Tnfaip2/exoc3‐driven lipid metabolism is essential for stem cell differentiation and organ homeostasis

Abstract Lipid metabolism influences stem cell maintenance and differentiation but genetic factors that control these processes remain to be delineated. Here, we identify Tnfaip2 as an inhibitor of reprogramming of mouse fibroblasts into induced pluripotent stem cells. Tnfaip2 knockout impairs differentiation of embryonic stem cells (ESCs), and knockdown of the planarian para‐ortholog, Smed‐exoc3, abrogates in vivo tissue homeostasis and regeneration—processes that are driven by somatic stem cells. When stimulated to differentiate, Tnfaip2‐deficient ESCs fail to induce synthesis of cellular triacylglycerol (TAG) and lipid droplets (LD) coinciding with reduced expression of vimentin (Vim)—a known inducer of LD formation. Smed‐exoc3 depletion also causes a strong reduction of TAGs in planarians. The study shows that Tnfaip2 acts epistatically with and upstream of Vim in impairing cellular reprogramming. Supplementing palmitic acid (PA) and palmitoyl‐L‐carnitine (the mobilized form of PA) restores the differentiation capacity of Tnfaip2‐deficient ESCs and organ maintenance in Smed‐exoc3‐depleted planarians. Together, these results identify a novel role of Tnfaip2 and exoc3 in controlling lipid metabolism, which is essential for ESC differentiation and planarian organ maintenance.

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Kind regards, Esther
Esther Schnapp, PhD Senior Editor EMBO reports Referee #1: In this manuscript, Deb and colleagues address the function of the Exocyst component Tnfaip2/Exoc3 in stem cell reprogramming and differentiation. Their findings include that the knockdown of TNFAIP in culture mouse cells increases fibroblast reprogramming efficiency and inhibits ES cell differentiation. These phonotypes correlate with reduced lipid droplet formation and the supplementation of storage lipid precursors rescues differentiation-associated Tnfaip loss of function phenotypes. In parallel, the authors examine the function of the planarian Exoc3 gene orthologue and report an apparent requirement in stem cell differentiation, which can also be rescued by FA supplementation. On basis of these findings, the authors conclude that they have identified an evolutionarily conserved pathway with important roles in stem cell differentiation and tissue regeneration.
I find the findings of the manuscript genuinely interesting, also for the broad readership of EMBO reports. The probing of deep evolutionary conservation of gene function via functional investigation of the planarian gene homologue adds a further interest dimension to this manuscript. However, some of the key conclusions are insufficiently supported by data. I am not an expert on vertebrate stem cell cultures, hence the predominant focus of my comments on the planarian experiments.
Major points 1) Tnfaip2 gene function in mouse: The broad involvement of the exocyst protein complex in many cellular functions requires additional evidence that the observed phenotypes are indeed all associated with lipid metabolism. The authors should present RNAseq data to support that the apparent FA-induced rescue of differentiation in Fig 6B-C is reflected in the rescue of the differentiation-associated gene expression profiles in Fig. 4A. In parallel, the effect of the FA supplementation on reprogramming efficiency could be examined.
2) The authors conclude deep evolutionary conservation of Tnfaip/Exoc3 gene function by inferring defects in stem cell differentiation in mouse stem cells and planarians. However, the planarian data in Fig 3 insufficiently supports a differentiation defect in planarians. Criticism: An increase in H3Ppositive cells alone could also reflect a mitotic arrest or an optical artefact resulting from the projection of an unchanged or even decreased density of mitotic stem cells onto the much reduced area of the strongly contracted animals (Fig. 3B); The phenotypes shown in Fig. 3E-G are either broadly associated with general defects in the planarian stem cell system (E, F) or not convincing (G-this image does not support a protonephridial defect); the supplemental RNAseq data is limited to a small number of inconclusive and obviously hand-picked genes (see below for detailed points). Therefore, additional data is required to support the conclusion that Exoc3 is required specifically for stem cell differentiation in planarians. Minimally, this requires the demonstration that stem cells accumulate at the expense of differentiating progenitors. Time course experiments would be particularly useful for providing a clearer view of direct versus indirect effects. If the authors cannot provide such data, the text needs to be generalized to state that Smed-exoc3 is required for stem cell driven tissue homeostasis in planarians, which might include effects on differentiation.
3) The authors conclude that Exoc3 in both systems affects stem cell differentiation via the modulation of lipid stores. However, the FA injection rescue (see below for additional criticism) provides the only tentative support for the link between Exoc3 and lipid storage in planarians. The authors therefore need to directly examine the effect of planarian Exoc3(RNAi) on planarian lipid storage. This isn't overly difficult, as protocols for lipid droplet visualization in planarians have been published or the authors could use their own mass spec assay in Fig. 5E to quantify storage lipid levels in control versus Exoc3(RNAi) animals. Such data are strategically important also as additional support for the validity of the rescue experiment.
4) The abstract and text claim the discovery of a new pathway. However, the majority of the data pertains to a single gene (Tnfaip2/Exoc3), the mechanistic connection to lipid droplets remains tentative and as stated in the text, lipid droplets have already been implicated in stem cell differentiation. The "new pathway" terminology is therefore a bit of an overstatement and the respective passages should be rephrased accordingly. Further, the text of the current manuscript is not in a submission-ready state and requires substantial editing (see below).

Minor points
Manuscript: 5) Multiple cut/paste scars persist and some sections are overly convoluted and hard to understand-please polish the text into a submission-ready state and edit for grammar mistakes. 6) The planarian data is poorly integrated, especially in the discussion. Please include some form of comparison between the two experimental systems used in the manuscript, the additional insights that the multi-pronged approach permits and some of the caveats regarding the interpretation of the planarian data (e.g., direct effects in stem cells (as implied by the text) vs. indirect effects of the organism-wide knock-down in other tissues (e.g., in the lipid storing intestine)).
Rescue assay: 7) The complete restoration of progeny cell marker levels after FA injection is remarkable. However, the low number of 3/19 animals that regenerated properly raises concerns in conjunction with the apparently small sample size (5?) for the qPCR assays. If the qPCR samples were to have been biased for "good looking" animals, the apparent rescue might actually be due to RNAi escapers. Please state explicitly how the samples for qPCR were selected and/or add additional experimental data points to alleviate such concern-this point is crucial in conjunction with the direct demonstration of Exoc3 effects on planarian lipid storage (point 3).
RNAseq analysis: 8) S Fig.3b generates the visual impression of a large number of analyzed genes that all follow the same pattern. However, this is NOT the case, as only 5 genes are analyzed. Please filter for isoforms of the same transcript (_1,...2,...3,...4 suffix-see PlanMine user manual) and display only 1 isoform per gene (common practice in the field is the use of the longest isoform). 9) In the case of "H2B" the authors likely analyze two separate genes (different transcript ID; see PlanMine usermanual). Please state which of the two gene sequences corresponds to the isoform that was functionally validated in the previous RNAseq isoforms. 10) Please include a range of bona-fide stem cell markers into the analysis/figure, i,.e., piwi-1, -2, pcna, bruli...; the more, the better). Rather than hand-picking a few genes, the point that stem cell markers are generally up and differentiation markers are generally down would be most strongly supported by querying one of the published global stem cell/progeny gene sets in this experiment.
Terminology: 11) Please do not use "endo/meso/ectoderm" in reference to planarian stem cell lineages-the implied homology has not been established. 12) "Smed-exoc-3(RNAi) displayed a good knock down efficiency"-please re-phrase with proper scientific terminology.
Referee #3: In this manuscript, the authors report that lipid metabolism mediated by Tnfaip2/Exoc3 plays the key role for stem cell differentiation and organ maintenance. Knockdown of Tnfaip2 was found to promote the iPSCs generation, however, suppression of Tbfaip2 or its planarian orthologure, Smed-exoc3, in stem cells abrogated differentiation and regeneration. Mechanistically, Tnfaip2 deficiency led to deregulation of TAG synthesis and reduced the expression of Vimentin that is responsible for lipid droplets formation. While the major findings in this study are potentially interesting, the author did not provide sufficient evidence to support the conclusion. 1. shTnfaip2s were found to enhance the somatic reprogramming process, however, the author also observed that Tnfaip2-deficient ESCs exhibited differentiation failure. It is not clear that Tnfaip2 functions to prevent or promote the acquirement and maintenance of pluripotency. The author should examine the expression level of Tnfaip2 change during somatic cell reprogramming and ESCs differentiation. 2. In this study, Tnfaip2 seemed to play the conflicting roles in somatic reprogramming and stem cell differentiation. It would be interesting to investigate whether the iPSCs that generated from MEFs with OSKM and shTnfaip2 induction exhibit differentiation deficiency. 3. As shown in Figure 6, the supplementation of palmitic acid and palmitoyl-L-carnitine rescued the differentiation defects of Tnfaip2/exoc3-deficient pluripotent stem cells in culture and in vivo in planarian. The authors concluded that Vim-dependent lipid droplet (LD) formation mediated by Tnfaip2/exoc3 was critical for ESC differentiation. However, they did not provide sufficient evidence to support the conclusion. Since Vimentin also is a regulator of EMT process that is well-known to be critical for somatic reprogramming or stem cell differentiation, the author may wish to go further to prove that Vimentin deficiency lead to failure in lipid droplet formation and then ESCs differentiation. 4. The authors claimed Tnfaip2 regulated the expression of Vimentin and Cpt1a. It's better to provide the results of qRT-PCR and western blot besides the proteomics data. 5. Another critical missing part is how Tnfaip2/exoc3 regulate the protein levels of Vimentin or Cpt1a. In Supplementary Figure 6E, the author claimed that there was no additive effect in the enhancement of reprogramming efficiency upon co-depletion of Tnfaip2 and Vim indicating that Tnfaip2 and Vim act especially in this regard. It is not surprising that knockdown of Vimentin, the regulator of EMT, significantly increased the somatic reprogramming efficiency. The author may wish to provide more evidence to claim Vimentin is the downstream effector of Tnfaip2. It is not clear whether Vimentin could rescue the differentiation defect of ESCs or somatic stem cells with Tnfaip2/exoc3. 6. In Figure 3C&D, Smed-exoc3 knockdown increased the numbers of proliferating stem cells in planarian. The authors explained that Smed-exoc3 knockdown led to the self-renewal versus differentiation of stem cells. Nevertheless, it is known that the asymmetrical and symmetrical division is essential for stem cell maintenance and tissue regeneration. The authors may wish to address whether stem cells with Smed-exoc3 knockdown prefer symmetrical division rather than asymmetrical division and whether the lipid drop formation affects the division pattern.
In this manuscript, Deb and colleagues address the function of the Exocyst component Tnfaip2/Exoc3 in stem cell reprogramming and differentiation. Their findings include that the knock-down of TNFAIP in culture mouse cells increases fibroblast reprogramming efficiency and inhibits ES cell differentiation. These phonotypes correlate with reduced lipid droplet formation and the supplementation of storage lipid precursors rescues differentiation-associated Tnfaip loss of function phenotypes. In parallel, the authors examine the function of the planarian Exoc3 gene orthologue and report an apparent requirement in stem cell differentiation, which can also be rescued by FA supplementation. On basis of these findings, the authors conclude that they have identified an evolutionarily conserved pathway with important roles in stem cell differentiation and tissue regeneration.
I find the findings of the manuscript genuinely interesting, also for the broad readership of EMBO reports. The probing of deep evolutionary conservation of gene function via functional investigation of the planarian gene homologue adds a further interest dimension to this manuscript. However, some of the key conclusions are insufficiently supported by data. I am not an expert on vertebrate stem cell cultures, hence the predominant focus of my comments on the planarian experiments.
Major points 1) Tnfaip2 gene function in mouse: The broad involvement of the exocyst protein complex in many cellular functions requires additional evidence that the observed phenotypes are indeed all associated with lipid metabolism. The authors should present RNAseq data to support that the apparent FA-induced rescue of differentiation in Fig 6B-C is reflected in the rescue of the differentiation-associated gene expression profiles in Fig. 4A. In parallel, the effect of the FA supplementation on reprogramming efficiency could be examined.
Response: We thank the reviewer for his/her positive comments on our manuscript and for the valuable comments, which we all addressed in the revised manuscript and which significantly improved our study.
We followed the reviewer's suggestion and included a new RNA-sequencing analysis on the rescue of differentiation of Tnfaip2 -/-EBs by fatty acid (FA) treatment. The analysis shows that FA treatment rescues the expression of stemness/differentiation regulating genes (new Figure  3G; new Expanded View 6A and 6B and new Appendix Table 10) that are dysregulated in Tnfaip2 -/-EBs versus WT EBs (revised Figure 2E-former Figure 4A and revised Expanded View 4A and 4Bformer Supplementary Figure 5B and 5C). We highlight the differentially regulated stemness/differentiation genes that were differentially expressed in Tnfaip2 -/-EBs versus WT EBs but rescued in Tnfaip2 -/-EBs by FA-treatment (marked with asterisk in new Figure 3G  We also addressed the role of FA in reprogramming Specifically, we show that supplementation of PA/PC during cellular reprogramming inhibits generation of iPSCs, which is in line with prodifferentiation influence of PA/PC. Similarly, we now demonstrated that treatment with Etomoxir, a CPT1A-specific inhibitor, improves reprogramming efficiency (new Figure 4F and 4G) (2 nd paragraph on page 21 of the result section).
2) The authors conclude deep evolutionary conservation of Tnfaip/Exoc3 gene function by inferring defects in stem cell differentiation in mouse stem cells and planarians. However, the planarian data in Fig 3 insufficiently supports a differentiation defect in planarians. Criticism: An increase in H3P-positive cells alone could also reflect a mitotic arrest or an optical artefact resulting from the projection of an unchanged or even decreased density of mitotic stem cells onto the much reduced area of the strongly contracted animals (Fig. 3B); The phenotypes shown in Fig. 3E-G are either broadly associated with general defects in the planarian stem cell system (E, F) or not convincing (G-this image does not support a protonephridial defect); the supplemental RNAseq data is limited to a small number of inconclusive and obviously handpicked genes (see below for detailed points). Therefore, additional data is required to support the conclusion that Exoc3 is required specifically for stem cell differentiation in planarians. Minimally, this requires the demonstration that stem cells accumulate at the expense of differentiating progenitors. Time course experiments would be particularly useful for providing a clearer view of direct versus indirect effects. If the authors cannot provide such data, the text needs to be generalized to state that Smed-exoc3 is required for stem cell driven tissue homeostasis in planarians, which might include effects on differentiation.
Response: We thank the reviewer for his/her insightful comment. We followed the suggestion of this reviewer and have included a new time course experiment analyzing the number of stem cells (X1 population) in Smed-exoc3 depleted planarians versus controls at different time points after starting the iRNA treatment (day-30, -34, and -38). In addition, we also monitored the expression of 5 stem cell marker genes in RNA extracted from total planarians at the same timepoints. The results show an increase in stem cell marker gene expression (new Expanded View 3A) and an increase in the number of X1 cells (new Expanded View 3B and 3C) in planarians that were injected with iRNA against Smed-exoc3 versus planarians injected with a control iRNA against gfp. This increase was gradually developing over time (1 st paragraph on page 11 of the result section). In addition, we also conducted new experiments to analyze whether the number of differentiated cells would decrease in Smed-exoc3-treated planarians versus control iRNA treated planarians. These experiments revealed a decrease in differentiated cells (new Expanded View 3B and 3D; page 12 of the result section) as well as a decline in the expression of 2 differentiation markers in response to Smed-exoc3 depleted planarians (new Appendix Figure 1C; 1 st paragraph on page 10 of the result section). The reviewer is right that a mitotic arrest could also lead to an increase in the X1 population. As X1 cells are mostly in G2/M and the number of X1 increases in Smed-exoc3 depleted planarian vs. wildtype, it is difficult to employ cell cycle analysis to discriminate G2/M arrest from an increase in X1 neoblast cells. However, an increase G2/M arrested cells usually coincides with an increase in apoptosis. We included an analysis of apoptosis (Annexin V positive cell) in the X1 population, which did not reveal any evidence for an increase in apoptosis (new Appendix Figure 3A) (3 rd paragraph on page 13 of the result section). Besides, the expression of the planarian homologue of mammalian p53/p63 a bona fide mitotic checkpoint genewas not found to be significantly decreased in our RNA-sequencing analysis of exoc3(RNAi)-treated planarians versus controls (page 14, first paragraph of the revised manuscript). Together, these data suggested that the increase in X1 population in exoc3(RNAi)-treated planarians reflected a differentiation failure rather than a mitotic arrest.
3) The authors conclude that Exoc3 in both systems affects stem cell differentiation via the modulation of lipid stores. However, the FA injection rescue (see below for additional criticism) provides the only tentative support for the link between Exoc3 and lipid storage in planarians. The authors therefore need to directly examine the effect of planarian Exoc3(RNAi) on planarian lipid storage. This isn't overly difficult, as protocols for lipid droplet visualization in planarians have been published or the authors could use their own mass spec assay in Fig. 5E to quantify storage lipid levels in control versus Exoc3(RNAi) animals. Such data are strategically important also as additional support for the validity of the rescue experiment.
Response: We thank the reviewer for this suggestion. We have taken a quantitative approach to measure the lipidomics profile of exoc3(RNAi) planarians compared to the control gfp(RNAi) counterparts (new Figure 5A). Like Tnfaip2 -/-EBs, exoc3(RNAi) planarians were found to exhibit profound loss of Triacylglycerides (TAG) in comparison to control counterpart (bottom of page 21 of the revise manuscript).
4) The abstract and text claim the discovery of a new pathway. However, the majority of the data pertains to a single gene (Tnfaip2/Exoc3), the mechanistic connection to lipid droplets remains tentative and as stated in the text, lipid droplets have already been implicated in stem cell differentiation. The "new pathway" terminology is therefore a bit of an overstatement and the respective passages should be rephrased accordingly. Further, the text of the current manuscript is not in a submission-ready state and requires substantial editing (see below).
Response: We thank the reviewer for this comment. Also, the other reviewers have asked us to delineate the Exoc3/Tnfaip2 -Vim pathway more clearly. We have done so and our new experiments indeed confirm that Tnfaip2 -Vim act epistatically in regulating lipid metabolism and stem cell maintenance and differentiation (see new Figure 4D-G, Appendix Figure 5A-C, see also responses to 3 rd and 4 th comments of reviewer 3 below). Based on these new data, we think that it is justified to describe these findings as a new pathway that regulates lipid droplet formation/lipid metabolism and stem cell differentiation (Page 19-21 of the result section).

Minor points
Manuscript: 5) Multiple cut/paste scars persist and some sections are overly convoluted and hard to understand-please polish the text into a submission-ready state and edit for grammar mistakes.
This has been corrected in the revised manuscript.
6) The planarian data is poorly integrated, especially in the discussion. Please include some form of comparison between the two experimental systems used in the manuscript, the additional insights that the multi-pronged approach permits and some of the caveats regarding the interpretation of the planarian data (e.g., direct effects in stem cells (as implied by the text) vs. indirect effects of the organism-wide knock-down in other tissues (e.g., in the lipid storing intestine)).
Response: We followed the reviewer's suggestion and have included following paragraph in the revised discussion: "Unlike in mouse or in other mammals, wherein pluripotent stem cells are restricted to early phases of embryonic development, pluripotent somatic stem cells are present in planarians throughout their life to ensure homeostasis and regeneration of all tissues of the worms. Thus, planarians provide a unique in vivo model system to study the function of genes and pathways in regulating pluripotent somatic stem cells in vivo. In vertebrates, organ regeneration and homeostasis are driven by organ specific, adult stem cells with restricted differentiation capacity, including multipotent, oligopotent, or unipotent stem cells. The current study shows that it is possible to use planarians to delineate conserved gene functions as well as gene specification that control the function of pluripotent, embryonic stem cells in culture as well as pluripotent, somatic stem cells at the organism level (in planarians). It seems promising to go on to use this approach to identify genetically controlled mechanisms that control the function of restricted, somatic stem cells in more complex organisms such as vertebrates and mammals." (bottom of page 24 of the revised manuscript).
Rescue assay: 7) The complete restoration of progeny cell marker levels after FA injection is remarkable. However, the low number of 3/19 animals that regenerated properly raises concerns in conjunction with the apparently small sample size (5?) for the qPCR assays. If the qPCR samples were to have been biased for "good looking" animals, the apparent rescue might actually be due to RNAi escapers. Please state explicitly how the samples for qPCR were selected and/or add additional experimental data points to alleviate such concern-this point is crucial in conjunction with the direct demonstration of Exoc3 effects on planarian lipid storage (point 3).
Response: We thank the reviewer for this suggestion. Initially the numbers in the figures depicted the number of planarians that retained the defective phenotype (indicating for example that 3/20 exoc3(RNAi)-treated planarians showed a defective phenotype instead of labeling it as 17/20 showing restored phenotype). We have corrected this labeling as suggested by the reviewer and also included additional animals for the analysis and hence now 20 out of 23 (20/23) exoc3(RNAi)-treated planarians exhibited restoration of differentiation potential. In addition, we have involved a second independent experimentalist to evaluate the number of animals showing the depicted phenotype. The corrected figures are included in the revised manuscript.
RNAseq analysis: 8) S Fig.3b generates the visual impression of a large number of analyzed genes that all follow the same pattern. However, this is NOT the case, as only 5 genes are analyzed. Please filter for isoforms of the same transcript (_1,...2,...3,...4 suffix-see PlanMine user manual) and display only 1 isoform per gene (common practice in the field is the use of the longest isoform). 9) In the case of "H2B" the authors likely analyze two separate genes (different transcript ID; see PlanMine usermanual). Please state which of the two gene sequences corresponds to the isoform that was functionally validated in the previous RNAseq isoforms. 10) Please include a range of bona-fide stem cell markers into the analysis/figure, i,.e., piwi-1, -2, pcna, bruli...; the more, the better). Rather than hand-picking a few genes, the point that stem cell markers are generally up and differentiation markers are generally down would be most strongly supported by querying one of the published global stem cell/progeny gene sets in this experiment.
Response: We thank the reviewer for the advice on improving the depiction of our RNAsequencing analysis of X1 cells from Smed-exoc3 depleted planarians and controls (gfp-RNAi treated). We now provide a completely unbiased analysis on the enrichment of stem cell and progenitor cell related genes in our data set. Specifically, we compared DEGs of Smed-exoc3 depleted X1 versus control treated (gfp-RNAi) planarians from our RNA-sequencing experiment with  Figure 1D and E and page 12 of the revised manuscript). These data support the interpretation that exoc3(RNAi) disturbs the differentiation of neoblast cells.
Terminology: 11) Please do not use "endo/meso/ectoderm" in reference to planarian stem cell lineages-the implied homology has not been established.
Review: As per the suggestion, we have removed these terms throughout the text and from the relevant figures (revised Figure 2A  12) "Smed-exoc-3(RNAi) displayed a good knock down efficiency"-please re-phrase with proper scientific terminology.
Response: We changed the text as follows "RT-qPCR analysis of total (whole body derived) RNA from exoc3(RNAi)-treated planarians compared to controls revealed a good knockdown of Smed-exoc3"(1 st paragraph on page 9 of the result section). Referee #2: The Manuscript by Deb et al entitled "Tnfaip2/Exoc3 driven lipid metabolism is essential for stem cell differentiation & organ homeostasis" involves an RNAi screen that aims at identifying genes that impair the generation of mouse induced pluripotent stem (iPS) cells, revealing Tnfaip2 to enhances formation of iPS cells. Tnfaip2 is a target of inflammatory TNFa and NFkB pathways. To study the function of the gene in a pluripotency context, a knockdown of the planarian Tnfaip2 orthologue exoc3 has been performed. Downregulation of exoc3 yielded an increased planarian neoblast stem cell pool whereas differentiation is blocked. Moreover, by CRISPR/Cas genome engineering a murine Tnfaip2 knockout ES cell line was generated. The study reports that these KO cells do show an aberrant differentiation phenotype as judged by embryoid body (EB) formation assay. Furthermore, the study aims at deciphering the putative mechanism of Tnfaip2 loos-of-function and claims that vimentin expression is controlled by Tnfaip2. This is stated to result in differentiation defects by impairing lipid metabolism. Supplementation of free fatty acids to the cells appears to rescue the differentiation defects of Tnfaip2 KO cells.
The study is clearly written and very well organized. The materials and methods section is sound and seems to be sufficiently comprehensive to allow reproduction of the results. Also the results are clearly structured and well presented. The study would have benefited from more comprehensive studies of mutants at cellular level. E.g. the Tnfaip2 KO is poorly described and no data on functional validation given. The differentiation analyses is based on EB formation and follows a very short protocol of 6 days only. In this respect Fig. 4A might be misleading. Global RNA-seq data is presented that is appropriately being analyzed. However, this has not been carried out in the rescue experiment (Fig. 6), which is solely based on EB formation and a quite rough quantification of morphological charactersitics (rosettes of surrounding EBs), representing a rather preliminary state of these findings. A more comrprehsive staining against germ layer markers enabling insight into particular differentiation defects would be more informative.
Response: We thank the reviewer for his/her positive comments on our manuscript and for the valuable comments, which we all addressed in the revised manuscript and which significantly improved our study.
We followed the reviewer's suggestion and included a new RNA-sequencing analysis on the rescue of differentiation of Tnfaip2 -/-EBs by fatty acid (FA) treatment (new Figure 3G; new Expanded View 6A and 6B and new Appendix Table 10; 1 st paragraph on page 19 of the result section). The analysis shows that FA treatment rescues the expression of stemness/differentiation regulating genes that are dysregulated in Tnfaip2 -/-EBs versus WT EBs (revised Figure 2Eand revised Expanded View 4A and 4B). We highlight the differentially regulated stemness-and differentiation-associated genes that were differentially expressed in Tnfaip2 -/-EBs versus WT EBs but rescued in Tnfaip2 -/-EBs by FA-treatment (marked with asterisk in new Figure 3G and new Expanded View 6A and 6B). We have also included immunostaining images with differentiation markers of ectoderm and mesoderm lineages like Brachyury (T) and SOX1 respectively to demonstrate repression of these markers at protein level in cells in Tnfaip2 -/-EBs as compared to WT EBs (new Expanded View 4C and page 15 of the revised manuscript). We conducted similar immunofluorescence analysis to demonstrate activation of the T and SOX1 upon supplementation of PA/PC to Tnfaip2 -/-EBs undergoing in vitro differentiation (new Expanded View 6C; 1 st paragraph on page 19 of the revised manuscript).
Furthermore, in response to reviewer-1, we also analyzed the role of FA in reprogramming. Specifically, we now show that in line with pro-differentiation influence of PA/PC, treatment with Etomoxir, a CPT1A-specific inhibitor, improves reprogramming efficiency (new Figure 4F and 4G) (2 nd paragraph on page 21 of the revised manuscript).
Altogether, we think that our revised data more clearly describe the phenotype of the mutant ES cells versus WT and provide further support for the role of In this manuscript, the authors report that lipid metabolism mediated by Tnfaip2/Exoc3 plays the key role for stem cell differentiation and organ maintenance. Knockdown of Tnfaip2 was found to promote the iPSCs generation, however, suppression of Tbfaip2 or its planarian orthologure, Smed-exoc3, in stem cells abrogated differentiation and regeneration. Mechanistically, Tnfaip2 deficiency led to deregulation of TAG synthesis and reduced the expression of Vimentin that is responsible for lipid droplets formation. While the major findings in this study are potentially interesting, the author did not provide sufficient evidence to support the conclusion. 1. shTnfaip2s were found to enhance the somatic reprogramming process, however, the author also observed that Tnfaip2-deficient ESCs exhibited differentiation failure. It is not clear that Tnfaip2 functions to prevent or promote the acquirement and maintenance of pluripotency. The author should examine the expression level of Tnfaip2 change during somatic cell reprogramming and ESCs differentiation.
Response: We thank the reviewer for his/her positive comments on our manuscript and for the valuable comments, which we all addressed in the revised manuscript and which significantly improved our study.
Following the insightful comment of the reviewer, we have analyzed the expression of Tnfaip2 during somatic cell reprogramming and ESC. The experiments show that the expression of Tnfaip2 increases at later time points during cellular reprogramming, while in iPSCs we detected a profound repression of Tnfaip2 (new Figure 1E; 2 nd paragraph on page 7 of the result section). On the other hand, Tnfaip2 was found to be activated upon formation of EBs and during the first days of in vitro differentiation (new Figure 2F; 1 st paragraph on page 15 of the result section).
2. In this study, Tnfaip2 seemed to play the conflicting roles in somatic reprogramming and stem cell differentiation. It would be interesting to investigate whether the iPSCs that generated from MEFs with OSKM and shTnfaip2 induction exhibit differentiation deficiency.
Response: The reviewer is right that it is possible that the knockdown of Tnfaip2 could also compromise the differentiation capacity of the iPSCs. However, the knockdown efficiency of Tnfaip2 was only 70-80% in our studies (revised Expanded View 1Bformer Supplementary Figure 1C). The abrogation of differentiation potential by Tnfaip2 knockdown may in fact be incomplete in these shRNA studies. We think that the experiments on Tnfaip2 knockout ESCs versus WT controls provide a cleaner experimental system to address this question. We have thus extended the molecular analysis of the Tnfaip2 KO ESCs vs WT ESCs to gain further insights in the functional role of Tnfaip2 in differentiation (see new Figure 3G; new Expanded View 6A and 6B and new Appendix Table 10 and page 19 of the revised manuscript). Figure 6, the supplementation of palmitic acid and palmitoyl-L-carnitine rescued the differentiation defects of Tnfaip2/exoc3-deficient pluripotent stem cells in culture and in vivo in planarian. The authors concluded that Vim-dependent lipid droplet (LD) formation mediated by Tnfaip2/exoc3 was critical for ESC differentiation. However, they did not provide sufficient evidence to support the conclusion. Since Vimentin also is a regulator of EMT process that is well-known to be critical for somatic reprogramming or stem cell differentiation, the author may wish to go further to prove that Vimentin deficiency lead to failure in lipid droplet formation and then ESCs differentiation.

As shown in
Response: We thank the reviewer for his/her insightful comment. To further substantiate the conclusion the Tnfaip2/Exoc3 act through Vim-mediated lipid metabolism contributes to the differentiation defect, we have now included a series of new experiments into the revised manuscript: (i) We analyzed triacyl glycerol (TAG) in Smed-exoc3-depleted planarians compared to controls. The experiment shows a profound reduction in TAGs as in Tnfaip2 deficient ESC compared to WT controls (new Figure 5A) (3 rd paragraph on page 21 of the result section), (ii) We conducted RNA-sequencing analysis on the rescue of differentiation of Tnfaip2 -/-EBs by fatty acid (FA) treatment (new Figure 3G; new Expanded View 6A and 6B and new Appendix Table 10). The analysis shows that FA treatment rescues the expression of stemness/differentiation regulating genes that are dysregulated in Tnfaip2 -/-EBs versus WT EBs (revised Figure 2E-former Fig. 4A and revised Expanded View 4A and 4Bformer Supplementary Figure 5B and 5C). We highlight the differentially regulated stemness/differentiation genes that were differentially expressed in Tnfaip2 -/-EBs versus WT EBs but rescued in Tnfaip2 -/-EBs by FAtreatment (marked with asterisk in new Figure 3G and new Expanded View 6A and 6B) (1 st paragraph on page 19 of the result section). (iii) We combined knockdown of Tnfaip2 with knockdown of Vim to analyze whether the knockdown of both genes would have additive effects in enhancing reprogramming of MEFs into iPSCs (revised Figure 4C and Expanded View 6F -Former Supplementary Figure 6E and 6F). This is not the case supporting the conclusion that Tnfaip2 dependent effect on Vim act in the same pathway to increase reprogramming of MEFs into iPSCs (2 nd paragraph on page 19 of the result section). (iv) We combined knockdown and overexpression of Vim and Tnfaip2 to analyze whether the two genes act epistatically in influencing reprogramming of MEFs into iPSCs. The experiment shows that, the knockdown of Tnfaip2 fails to enhance reprogramming when combined with overexpression of Vim. In contrast, the knockdown of Vim enhances reprogramming in the presence of Tnfaip2 overexpression (new Figure 4D and 4E). These results support the conclusion that Vim acts downstream of Tnfaip2 in inhibiting reprogramming (page 20 of the result section).
Together, our new data support the conclusion the Tnfaip2/Vim mediated lipid metabolism impairs reprogramming of MEFs into iPSCS and is required for differentiation of ESCs in EB cultures. We agree with the reviewer that Vim mediated EMT may also be involved in the observed phenotypes. It could even be possible Tnfaip2/Vim mediated lipid metabolism is involved in the regulation of EMT by Vim. We included this thought in the revised discussion (3 rd paragraph on page 23 of the discussion section).
4. The authors claimed Tnfaip2 regulated the expression of Vimentin and Cpt1a. It's better to provide the results of qRT-PCR and western blot besides the proteomics data. Figure 4B; 2 nd paragraph on page 16 of the result section). Moreover, we included the expression profile of Vim and Tnfaip2 upon depletion of Tnfaip2 and Vim respectively (new Appendix Figure 5A and 5B). The experiments show that knockdown of Tnfaip2 led to suppression of Vim (new Appendix Figure 5A), whereas the knockdown of Vim didn't have a significant effect on Tnfaip2 expression (new Appendix Figure 5B; page 20 of the result section).

Response: We followed the reviewer's suggestion and analyzed Vim expression in Tnfaip -/-EBs during EB differentiation by RT-qPCR (new Appendix
We have also included RT-qPCR based analysis of Cpt1a expression in Tnfaip2 -/-EBs during EB differentiation (new Expanded View 5D). The analysis shows a reduction of Cpt1a expression (new Expanded View 5D, 3 rd paragraph on page 18 of the result section). Overall these data support the conclusion that Tnfaip2 regulates Vim and Cpt1a expression.
In addition, the revised manuscript also provides functional prove for Cpt1a dependent effect of Tnfaip2 on enhancing the reprogramming efficiency of MEFs into iPSCs. A new experiment was conducted to treat shScr infected MEFs and shTnfaip2 infected MEFs that were also infected with a reprogramming vector with Etomoxir, a specific Cpt1a inhibitor. While treatment of shScr MEFs with Etomoxir enhanced reprogramming efficiency to a similar extent as the shTnfaip2infection of MEFs, treatment with Etoxomir had no effect on the reprogramming of shTnfaip2infected MEFs (new Figure 4F and 4G). Together, these data support the conclusion that the induction of Cpt1a contributes to Tnfaip2-mediated suppression of reprogramming (2 nd paragraph on page 21 of the result section).
5. Another critical missing part is how Tnfaip2/exoc3 regulate the protein levels of Vimentin or Cpt1a. In Supplementary Figure 6E, the author claimed that there was no additive effect in the enhancement of reprogramming efficiency upon co-depletion of Tnfaip2 and Vim indicating that Tnfaip2 and Vim act especially in this regard. It is not surprising that knockdown of Vimentin, the regulator of EMT, significantly increased the somatic reprogramming efficiency. The author may wish to provide more evidence to claim Vimentin is the downstream effector of Tnfaip2. It is not clear whether Vimentin could rescue the differentiation defect of ESCs or somatic stem cells with Tnfaip2/exoc3.
Response: We thank the reviewer for his/her insightful comment. We included a new experiment on reprogramming to functionally address this question. We combined the knockdown of Vim or Tnfaip2 with the overexpression of Tnfaip2 or Vim to analyze whether the two genes act epistatically and which one is upstream in influencing reprogramming of MEFs into iPSCs. The experiment shows that Vim acts downstream of Tnfaip2 in inhibiting reprogramming (new Figure  4D and 4E). Specifically, the knockdown of Tnfaip2 fails to enhance reprogramming when combined with overexpression of Vim. In contrast, the knockdown of Vim enhances reprogramming in the presence of Tnfaip2 overexpression (new Figure 4D and 4E). These results support the conclusion that Vim acts downstream of Tnfaip2 in inhibiting reprogramming (page 20 of the result section).
6. In Figure 3C&D, Smed-exoc3 knockdown increased the numbers of proliferating stem cells in planarian. The authors explained that Smed-exoc3 knockdown led to the self-renewal versus differentiation of stem cells. Nevertheless, it is known that the asymmetrical and symmetrical division is essential for stem cell maintenance and tissue regeneration. The authors may wish to address whether stem cells with Smed-exoc3 knockdown prefer symmetrical division rather than asymmetrical division and whether the lipid drop formation affects the division pattern.
Response: This is an excellent point and a very interesting question. However, the current technology in the field of planarian research does not allow us to address this question. The reviewer has sparked our interest in this question, and we are currently planning to move into genetic mouse studies to address this question. In mice, it is possible to get definitive answers on asymmetric vs. symmetric stem cell division by using transplantation assays, such as the paireddaughter assay making use of single cell transplants of hematopoietic stem cell that have undergone one cell division in culture. We are interested in doing this but we cannot conduct this under the time frame of the current study and hope for the understanding of the reviewer. 1st Revision -Editorial Decision Dear Lenhard, Thank you for the submission of your revised manuscript. We have now received the comments from both referees and I am happy to tell you that both are overall happy with the revisions of your study.
However, referee 1 still raises several important points that will all need to be addressed for publication of your manuscript here. I would thus like to invite you to address these remaining concerns and send us a newly revised manuscript as soon as possible.
A few other changes will also be required: Given that important data seem to be present in the supplementary information, I suggest that you add more main figures and change the format of your manuscript to a full-length Article with separate results and discussion sections.
We do agree with referee 1 that the possibilities for adding extra/supplementary information to our manuscripts might be confusing, and we are currently working on this issue. I suggest that your excel files with new data in it should be called Dataset EV1, etc. You can have as many EV Datasets, tables and movies as required, but the number of EV figures is restricted to a maximum of 5. Also, you can move all other supplementary data to the Appendix pdf file, but they need to fit into a pdf file, and no large excel tables can be included. The Appendix also needs a table of content with page numbers and the figures are called Appendix Figure S1, etc. Please correct all figures names and cite all items in the main manuscript file. You can also find all info about our file types in our guide to authors online.
Please reduce the list of keywords to 5.
In the authors contributions there are 2 AGs, which should be differentiated by using the 2nd letter of the surname.
Our reference format has changed to Harvard style, please correct.
I attach to this email a related manuscript file with comments from our data editors. Please address all comments in the final manuscript.
All publicly deposited data -such as the shRNA sequences data -must be accessible before the online publication of your study.
Please avoid overstatements in the abstract.
EMBO press papers are accompanied online by A) a short (1-2 sentences) summary of the findings and their significance, B) 2-3 bullet points highlighting key results and C) a synopsis image that is 550x200-600 pixels large (the height is variable). You can either show a model or key data in the synopsis image. Please note that text needs to be readable at the final size. Please send us this information along with the revised manuscript.
I look forward to seeing a final form of your manuscript when it is ready.

Best regards, Esther
Esther Schnapp, PhD Senior Editor EMBO reports Referee #1: The revised version of the manuscript includes substantial amounts of new data and text revisions. Important improvements include the additional cell culture RNAseq data, the quantification of triglycerides in exoc3(RNAi) planarians and text changes that better integrate the two model systems. However, the manuscript is still not publication-ready. Remaining issues include: 1) The split of the supplemental material into "Expanded View" and "Appendix Figures" is entirely unserviceable to both reviewers and potential readers-irrespective of whether this originates with the journal or the authors, the final version of this manuscript needs to have a single, consolidated list of supplemental figures.
2) I remain skeptical that the claim of "new pathway discovery" is justifiable on basis of the data. To me at least, a "pathway" is a chain of multiple cause-consequence interactions that collectively explain a cellular phenotype. To claim a pathway on basis of two proteins seems a bit of a stretch, especially if their mechanistic interplay is entirely obscure and quite possibly indirect, as in the case of vim and tnfaip2.
The conceptual lynchpin of the planarian section, namely the demonstration of a stem cell differentiation failure in exoc3(RNAi) planarians, still remains rather circumstantial. The added analyses are lacking important controls or are poorly presented and the bits of relevant data are buried deeply within the supplement. Specific problems include: 3) The FACS quantification that the authors added to corroborate the accumulation of stem cells at the expense of stem cell progeny/differentiated cells is lacking essential controls. Without an irradiation control to verify the positioning of the X2/Xins gates and the low number of replicates relative to the inter-replicate variation, apparent population shifts are just as likely to reflect the shift in the X1 population or technical noise, rather than a depletion of "differentiated" cells. The apparently 7.3% Xins cells in exoc3(RNAi) animals in the day 34 FACS plot shown are a case in point, as an animal comprised of ~93% stem cells and early progeny would hardly be able to maintain the degree of anatomical organization evident in Fig. 1G or 2A-C. Bottom line: As is, the data insufficiently supports the claimed decrease in "differentiated" cells. Instead, the authors might want to consider integrating some of the FACS data into figure 1 as additional evidence for the relative accumulation of stem cells.

4)
Although the qPCR analysis of progeny markers NB.21.11e (now generally referred to as prog-1 by the community-the authors might want to cite the relevant study) indeed documents an initial decrease of prog-1 expression, expression recovers at later time points in spite of the continued increase in stem cell marker genes -this is inconsistent with the assumed depletion of differentiating cells at the expense of stem cells and this caveat needs to be acknowledged.

5)
The new experiments meant to support that the increased number of H3P-positive cells reflects an increase in the numbers of normally cycling stem cells rather than abnormal cycling of an unchanged number of stem cells, are inconclusive at best. The level of apoptosis within the mitotic cell population can hardly provide strong evidence for one or the other and without a positive control, it remains doubtful whether the author's FACS assay would have sufficient sensitivity for picking up a small and transient fraction of apoptotic cells amongst the sorted X1 cells. Similarly, the lack of upregulation of the "bona-fide mitotic checkpoint gene p53/p63" simply cannot be cited as evidence without a prior demonstration that planarian p53/p63 is indeed upregulated in mitosis. In short: These data weaken the manuscript, rather than strengthening it.
6) Once again, please drop the claim of reduced protonephridial density on basis of the images in Fig. 2B and appendix Fig. 2-protonephridia remain present and the difference in protonephridial density in the shown images are within the variation range that can be expected on basis of technical noise/inter-animal variation or that can be attributed to the evident head regression. Plus, protonephridial defects tend to manifest in edema, which is not evident in Fig. 1G. Bottom line: Either remove this data or present quantitative evidence in the sense of numbers of protonephridial units/projected area.

7)
Possibly the strongest evidence for a stem cell differentiation defect in exoc3(RNAi) is buried within the expanded RNAseq analyses in the appendix figure and the appendix tables. I write possibly, because as is, the data is imply shard to fathom. Specifically: a. Please specify whether the exoc3(RNAi) X1 fractions were compared to X1 fractions of GFP(RNAi) controls or to whole GFP(RNAi) animals-this remains unclear and evidently important for the interpretation of the data. b. Please label the bar graphs with the specific gene category, rather than the study authors (the refs should go in the figure legend). Moreover, the text remains murky regarding the original definition of the "differentiation gene sets"-this is essential information for judging the relevance of the analysis and the authors cannot expect readers to scrutinize the original publications for this information. c. Provided that the analyzed gene sets are indeed enriched for bona-fide differentiation associated genes, the authors need to present the data in a way that directly visualizes the downor upregulation of specific genes (rather than merely the fraction of differentially expressed genes within the set, as in the present figures). The heat maps in the stem cell sections of the manuscript provide one example of how to accomplish this. d. I remain puzzled as to why the authors restricted their analysis to FACS sorted neoblasts, rather than simply comparing gene expression differences between whole exoc3(RNAi)/GFP(RNAi) animals. This would have afforded a direct opportunity to quantify global up/down regulation of stem cell genes and/or progeny markers, while the present X1 analysis can only detect relative shifts within the population. e.g., differences in cell cycle progression...
Though the link between exoc3 and planarian stem cell differentiation remains weak, the quantitative depletion of triglycerides and the phenotypic rescue by FA supplementation nicely complements the vertebrate stem cell data. I can therefore cautiously recommend publication of a substantially revised manuscript, provided that the authors i) re-work their added planarian exoc3(RNAi) RNAseq analysis to incorporate the above concerns; ii) ensure that the salient bits of data are in the main figures and not in supplement; iii) omit the inconclusive support pertaining to a lack of cell cycle effects (p53/p63; annexin FACS plots); iv) undertake appropriate text revisions to ensure congruency between data shown and claims made (e.g., already in the abstract: "...Smed-exoc3 abrogates in vivo differentiation of somatic stem cells...").
Minor point: 8) Once again, my request for "Scientific terminology" refers to QUANTITATIVE statements-please replace "good knock-down" by something along the lines of "... reduced mRNA levels by > 70 % (reference to figure) in comparison to untreated controls". 9) Please omit the statement that you carried out a "lipidomic" analysis-the targeted quantification of triglycerides is not a lipidome analysis.
Referee #3: The authors have addressed all my concerns. The reviewer has no more question.
The revised version of the manuscript includes substantial amounts of new data and text revisions. Important improvements include the additional cell culture RNAseq data, the quantification of triglycerides in exoc3(RNAi) planarians and text changes that better integrate the two model systems. However, the manuscript is still not publication-ready. Remaining issues include: 1) The split of the supplemental material into "Expanded View" and " 2) I remain skeptical that the claim of "new pathway discovery" is justifiable on basis of the data. To me at least, a "pathway" is a chain of multiple cause-consequence interactions that collectively explain a cellular phenotype. To claim a pathway on basis of two proteins seems a bit of a stretch, especially if their mechanistic interplay is entirely obscure and quite possibly indirect, as in the case of vim and tnfaip2.
Response: We have addressed this point throughout the manuscript by: (i) referring to a "new mechanism" instead of a "new pathway" that controls ES cell differentiation and organ maintenance in planarians (see yellow highlights in the abstract and 2 nd paragraph and throughout the manuscript). and (ii) avoiding statements on stem cell differentiation in planarians by rather referring to defects in organ maintenance (see yellow highlights in the abstract and 2 nd paragraph and throughout the manuscript). The reviewer is right that cell cycle arrest could also affect the production of differentiated cells. As these two processes are interconnected (proliferation and differentiation) and since we cannot exclude the possibility of cell cycle being involved in the observed phenotypes, we have reworded the manuscript throughout.
The conceptual lynchpin of the planarian section, namely the demonstration of a stem cell differentiation failure in exoc3(RNAi) planarians, still remains rather circumstantial. The added analyses are lacking important controls or are poorly presented and the bits of relevant data are buried deeply within the supplement. Specific problems include: 3) The FACS quantification that the authors added to corroborate the accumulation of stem cells at the expense of stem cell progeny/differentiated cells is lacking essential controls. Without an irradiation control to verify the positioning of the X2/Xins gates and the low number of replicates relative to the inter-replicate variation, apparent population shifts are just as likely to reflect the shift in the X1 population or technical noise, rather than a depletion of 27th Oct 2020 2nd Authors' Response to Reviewers "differentiated" cells. The apparently 7.3% Xins cells in exoc3(RNAi) animals in the day 34 FACS plot shown are a case in point, as an animal comprised of ~93% stem cells and early progeny would hardly be able to maintain the degree of anatomical organization evident in Fig. 1G or 2A-C. Bottom line: As is, the data insufficiently supports the claimed decrease in "differentiated" cells. Instead, the authors might want to consider integrating some of the FACS data into figure 1 as additional evidence for the relative accumulation of stem cells.
Response: We followed the reviewer's suggestion and integrated the FACS data on the relative number of neoblast stem cell increases in Smed-exoc3-depleted planarians into the main figure ( Figure 3C). Other FACS data were removed. 4) Although the qPCR analysis of progeny markers NB.21.11e (now generally referred to as prog-1 by the community-the authors might want to cite the relevant study) indeed documents an initial decrease of prog-1 expression, expression recovers at later time points in spite of the continued increase in stem cell marker genes -this is inconsistent with the assumed depletion of differentiating cells at the expense of stem cells and this caveat needs to be acknowledged.
Response: We followed the reviewer's suggestion: In the revised manuscript NB.21.11e and NB.32.1g are referred to as prog-1 and prog-2, respectively. Regarding the statement on the expression level of prog-1 (NB.21.11e) at later time points (Appendix Figure 1C now Figure  3F), we respectfully disagree with the reviewer indicating that this result would be inconsistent with our interpretation of the data. We agree that there is an increase in the expression of prog-1 at later time point after RNAi (day 38). However, the level of prog-1 expression in Smed-exoc3-depleted worms at this timepoint still remains significantly lower than the level in control worms (p=0.0096, new Figure 3F). Thus, the result on the impaired expression of this marker gene of differentiation stands very much in line with reduced generation of differentiated cells in Smed-exoc3-depleted worms versus controls.

5)
The new experiments meant to support that the increased number of H3P-positive cells reflects an increase in the numbers of normally cycling stem cells rather than abnormal cycling of an unchanged number of stem cells, are inconclusive at best. The level of apoptosis within the mitotic cell population can hardly provide strong evidence for one or the other and without a positive control, it remains doubtful whether the author's FACS assay would have sufficient sensitivity for picking up a small and transient fraction of apoptotic cells amongst the sorted X1 cells. Similarly, the lack of upregulation of the "bona-fide mitotic checkpoint gene p53/p63" simply cannot be cited as evidence without a prior demonstration that planarian p53/p63 is indeed upregulated in mitosis. In short: These data weaken the manuscript, rather than strengthening it.
Response: We followed the reviewer's suggestion and removed this part of the FACS analysis as well as the p53 data from the revised manuscript. 6) Once again, please drop the claim of reduced protonephridial density on basis of the images in Fig. 2B and appendix Fig. 2-protonephridia remain present and the difference in protonephridial density in the shown images are within the variation range that can be expected on basis of technical noise/inter-animal variation or that can be attributed to the evident head regression. Plus, protonephridial defects tend to manifest in edema, which is not evident in Fig. 1G. Bottom line: Either remove this data or present quantitative evidence in the sense of numbers of protonephridial units/projected area.
Response: We followed the advice of the reviewer to remove this part of the analysis of impaired organ homeostasis in Smed-exoc3-depleted planarians, since the remaining data on the depletion of neuronal cells (new Figure 4A) and photosensitive cells (new Figure 4B) are fully sufficient to make this point. 7) Possibly the strongest evidence for a stem cell differentiation defect in exoc3(RNAi) is buried within the expanded RNAseq analyses in the appendix figure and the appendix tables. I write possibly, because as is, the data is imply shard to fathom. Specifically: a. Please specify whether the exoc3(RNAi) X1 fractions were compared to X1 fractions of GFP(RNAi) controls or to whole GFP(RNAi) animals-this remains unclear and evidently important for the interpretation of the data.
Response: We clarified the description that we indeed compared exoc3(RNAi) X1 fractions to gfp(RNAi) X1 fractions in these experiments. We have now also included this information to the figure legends of the new Figure 5 and new EV2.
b. Please label the bar graphs with the specific gene category, rather than the study authors (the refs should go in the figure legend). Moreover, the text remains murky regarding the original definition of the "differentiation gene sets"-this is essential information for judging the relevance of the analysis and the authors cannot expect readers to scrutinize the original publications for this information.
Response: We have followed this suggestion from the reviewer. The information was given in the result text but we have now included the original names of the gene sets along with the publication in the revised Figure labeling (see new Figure 5A). We agree that this is much clearer.
c. Provided that the analyzed gene sets are indeed enriched for bona-fide differentiation associated genes, the authors need to present the data in a way that directly visualizes the down-or upregulation of specific genes (rather than merely the fraction of differentially expressed genes within the set, as in the present figures). The heat maps in the stem cell sections of the manuscript provide one example of how to accomplish this.
Response: This was a very good suggestion and we have followed the reviewer's advice. In the revised manuscript we have included heatmaps depicting the overlap of genes that (i) are . These heatmaps depict whether the genes in the overlap are up or downregulated in response to Smed-exoc3-depletion. For readability we have focused the heatmap depiction on those genes that show the strongest difference in expression in response to Smed-exoc3-depletion (log(2)fold-change) > 0.5 or < -0.5 (new Figure 5B-E; last paragraph on page 10 to 1 st paragraph on page 12 of the result section ). In addition, the full gene list of the overlaps is provided in the Dataset EV 4 and 6. In these datasets, we have included an analysis of the literature highlighting the function of the Smed-exoc3-regulated genes in stem cells and differentiation (Column R of the dataset). This literature analysis was focused on those genes showing the strongest difference in expression in response to Smed-exoc3-depletion (log 2 fold-change) > 0.5 or < -0.5 as depicted in Dataset EV 4 and 6. We think that this new depiction is much better and very interesting for the readers. While all of the differentiation marker genes (from the Zhu et al.) were downregulated in Exoc3-depleted planarians vs controls ( Figure 5E), the neoblast related genes were both up-and downregulated in Exoc3-depleted planarians. We think that this finding stands in line with the possibility (suggested by this reviewer) that the neoblast cells are possibly arrested at certain transitional states between stemness and differentiation thus leading to an up-or downregulation of the respective genes that characterize these transition stages. We included this thought in the result description (highlighted on page 11 of the revised manuscript).
d. I remain puzzled as to why the authors restricted their analysis to FACS sorted neoblasts, rather than simply comparing gene expression differences between whole exoc3(RNAi)/GFP(RNAi) animals. This would have afforded a direct opportunity to quantify global up/down regulation of stem cell genes and/or progeny markers, while the present X1 analysis can only detect relative shifts within the population. e.g., differences in cell cycle progression...
Response: We agree with the reviewer that RNA-seq on whole animals would have helped to confirm the accumulation of stem cells in Smed-exoc3-depeleted planarians. However, this kind of an analysis was already included (new Figure 2C and 3E,F). In our analysis on transcriptome changes, we actually intended to analyze gene expression in neoblast stem cells to see whether defects in the generation of differentiated cells were associated with changes in gene expression in neoblast cells from Smed-exoc3-depeleted vs. wildtype worms, which turned out to be the case. We think that the new depiction (see above response) makes this analysis even more interesting as it shows that Smed-exoc3-depletion leads to a deregulation of genes that have a role in stem cell function and differentiation. We agree with the reviewer that this deregulation could have multiple reasons including effects of Exoc3-deletion on cell cycle progression of neoblast stem cells.
Though the link between exoc3 and planarian stem cell differentiation remains weak, the quantitative depletion of triglycerides and the phenotypic rescue by FA supplementation nicely complements the vertebrate stem cell data. I can therefore cautiously recommend publication of a substantially revised manuscript, provided that the authors i) re-work their added planarian exoc3(RNAi) RNAseq analysis to incorporate the above concerns; ii) ensure that the salient bits of data are in the main figures and not in supplement; iii) omit the inconclusive support pertaining to a lack of cell cycle effects (p53/p63; annexin FACS plots); iv) undertake appropriate text revisions to ensure congruency between data shown and claims made (e.g., already in the abstract: "...Smed-exoc3 abrogates in vivo differentiation of somatic stem cells...").
Response: We thank the reviewer for being supportive for publication and we cautiously addressed all his/her comments as outlined in the above responses and as highlighted throughout the revised manuscript.
Minor point: 8) Once again, my request for "Scientific terminology" refers to QUANTITATIVE statements-please replace "good knock-down" by something along the lines of "... reduced mRNA levels by > 70 % (reference to figure) in comparison to untreated controls".
Response: We have specified this statement as suggested (1 st paragraph on page 9). 9) Please omit the statement that you carried out a "lipidomic" analysis-the targeted quantification of triglycerides is not a lipidome analysis. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
Is there an estimate of variation within each group of data?

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