliferation upon injury, 2) caused by unintentional damage to cells associated with the pharynx, or 3) a response to the loss of FoxA+ progenitor populations that surround the pharynx rather than the loss of the differentiated organ. The authors have the opportunity to revisit this published observation by quantifying the FoxA+ progenitor response during flank resection +/- pharynx. Without these data, this story is incomplete and therefore the conclusion of a targeted regeneration response is not yet convincing.FoxA+piwi-1+ cells after flank resection , which proves that injuries outside of the pharynx do not trigger an increase in pharynx progenitors. Additionally, we administered EdU at different times (0hr or 24hr) after head or pharynx amputation . We found that cells generated immediately (0hr) after amputation are incorporated broadly, reinforcing the  result that flank resection increases BrdU labeling into the uninjured pharynx. Importantly, by administering EdU 1 day after amputation, we found that cells generated at this time were channeled specifically into regenerating organs, providing evidence supporting a targeted mechanism for regeneration that selectively produces progenitors of missing tissues.We appreciate the reviewer’s suggestion to include this important experiment. We now include quantification of 12. The negative results that proliferation and ERK signaling are dispensable for eye regeneration in Figure 6 are weak and unconvincing. The regenerated eyes appear smaller; this should be quantified (number of PRNs per eye). If the small pharynges that form in Figures 3D and 4C are considered a deleterious phenotype, why is the same standard not applied to the eye? Also, existing eye progenitors could have been sufficient for eye regeneration in these drug treatments. Furthermore, eyes did not regenerate after nocodazole treatment 50% of the time. Is it not more likely that the observations reported are dosage and timing artifacts? How has proliferation been affected? These observations do not live up to the claims made.We have addressed potential issues with dosage and timing by standardizing the exposure times for nocodazole experiments for 2 days, increasing animal numbers, and improving the quantification. Proliferation and ERK activity are only required for eye regeneration in the context of larger amputation, but not after eye resection alone . Regarding the pharynx, laminin is expressed strongly in the pharynx and mouth, and weakly in the body where the pharynx attaches. Even though there is residual laminin staining 7 days after amputation with PD, UO, or nocodazole exposure , this is not at all a ‘pharynx’. The normal architecture, which is apparent in the DMSO controls and in the supplemental figures associated with Figures 4 and 5, is completely lost after these treatments. We have clarified this in the writing (line 290-296). Conversely, eye regeneration in drug-treated animals is undoubtedly more comparable to controls .Eye regeneration is mediated by homeostatic turnover, but whether existing eye progenitors are sufficient to regenerate these small structures after drug treatments remains unclear. We have added text in this section to address this possibility (line 450-453).13. The authors claim that ovo+ cells are not proliferative (H3P+) even in cases where there is eye progenitor amplification (head amputation), but the data are not shown (line 321). They should be. Indeed, previous publications have never shown that ovo+ cells proliferate. This might mean that there are proliferating eye progenitors that precede expression of ovo. The authors should discuss this alternative.ovo+ H3P+ cells in intact animals and after head amputation with or without nocodazole treatment to enrich for cells in mitosis . We were only able to detect dividing ovo+ cells after nocodazole treatment, but did not observe any increase after head amputation as compared to intact nocodazole treated controls. As suggested, we added discussion of the possible explanations to this section (line 265-267).We have now included images for + cells into the regenerating pharynx should be higher than in uninjured controls. Conversely, the rate of BrdU incorporation into the regenerating eye should remain unchanged between injured (i.e. eye-resected) and control animals . Once the authors have established the prediction above, they have the opportunity to show the effects of nocodazole, PD, and U0126 on BrdU incorporation in the regenerating eye vs pharynx following eye resection and pharynx amputation in the same animal. This way, the authors can directly test the requirement for proliferation and/or ERK signaling in both tissues.14. The authors claim that eye regeneration does not require proliferation or ERK signaling but pharynx regeneration does. This conclusion hinges on the gross observation that eyes can regenerate in the presence of nocodazole and PD (see point 12 above). These data are coarse and the interpretations are unconvincing. Instead, their model can be directly tested using BrdU pulse-chase experiments. According to the authors' model, one would predict that following pharynx amputation, the rate of incorporation of BrdU+ cells into either the regenerating pharynx or the brain . However, we have reserved the pharmacological perturbations for future work.We agree with the reviewer that this is an important experiment, and we have now addressed the different contributions of EdUReviewer #2:Bohr, Shiroor, and Adler investigate how stem cells respond to the loss of specific tissues in planarians. The planarian stem cell population (neoblasts) are distributed throughout the planarian body and include pluripotent stem cells and a wide range of lineage-committed progenitor cells. How this heterogenous pool of cells behave post-injury or amputation is incompletely understood. The discovery of markers to label stem cell progeny have opened the door to investigate stem cells respond to tissue loss. However, the anatomy of planarians makes it difficult to surgically remove or damage specific organs. The PI of this study developed an assay to remove the pharynx by "chemical amputation" to study the mechanisms underlying regeneration of this organ without drastically perturbing or injuring other tissues. Using this approach, this paper investigates how a well-defined population of FoxA+ progenitors respond to pharynx removal at early time-points during the regeneration. Their data suggest that stem cells are able to detect loss of the pharynx and respond by generating significantly more cells fated to become pharynx, whereas amputation of non-pharyngeal tissues does not have an obvious effect on pharynx progenitor specification dynamics. In addition, using pharmacological treatments, the authors show that cell proliferation and ERK signaling are required for the expansion of pharynx progenitors and cell differentiation. In contrast, other cell types in the planarian eye do not appear to require proliferation or ERK signaling, suggesting that stem cell responses "target blind" as suggested in a previous study, but are rather tuned to specific missing tissues.This work has the potential to make a significant contribution to the field by advancing our understanding of how the planarian heterogenous stem cell population responds to the loss of a specific organ. However, the report is preliminary as presented. It appears that the authors performed many experiments a single time. In addition, description of the methods is insufficient. Consequently, before this work can be considered for publication, the authors need to chiefly demonstrate the reproducibility of the data and robustness of the observations.1. The authors need to replicate experiments to increase the sample size for most experiments.We have addressed this concern, as detailed in the overview above. All experiments have been repeated, increasing the sample sizes across the board. We have also included a table detailing biological and technical replicates for each relevant figure.2. Details for imaging and quantification should be explicitly stated in the methods, and the reported cell count numbers should be normalized as appropriate for each set of experiments.We have addressed this concern, as detailed in the overview above. Briefly, imaging and quantification methods are now described in detail within the figure legends and methods section entitled Image acquisition, quantification and statistical analysis (beginning on line 759). Regions analyzed were either of standard area or normalized to area, and for a given confocal imaging experiment, the numbers of z-sections were kept consistent.-1 (which was only examined at 1 dpa). Could the authors clarify what they think FoxA+ stem cells represent? Are these self-renewing pluripotent stem cells or lineage-committed progenitors? Can the authors get some insight by scanning their images of PH3+ cells expressing FoxA visibly undergoing metaphase? Are daughter cells uniformly FoxA+ as reflected in the model? At least in one of the cells shown in the nocodazole treated controls it appears that both daughter cells express FoxA . I suggest showing some higher magnification images to support the interpretations/conclusions. Others have posited , that not every dividing cell may be a long-term self-renewing stem cell and whether a transient amplifying cells exist or contribute to regeneration in planarians is unknown. Adler and Sánchez Alvarado (2015) discuss the role of transient states and how the transcriptional profiles change in response to regeneration. It wasn't clear to me how the authors think about these cells based on the limited number of experiments and analysis, and there are a few places where the terminology is inconsistent, especially in reference to proliferating ovo+ progenitors (P. 14). The authors need to be clear, and it might be helpful to illustrate their model in one of the early figures or to include it in the final model, which omits tgs-1 due to the limited number of experiments performed with this marker gene .3. Although the authors mention "lineage-tracing" experiments, they do not perform DNA analog pulse-chase experiments to analyze a temporal progression and spatial localization of stem cells to FoxA+ progenitors after pharynx removal. The authors rely on PH3-staining in conjunction with FoxA, and supplementary experiments using the pluripotent stem cell marker tgsWe appreciate the suggestion to clarify our language surrounding stem cells, pluripotent stem cells, and progenitors. To address the important point regarding the temporal progression of pharynx progenitors, we now include DNA analog (EdU) pulse-chase experiments to analyze the output of stem cells generated either immediately after amputation, or 1 day later when we see a specific increase in pharynx progenitors. This experiment  shows that stem cells produced 1 day after amputation are selectively incorporated into the regenerating pharynx. As addressed in the overview above, point 4, we have made our terminology throughout the manuscript more consistent.tgs-1 data, we removed this figure and any claims about pluripotent stem cells from this version of the manuscript. Although we do not have data to definitively indicate the potency of FoxA+ progenitors, we now include images of anaphase cells that suggest that FoxA can segregate into daughter cells both symmetrically and asymmetrically . We have added a section addressing these issues to the discussion, because we agree that investigating these questions is important (lines 511-528).We agree with the reviewer regarding the complexity of the stem cell population, which includes both long-term self-renewing cells and progenitors. Because we could not make clear conclusions based on the 4. The pharynx is complex and there is no data to assess what the contribution of other progenitor populations might be. I don't think or think it is unlikely that FoxA+ progenitors are solely responsible for reconstructing the pharynx. The authors should examine how other progenitor populations behave during the process of pharynx regeneration by extending the timeline of progenitor cell analysis. This would reveal if there is fluctuation in progenitor dynamics as animals regenerate the pharynx or re-scale proportions after pharynx regeneration. For example, can the authors test if they are able to detect a contribution of neural progenitors to regeneration of the pharyngeal nervous system? And if so, when during the regeneration process does it take place in the context of their study?FoxA+ progenitors are the sole source of pharyngeal tissue or only generate a subset of critical cells. The pharynx is indeed a complex organ and we would love to know whether FoxA is a master regulator of them all. To address this point, we have knocked down every progenitor included in this paper and every proposed pharynx progenitor from the literature  and none of them seem to play any role in pharynx regeneration . The lack of a detectable phenotype from these markers has made us reluctant to draw conclusions from their dynamics during pharynx regeneration.The reviewer is correct in noting that we still lack definitive proof of whether Reviewer #3:In this manuscript, Bohr et al. examine how the pluripotent stem cell system of planarians responds to organ-specific damage. If and how the differentiation of specific cell types is dynamically regulated is a conceptually fascinating problem in planarians and in general stem cell research. The authors address this problem by comparing the stem cell response between a single-organ amputation (the pharynx) versus broad tissue loss (decapitation). Their findings indicate that only the removal of the pharynx triggers the increased differentiation of pharyngeal cell types, while the loss of non-pharyngeal tissues upregulates the differentiation of progenitors of multiple organs, but not the pharynx. Further, the authors implicate temporally restricted ERK signaling as a regulatory component in the differentiation of pharyngeal cell types. These observations are also important because they contrast with the previously proposed "target blind" model  that posits the differentiation of different cell types at constant relative proportions, with the rate of stem cell divisions as global production rate regulator. In contrast, the observations by Bohr et al. provide further evidence for more flexibility and specificity within the planarian stem cell system ("target consciousness") in the sense of lineage-specific adjustments in the production rates of specific cell types.That said, the manuscript generally suffers from an overly narrow focus. Important questions remain regarding the specificity of the stem cell response to pharynx amputation and multiple experiments lack important controls (see below). Moreover, the authors have overlooked that a "target conscious" progenitor response has already been demonstrated by the selective proliferation of protonephridial marker expressing neoblasts in response to protonephridial damage by RNAi . As a result, the current manuscript would require substantial additional experimentation to consolidate its findings sufficiently for publication in eLife.We appreciate the reviewer’s recognition of our work and their thoughtful criticisms. In the revised version we have added new controls, included the important reference of Vu et al., 2015, and extensively discussed the conflicting models for regeneration (referred to in our manuscript as ‘targeted’ vs ‘non-targeted’). Addressing these criticisms has resulted in a substantially more clear manuscript that adds to our understanding of the mechanisms underlying regeneration.1. Specificity of the stem cell responseThe central premise of the paper is the selective amplification of pharyngeal progenitors in response to pharynx amputation. This the authors conclude on basis of i) an increase in the absolute number of foxA+/piwi-1+ cells in a specific area, while ii) de-capitation has no effect on the absolute number of foxA+/piwi-1+ cells in the same area. This approach is an insufficient demonstration of specificity, as the known phenomenon of wound-induced stem cell activation might also change the absolute number of specific neoblast subclasses and might do so in an injury-dependent manner.To account for this important caveat, the authors need to i) quantify RELATIVE proportions of foxA+/piwi-1+ cells out of total piwi cells  and ii) they need to include other organ progenitors in the initial analysis. The latter is also critical because the pharynx is a complex organ comprising descendants of multiple lineages  and it is not clear whether the foxA+/piwi-1+ cells indeed serve as a singular origin of all constituting lineages (as assumed by the authors), or if they only provide a subset of pharyngeal cells with a rate-limiting role in pharynx assembly . In face of such uncertainty, iii) the quantification of new cell incorporation into the pharynx versus other tissues via BrdU labeling would be necessary to address this caveat and to provide a global perspective on the specificity of the response independent of incompletely characterized marker genes.In addition, the following experimental design problems need to be addressed or better documented, including:• The authors provide insufficient methodological detail on progenitor quantifications, even though the entire manuscript rests on this assay. What are their criteria for scoring a piwi-1+ cell as double-positive for the often weak and noisy lineage labels? If done "by eye", was double-blind scoring used? Were all cells in a given Z-stack counted or only specific planes? If the latter, by which criteria were image planes selected for quantification? How were the specific specimens out of an experimental cohort selected for imaging/quantification? Though not necessarily a unique shortcoming of this particular study, these points simply need to be adequately addressed in order to rigorously support quantitative differences between experimental conditions .• The authors appear not to distinguish at all between technical replicates  versus biological replicates . This is significant, because i), the use of the standard error of the mean (SEM) that the authors employ throughout is not really an appropriate measure for a single biological replicate with 3 animals – the standard deviation (SD) would seem a more appropriate measure in this context (SD). ii), the number of worms quantified for each experiment is generally low  given the observed variability in the data . Similarly, for kinetic experiments as in Figure 1H or 2C, it is simply crucial to ensure that the error bars include the variation in response dynamics between multiple replicates due to the drift in the baseline fraction of H3P+ cells or varying staining efficiencies , rather than the technical variation in a single experimental cohort only. Please address these concerns by adding more specimens and a thorough description of the experimental design.We thank the reviewer for highlighting these important caveats with our interpretation of our data. In point 2 of the introduction to this response, and in Author response figures 1 and 2, we have included a side-by-side comparison of absolute vs relative quantification and show that for key figures in our manuscript, the two strategies yield very similar results. In addition, because we are comparing different wounding paradigms that induce general wound responses, our experiments are internally controlled for wound-induced proliferation.FoxA+ progenitors are the sole source of pharyngeal tissue or only generate a subset of critical cells . To address this point, we have knocked down every progenitor used in this paper and every proposed pharynx progenitor from the literature . None of them cause defects in pharynx regeneration . In the current version of the manuscript, we also include pulse-chase labeling with EdU to demonstrate that stem cells are indeed selectively incorporated into the regenerating pharynx, depending on when they proliferate relative to tissue removal . However, we acknowledge the caveat that FoxA may only control the production of a subset of cells. Ongoing work not included here is aimed at addressing this caveat.The reviewer is correct in noting that we still lack definitive proof of whether We have also corrected all of the flaws the reviewer has pointed out related to replicates and statistical analysis in this version as outlined in the introduction to this response, point 1. Briefly, these include: 1) clarifying and standardizing methods for imaging and quantification, 2) distinguishing biological vs technical replicates in all experiments, 3) replaced error bars representing SEM with error bars representing standard deviation, and 4) increased number of animals in every experiment.2. Timeline of pharynx regenerationThe pharynx regeneration timeline and associated events that the authors present are insufficiently supported by experimental data. The conclusion in line 215 "that proliferation in a critical window of 1 to 2 days after pharynx amputation produces a population of progenitors that are likely essential for pharynx regeneration" rests i) on the diagnosis of a "proliferative peak of FoxA+ stem cells that occurs after pharynx amputation " (line 202). However, rather than a "proliferative peak", Figure 2C shows a broad "proliferative plateau" of FoxA+ stem cells between 6h and 3 days after amputation. Similarly, the foxA+/H3P+ quantification after pharynx amputation in Figure 4G also displays a lack of a peak of foxA+/H3P+ from 1d to 2d after amputation. Ii), the associated nocodazole experiments suffer from the fact that the authors did not quantify the impact of the drug on the abundance of foxA+/piwi-1+ cells during treatment intervals from 0-1d and 2-3d after pharynx amputation. Therefore, the authors cannot rule out that nocodazole treatment might have similar effects on the abundance of foxA+/piwi-1+ cells throughout the 1-3 d post-amputation time interval, with the more severe organ-level phenotype of the 1-2d treatment window being caused by some other effect of the drug . Similar concern apply regarding the statement in line 242, "… a window 1-2 days after amputation in which activation of ERK signaling is important for pharynx regeneration." Here, i) the quantification of the end-stage phenotype of drug treatment during the 1-2d time interval (regain of feeding ability) is missing. ii) Similarly, the examination of the consequences of PD treatment on foxA+ expression in piwi-1+ cells in panel 4D-H employs drug soaking for 3 days, yet the corresponding end-stage phenotype of 3-day drug treatment is not shown. iii) the implications of ERK in pharynx regeneration are tentative. Even though the PD compound is initially correctly introduced as "MEK inhibitor", the authors subsequently switch to the factually wrong "ERK inhibitor" designation . Further, additional experimental evidence for the assumed Erk inhibition as the cause of the observed phenotypes would be desirable to rigorously support the conclusion.These caveats need to be addressed if a cell biological timeline is to remain part of this manuscript.FoxA+ H3P+ peak may more closely resemble a shield volcano than a Matterhorn, regardless of terminology, there is an obvious and significant elevation in proliferation that occurs within two days of pharynx amputation. This conclusion has been strengthened by reviewers’ suggestions to boost sample size and alter statistical analysis methods. We now include additional characterization of FoxA+piwi-1+ pharynx progenitors after nocodazole treatment 0-1 and 2-3 days after pharynx amputation , reinforcing our claims that proliferation occurring 1-2 days after amputation is critical for pharynx progenitor production. Additionally, this 1-2 day window is concurrent with the timing of selective incorporation of EdU+ into the regenerating pharynx, supporting our conclusion that this 1-2 day proliferative window is essential for pharynx progenitor production and subsequent regeneration.We thank the reviewer for their insightful criticisms. While we agree that the Regarding ERK experiments, we have addressed every point raised by the reviewer by expanding our MEK inhibitor experiments to more thoroughly test when ERK is required during pharynx regeneration. This includes standardizing the length of MEK inhibitor treatments and performing many additional controls to show that multiple MEK inhibitors both prevent phosphorylation of ERK and lead to the same outcomes in regeneration. To bolster evidence supporting our timeline, we have done the following:We now include data for the end-stage phenotype following both 1-2 day and 0-3 day PD exposure , which both show a delay in regeneration. We also exposed animals to PD or the alternative MEK inhibitor U0126 for 24 hour intervals from 0-1, 1-2 or 2-3 days after pharynx amputation, and analyzed pharynx progenitor abundance. These experiments show that ERK inhibition between 1-2 days after amputation specifically impacts the increase of pharynx progenitors . Together, these experiments have strengthened our argument that ERK is required for pharynx progenitor production and pharynx regeneration.Figure 5—figure supplement 1B).We have modified our language to refer to the drug inhibitors as MEK inhibitors, but continue to refer to ERK inhibition, as application of MEK inhibitors does inhibit ERK activity  and phosphorylation, as shown in our western blot . As such, this study already demonstrates the main point of Bohr et al., namely that the planarian stem cell system is capable of "target conscious" progenitor provision. In the very least, the authors should credit these results as additional evidence for their model. A further finding that they should discuss is the demonstration by LoCascio et al.  that flank region cut-outs cause a significant increase in pharynx cell incorporation over baseline, despite the absence of injury to the pharynx. How do the authors reconcile the discrepancy between these data and their own? In general, the discussion would benefit greatly from a more explicit comparison between the "target blind" model versus their data, as well as a broader perspective on the regulation of stem cell homeostasis.We thank the reviewer for this suggestion, and apologize for this oversight of our colleagues’ work. In the revised version, we have more thoroughly described and contextualized our findings with the existing literature. We added sections in the introduction, discussion, and figures that specifically address the contrast between existing models for regeneration (referred to in our manuscript as targeted vs non-targeted).FoxA+piwi-1+ cells after flank resection , but do not observe any increase. We have reconciled this finding with the existing literature by performing EdU labeling after different amputations, somewhat similar to those performed in , but at different times. These experiments reveal that a targeted mechanism channels cells towards missing tissues one day after amputation , but that a non-targeted mechanism also contributes to regeneration, potentially if small amounts of tissues have been damaged (like eye resections). The first section of the discussion extensively describes these two models and how they may be utilized in planarian regeneration.We have also included experiments analyzing