How epithelial cell behaviors are coordinately regulated to sculpt tissue architecture is a fundamental question in biology. Kupffer’s vesicle (KV), a transient organ with a fluid-filled lumen, provides a simple system to investigate the interplay between intrinsic cellular mechanisms and external forces during epithelial morphogenesis. Using 3-dimensional (3D) analyses of single cells we identify asymmetric cell volume changes along the anteroposterior axis of KV that coincide with asymmetric cell shape changes. Blocking ion flux prevents these cell volume changes and cell shape changes. Vertex simulations suggest cell shape changes do not depend on lumen expansion. Consistent with this prediction, asymmetric changes in KV cell volume and shape occur normally when KV lumen growth fails due to leaky cell adhesions. These results indicate ion flux mediates cell volume changes that contribute to asymmetric cell shape changes in KV, and that these changes in epithelial morphology are separable from lumen-generated forces. In an embryo, epithelial cells undergo tightly regulated shape changes to drive tissue remodeling and organ development. Changes in epithelial cell morphology can be mediated by intrinsic mechanisms, such as rearrangements of the actomyosin cytoskeleton that often occur in response to biochemical signaling cascades . ExtrinsTg(sox17:GFP-CAAX), provides bright labeling of KV cell membranes with green fluorescent protein (GFP) that shows the AP asymmetric architecture of the whole organ  as a model organ to investigate mechanisms that regulate shape changes of single cells during epithelial morphogenesis. KV, which functions as a ‘left-right organizer’ to determine left-right asymmetry of the zebrafish embryo , is a trle organ  and the le organ . KV cellle organ   transgenic zebrafish using a sox17 promoter Tg(sox17:Cre zebrafish that expresses Cre recombinase in the KV cell lineage that has inducible activity through the addition of 4-hydroxytamoxifen (4-OHT) (ERT2)Tg(sox17:Cre fish were crossed with a previously described Tg(ubi:Zebrabow) (‘zebrabow’) strain (ERT2); Tg(ubi:Zebrabow)Tg(sox17:Cre embryos were briefly treated with 4-OHT from the dome stage (4 hr post-fertilization or hpf) to the shield stage (6 hpf) (+ KV cells with boundaries that are easily distinguished from surrounding RFP+ cells (ERT2); Tg(ubi:Zebrabow)Tg(sox17:Cre embryos were volume-rendered using Imaris (Bitplane) software to generate 3D reconstructions of the lumen and KV cells  . Transge) strain  that can) strain . Double  (6 hpf)  to induc (6 hpf) . This stP+ cells . Images KV cells . To asseKV cells . The comERT2); Tg(ubi:Zebrabow)Tg(sox17:Cre embryos from the 2-somite stage (2 ss) when the lumen first forms to the 8 somite stage (8 ss) when the lumen is fully expanded  and then individual cells at the middle plane of the organ were volume-rendered . We meas3 to 2547 ± 693 μm3 (shown are mean ±one standard deviation) and KV-post cell decreased volume from 2180 ± 1034 μm3 to 1564 ± 539 μm3  transgenic fish were crossed with Tg(actb2:myl12.1-MKATE2) zebrafish that have enriched apical membrane expression of the fluorescent mKate2 protein fused to Myl12.1 (myosin light chain 12 genome duplicate 1) in KV cells. Double transgenic Tg embryos were used to visualize both KV lumen and KV cells in living embryos (Tg(actb2:myl12.1-MKATE2) expression and then divided into two equal halves  along the AP axis (Tg(sox17:GFP-CAAX) expression  or the cystic fibrosis transmembrane conductance regulator (Cftr). Both have previously been shown to play a role in KV lumen expansion (ERT2); Tg(ubi:Zebrabow)Tg and then imaged KVs at 6 ss (12 hpf). However, these brief treatments did not reduce KV lumen expansion    to reduccftr MO)  as expeccftr MO) . To testcftr MO) , KV-post 483 μm3 . Reducinisrupted . In cftrectively . This su+/K+-ATPase or Cftr activity as they did in controls  of KV cells did not increase between 2 ss and 8 ss in embryos treated with inhibitors of Nacontrols , which icontrols  failed tcontrols . These rmCherry-CAAX) in Tg(sox17:GFP-CAAX) embryos that allowed us to simultaneously visualize both KV cells at the middle plane of KV and the surrounding external cells /√]) to define surrounding cell morphology (mCherry-CAAX labeling) that were positioned either anterior or posterior of KV indicated that there was no significant difference in cell shapes at 2 ss or 8 ss stages between control or ouabain treated embryos  (Tg(sox17:GFP-CAAX) embryos  . Immunofs (DFCs)  indicate embryos . Thus, wjupa pre-mRNA splicing (jupa MO-1) or a previously reported MO that blocks translation (jupa MO-2) of jupa mRNA  to block upa mRNA . Injectintrol MO . Reductintrol MO —that was embryos . Interfe embryos  as predi embryos . Deliverthe DFCs  that givthe DFCs , indicatupa mRNA , indicat disease . This muebrafish . Similar defects . Importacontrols , which d- ion flux and can over-inflate the KV lumen  along KV cell lateral domains relative to controls , which iKV lumen . TreatmeKV lumen . This suKV lumen . TogetheERT2); Tg(ubi:Zebrabow)Tg  . 3D anal692 μm3) , KV-ant 692 μm3) . These r3 to 2329 ± 847 μm3 and KV-post cells decreased volume from 2127 ± 287 μm3 to 1617 ± 336 μm3) and normal asymmetric cell shape changes during KV remodeling  protein . Similarmodeling . Taken tmodeling , these rThe collective behavior of epithelial cells plays a key role in determining the architecture of tissues and organs. Studies of developmental processes in animal models have provided important insights into the biochemical signals and mechanical forces that regulate epithelial morphogenesis . The zebThe finding that KV cells change volume during development is insightful for thinking about mechanisms of epithelial morphogenesis in KV since previous analyses  that wer+/K+-ATPase or Cftr inhibitors between the bud stage and 8 ss, KV-post cells did not shrink (but swelled) and the lumen failed to expand. This finding is consistent with a model in which intraepithelial fluid movement directly from KV-post cells into the lumen promotes lumen expansion. Since the amount of volume lost by KV-post cells does not fully account for the increase in lumen size, we propose ion flux in KV establishes both transepithelial flows and intraepithelial flows from KV-post cells to fill the lumen. Interestingly, brief treatments with the Na+/K+-ATPase inhibitor ouabain between 4–6 ss did not block lumen expansion. This may be because ouabain needs more time to penetrate deep inside the embryo to effectively block ion channel function in KV. Alternatively, these results may suggest ion channel function early in KV development (between bud stage and 4ss) is sufficient for lumen expansion or there are additional mechanisms independent of ion flux that contribute to lumen expansion and cell volume changes.The decrease in KV-post cell volume is mediated by ion channel activity that regulates fluid movement. Decrease in cell volume has also been observed during morphogenesis of zebrafish otic vesicle , where iDrosophila showed that contractile force induced cell shape changes are instituted via cell volume reduction  in non-dividing cells . Intereseduction , which ieduction . A receneduction . Stretcheduction . This suOur vertex model simulations suggest that asymmetric volume changes are not alone sufficient to fully induce KV cell shape changes. In addition to cell-intrinsic mechanisms, biophysical forces likely guide the formation of the KV epithelium. These mechanical forces can also arise from extrinsic sources that stem from the mechanical properties of surrounding tissues or extracellular matrix (ECM) . LocalizOther mechanisms, which remain unexplored, are mechanical forces generated by cells surrounding the KV as it advances towards the tailbud via convergent extension movements. Tissue fluidity in the tailbud plays an important role in controlling body elongation in zebrafish  and may Lumen expansion occurs synchronously with changes in epithelial cell shapes during KV morphogenesis , which rWe propose that luminal forces have a nominal impact on KV cell shape changes. Previous experimental results support this idea. First, KV cells can fail to change shape even when lumen expansion is normal. Inhibiting Rock2b function or non-muscle myosin II activity had no effect on KV lumen expansion, but prevented cell shape changes during KV remodeling . This inDrosophila uncovered that during dorsal closure cells within the amnioserosa lose their volume by ~30% and change their shape  (Tg(sox17:GFP)  (this study), sny102Tg(actb2:myl12.1-MKATE2) (this study), ERT2)sny120Tg(sox17:Cre (this study). Embryos were staged as described  , Tg(sox1x17:GFP) , Tg(sox1escribed .ERT2)Tg(sox17:Cre and Tg(sox17:GFP-CAAX) transgenics, gateway cloning was performed by combining p5E-sox17 (a generous gift from Stephanie Woo) (ERT2)sny120Tg(sox17:Cre or sny101Tg(sox17:GFP-CAAX) F0 founders. Adult F0 animals were then crossed with wild-type fish to generate F1 heterozygotes. ERT2)sny120Tg(sox17:Cre fish were then crossed with homozygous Tg(ubi:Zebrabow) (ERT2); Tg(ubi:Zebrabow)Tg(sox17:Cre transgenic strain. To generate a Tg(actb2:myl12.1-MKATE2) transgenic fish, p5E-actb2 (Tol2 Kit v2), pME-myl12.1-MKATE2 (see below), and p3E-SV40-polyA (Tol2 Kit v2) plasmids were recombined into pDestTol2CG4 destination vector as described above. Wild-type TAB embryos were injected with verified constructs and Tol2 Transposase mRNA to generate sny102Tg(actb2:myl12.1-MKATE2) F0 fish. Adult sny102Tg(actb2:myl12.1-MKATE2) F0 fish were crossed with wild-type TAB to to generate F1 heterozygotes. Tg(actb2:myl12.1-MKATE2) fish were crossed with homozygous Tg(sox17:GFP-CAAX) animals to generate a double Tg(actb2:myl12.1-MKATE2); Tg(sox17:GFP-CAAX) transgenic strain.Transgene constructs were generated using the Gateway-based Tol2 kit . To genenie Woo) , pENTR/Dnie Woo)  or pME-Gebrabow)  animals +MKATE2 vector using BamH1 and EcoR1 restriction enzymes to construct pCS2+ myl12.1-MKATE2 plasmid. The myl12.1-MKATE2 construct was PCR amplified from pCS2+ myl12.1-MKATE2 plasmid using following primers- attB1: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTCGAGCAAACGCGCCAA-3′ and attB2: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTCATCTGTGCCCCAGTTT-3′. The PCR amplified myl12.1-MKATE2 construct was then cloned into pDONR221 vector using BP recombination to generate the middle entry pME-myl12.1-MKATE2 vector.The myl12.1 ORF was PCR amplified from cDNA pool generated from 8 ss zebrafish embryos using following primers- myl12.1F: 5′-ATTAATGGATCCATGTCGAGCAAACGCGCCAA-3′ myl12.1R: 5′-ATTAATGAATTCTGCATCGTCTTTGTCTTTGGCTC-3′. The PCR amplified myl12.1 ORF was sub-cloned into pCS2jupa was kindly provided by Maura Grealy’s lab   to detect jupa expression by in situ hybridization. RNA in situ hybridizations were performed as described  . It was escribed .jupa MO-1 (5′-TTATGATTGTGTCTTCTCACCTGCA-3′) to interfere with jupa pre-mRNA splicing of exons 2 and 3. jupa MO-2 (5′-GAGCCTCTCCCATGTGCATTTCCAT-3′) designed to block jupa mRNA translation was previously described (cftr MO (5′-CACAGGTGATCTCTGCATCCTAAA-3′) (lgl2 MO-1 (5′-GCCCATGACGCCTGAACCTCTTCAT-3′)  were obtained from Gene Tools, LLC . We designed escribed . Other pTAAA-3′) , lgl2 MOTCAT-3′)  and a st cftr MO , 4.4 ng gl2 MO-1  and 2.5 gl2 MO-1 . To cond protein  was a kijupa MO-1 treated Tg(sox17:GFP-CAAX) embryos were dechorionated and mounted in 1% low melting agarose at 6 ss. KV lumens were microinjected with ~1 nL of 10 kDa dextran, Alexa Fluor-568  at 6 ss and imaged using a Zeiss Imager M1 microscope immediately (at 6 ss). Successfully injected embryos were then incubated at 28.5 degrees for one hour and then imaged again at 8 ss.Control MO or For whole mount immunofluorescent staining experiments, embryos were fixed in 4% paraformaldehyde in 1X PBS with 0.5% Triton X-100 at 4°C overnight and then dechorionated in 1X PBS. Embryos were permeabilized in blocking solution containing 1X PBS, 0.1% Triton X-100, 0.1%DMSO, and 5% goat serum for 4 hr. Primary antibodies were diluted in fresh blocking solution and incubated with embryos at 4°C overnight. Primary antibodies used: mouse anti-junction plakoglobin , mouse anti-ZO-1 , mouse anti-E-cadherin , mouse anti-acetylated tubulin , mouse anti-GFP , rabbit anti-GFP  and rabbit anti-fibronectin . Embryos were then washed in 1X PBS with 0.1% Triton X-100, 0.1% DMSO, and 1% BSA at room temperature. AlexaFluor 488- and 568- conjugated anti-rabbit and anti-mouse secondary antibodies  were used at 1:200 dilutions in blocking solution overnight. Stained embryos were then washed in 1X PBS with 0.1% Triton X-100, 0.1% DMSO, 1% BSA at room temperature. Embryos were imaged using either Zeiss Imager M1 microscope or a Perkin-Elmer UltraVIEW Vox spinning disk confocal microscope. Quantification based on fluorescent immunostaining was performed using ImageJ software. KV lumen areas were measured using maximum projections of ZO-1 staining. E-cadherin levels at KV cell junctions were measured by determining the mean gray level (per pixel) along KV cell lateral membranes as described . This meProtein extracts from approximately 30 zebrafish embryos at 8 ss were prepared as described . 30 μL oERT2; Tg(ubi:Zebrabow)Tg in 0.1% DMSO from the dome stage to the shield stage. To inhibit ion transport, embryos were either treated with a working concentration of 1 mM ouabain (Sigma) dissolved in water or 30 μM CFTRinh-172  in 0.1% DMSO from the bud stage to 2 ss or 8 ss. To activate Cftr channels, control MO and jupa MO injected embryos were treated with a working concentration of 10 μM CFTRact-09  from the bud stage to 8 ss. 0.1% DMSO was used as a vehicle control for all experiments. After pharmacological treatments, embryos were thoroughly washed with embryo medium, mounted in 1% low melting agarose and imaged using either a Perkin-Elmer UltraVIEW Vox spinning disk confocal microscope or a Zeiss Imager M1 microscope.To induce low levels of Cre recombinase activity in To image live KV cells, embryos were dechorionated and mounted in 1% low-melting point agarose on a glass-bottom MetTek dish at specific stages. Time-lapse imaging of KV was performed using 2 μm step-scan captured at 5 min. intervals for 105 min. using a Perkin-Elmer UltraVIEW Vox spinning disk confocal microscope. The acquired 3D datasets were processed and volume rendered using surface evolver tool in Imaris . Imaris was used to measure the length, width and height of reconstructed KV cells. The surface of the lumen was used to establish the axes of KV cells such that lateral axis (cell width) is parallel to the tangent of the curved lumen surface. To measure KV-lumen, KV-ant and KV-post cell cross-sectional areas, captured 3D images were oriented and maximum cross-sectional area from the middle plane perpendicular to the DV axis of individual cells were measured using clipping plane function in Imaris (Bitplane).+) in the KV was 3D reconstructed using ‘Create Surface’ tool in Imaris (Bitplane) software. From 3D reconstructed cells the ‘cell volume’ was measured. To measure total KV cellular volume, double transgenic Tg(actb2:myl12.1mKATE2); Tg(sox17:GFP-CAAX) embryos were used to 3D reconstruct the KV lumen and the total KV cellular component. The 3D lumen was split into equal anterior and posterior halves and the cellular component associated with the two halves of the lumen were defined as the ‘total KV-ant cellular volume’ and ‘total KV-post cellular volume.’Single mosaic labeled cells (YFPmCherry-CAAX) was injected into Tg(sox17:GFP-CAAX) embryos at the 1 cell stage. Confocal images were captured from live embryos at 2 ss and 8 ss. The cell shape index, q = [/√] (55 pg of mRNA encoding a membrane-targeted mCherry (l area)]  was usedWe simulate KV morphogenesis using the Vertex Model with periodic boundary conditions . BecauseHere, the first sum is over all cells. The first term in it describes a cell area elasticity, where For our simulations, we choose the values of se cells . We set The values for the line tensions depend on both involved cell types. We have set the line tension between any two KV cells https://www.gnu.org/software/gsl/) for the energy minimization ’ = {(y – x)/x}*100. The standard deviations can be used as the uncertainty in the measured values. Thus, the uncertainty δz in z can be represented as:For the ion channel inhibition in For the perturbation of epithelial junctions in  at 8 ss . ResultsThe significance of pairwise differences between groups of biological data was computed by Welch’s two-tailed t-test. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Senior/Reviewing Editor.Thank you for submitting your work entitled "Asymmetric cell volume changes regulate epithelial remodeling of the left-right organizer" 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 eLife to reject papers if it would take longer than two months to make the necessary revisions. Therefore, we have no choice but to decline the present version of your manuscript. However, if you feel you can address the comments of the reviewers, detailed below, we would be happy to consider a new version of the paper as a new submission and every effort would be made to return the paper to the same reviewers. I hope you find the attached reviews useful in revising the manuscript.Specifically, all three reviewers were enthusiastic about many aspects of your manuscripts but also asked for rather detailed changes that are likely to take considerable time. This includes improving the illustrations, improving the quality of the results, increasing statistical significance, and improving the writing. It is the policy of Reviewer #1:This paper describes experiments and a model exploring the relationships among geometric changes and the forces of morphogenesis, an understudied topic that deserves more exposure. I believe this paper could be a model of an important way to study morphogenesis. My recommendation is for this elegant series of hypothesis-driven experiments, which combine mechanics with molecular tools and mathematical hypothesis generation, to be published with some revision.Strengths: Adding the third dimension to studies of morphology changes raises the bar in an important way. The study itself examines interactions among the players, including an epithelium, a lumen, and the surrounding cells, rather than characterizing a single component of the system, such as one ion channel. The trajectory of the paper, from observations of 3D morphology, to a model used to generate hypotheses about the underlying mechanisms including force generation and material properties, to explicit tests of those hypotheses using modern molecular techniques, is a good model of how these types of experiments can be conducted.Weaknesses: the illustrations are not as clear as they need to be, and labels are inconsistent making it difficult to follow, particularly the first half, (up through the description of the model). The first half of the document is difficult to read in a way that I associate with an author who knows a subject so well that she or he forgets to provide the reader with the "obvious" background needed.1) There is no discussion of total system volume. I wanted to know what is getting pushed where as the lumen increases in volume. I quickly counted the number of external cells in 2) I think it is imperative that they add a description of the effect of the drugs and the MOs on the rest of the embryo, especially the external cells. That, plus the differences in the apparent extent of the KV in 3) It was not clear to me why knocking out E-cadherin directly is lethal, but knocking out its accumulation is not. Is it just a dosage effect, or is something else going on?4) The manuscript should be revised one more time, preferably by someone not intimately familiar with the project, who can identify where and why clarity is lacking. I've made some specific suggestions below, but it needs to be revised for consistency in terms  and illustrations . The section describing the model, in particular, needs to include more information up front – even with long experience interpreting models, I had to go back and forth to the supplements many times before finding the background I needed to understand the model and its predictions.Highly recommended: Since the authors have the 3D data, I was disappointed that the model was nevertheless simplified to 2D. I have to assume the authors are heading in that direction, so I was torn about whether to list this under required. I chose to recommend it highly on the basis that the model as is clearly provides useful testable predictions, which are cleverly explored experimentally in the latter half of the paper. But, it would be great if the model could be that much more representative of the actual geometry.Reviewer #2:The study by Dasgupta and colleagues show for the first time asymmetric volume changes in the KV and argue that this asymmetry contributes to the asymmetric cell shape changes contribute to KV cell shape changes. They also provide experiments to show that inhibiting lumen expansion does not affect cell shape changes associated with KV morphogenesis. Although asymmetric volume change represents an interesting potential mechanism, the paper is severely hampered by the quality of the results, and specifically, the huge standard deviations for some experiments. I also have some doubts about the impact of the study considering that the described process lead to change in 20% of the size of the KV and that the Amack group showed that left-right patterning occurs normally even though KV size can change vary more than 30% between embryos .First main point of paper: There are asymmetric cell volume changes that correlate with asymmetric cell shape changes.The presented data are not completely convincing. The reason is the approach used by the authors is not validated. For example the authors never made sure that the results are not simply due to photobleaching, changes in depth, etc. The authors should perform alternative cell volume measurements in KVs where cells are labeled in a mosaic with a membrane marker either by late mRNA injection, DNA injection or even cell transplantation and assess if the signal and therefore the cell volume measurements affected by the location of cells within the KV or by the stage of the embryos (the organ is deeper?) or by photo-bleaching (prolonged imaging). These points need to be addressed since the volume change reported here is only of about 20% between 2 and 8 ss. Overall the best would be to provide a comprehensive analysis of cell surface or volume and present convincing results  of cell volume changes throughout the KV. At present the authors are dependent on mosaic experiments where the cells location is random and we are left with data presenting huge variability with scales on the plots that are not consistent and comparable between conditions .Given that their simulations are in 2D, it might help to also perform a simpler experiment where they label cells exactly as they did in Wang 2012, and see if they see the expected differences in cross-sectional area between anterior and posterior cells. They could also see if their cross-sectional area measurements match up with their volume data .In addition, the authors need to clarify the following points:- Sample size and number of independent experiments;- Experimental design; e.g. cell volume changes between 2 and 8 ss: did they follow the same cells overtime and therefore show single cell growth or shrinkage or simply measured the volume of different cells in embryos at different stages and compared average cell volumes?- Explain better the statistical tests and how the analysis was done; e.g. How was the cell volume change quantified?They write in the Discussion:"The finding that KV epithelial cells change volume during development is surprising and insightful since previous 2D analyses had not identified any statistically significant changes in KV cell size."Their previous study showed that cells reduced their cross-sectional area during morphogenesis (Wang 2012), suggesting that cells reduced their volume, but this study shows for first time that these volume changes are asymmetric. This point should be clarified.Second main point of paper: Inhibiting ion channels inhibits cell shape changes.Large error bars and inconsistent scales are a problem.Third main point of paper: In simulations, asymmetric cell volume changes could account partially for cell shape changes, provided that the mechanical properties of the cells satisfy certain conditions.The issue about mechanical properties of cells is important. It is unclear if the anterior and posterior mechanical properties of the KV environment are different and how this contributes to KV morphogenesis. If the mechanical properties of surrounding cells are important, could they simulate what happens when there are asymmetric differences in the environment , or when there are asymmetric differences in the KV cells .Given that the effect of cell volume changes relies on the condition that surrounding cells are solid like, it would be important to perform an experiment to test that. It brings a different light to the Wang et al., 2012 paper, which used blebbistatin and MOs to disrupt mechanical properties of KV cells. Could some of the effects they saw be due to the changes in mechanical properties of surrounding cells rather than the KV cells themselves?Given that their simulations show that cell volume changes alone could not account for cell shape changes, it is weird that in Is cell volume change somehow a prerequisite for cell shape changes via actomyosin contractility? They suggest there could be a link in the Discussion, referencing Saias et al., 2015.Considering the importance of actomyosin in the process of KV morphogenesis, it is important to show what happens to KV cell volume (anterior and posterior) when myosinII/rock2b is inhibited. It might be also good to check phosphorylated myosin via antibody staining (as they did in Wang 2012) to see if inhibiting ion channels somehow affects actomyosin contractility.Repeat experiments in Figure.5 with DFC specific knockdown and possibly in lgl mutant, which is available.Fourth main point: Lumen expansion does not affect KV cell shape change.This is Fifth and concluding point: Inhibiting cell volume changes disrupts cell shape changes.Here key experiments are missing. The authors never demonstrate that asymmetric (anterior-posterior) cell volume changes are necessary or contribute KV morphogenesis. The authors should directly test the impact of cell volume change on KV morphogenesis and in particular the impact of asymmetric volume change between anterior and posterior. Photomorpholinos could provide temporal and spatial specificity. If they can get it to work, blocking cftr in anterior vs. posterior cells, and blocking cftr in left vs. right cells to see if an additional axis of asymmetry arise  would be necessary.Finally, the authors need to rethink their final drawing because it is really misleading.Reviewer #3:The work of Dasgupta et al. uses the Kupffer's vesicle of zebrafish as a model to study the epithelial morphogenesis, in particular the possible role of programmed cell volume changes and the relationship between lumenogenesis and epithelial remodeling.Positive aspects of the manuscript include: Methodology: the use of an elegant mosaic labelling approach to perform 3D volumetric and shape analysis of single epithelial cells in vivo. Results: (a) The description of AP asymmetry in cell volume in the KV, with A cells increasing while P decreasing volume between 2 and 8 somites . (b) The finding that ion flux is required not only for KV lumen expansion (as previously described) but also for the establishment of AP polarity in cell volume. (c) The experimental separation of AP changes in cell shape and volume from lumen expansion, which indicate that the former changes might be autonomous and not a response to external forces generated during lumen expansion. Results from a-c led show that changes in KV cell volume might contribute to epithelial remodeling independent from the forces generated by lumen expansion.My main concerns of the paper are:Methodology:a) The authors mix results and discussion of experimental data of cell shape (measured in 2D) with volume changes (in 3D). This is not correct and could lead to wrong interpretations. For making a clearer connection between the two, parameters of cell shape should incorporate the 3 principal axes. Also, the authors equal the term "epithelial remodeling" with the changes in cell shape in 2D. The authors should restrict the use of the term epithelial remodeling when addressing the epithelial changes in a broader manner, for example, in the introduction and discussion, and use "changes in cell shape" in the other cases.b) The authors developed a mathematical approach using a 2D vertex model to test the relationship between changes in cell shape and volume and lumen expansion. The model works well for testing the relationship between cell shape changes and lumen expansion in 2D. However, the authors extend the interpretation of the results to the changes in cell volume, assuming that changes in cell cross sectional areas (2D) are equivalent to the changes in cell volume (3D). However, this is not necessarily the case, and thus all the conclusions and predictions that involve changes in cell volume are not valid. The authors should be very careful and make more explicit the limitations of the model. Although the use of 2D models for 3D data is in many cases useful, it is not in this particular case, where the changes of cell volume are central for the mechanisms.Results:It remains unclear what the main conclusions of the paper are. The authors propose that changes in cell volume work upstream of the changes in tissue remodeling but there is no direct evidence of this, nor they explore the mechanisms that could mediate this process.Also, it remains unexplored what makes the changes in cell volume to be AP asymmetric, and whether these changes are really "programmed autonomously" or if the mechanisms that control cell volume are symmetric but modulated in an asymmetric manner by non-autonomous forces (e.g. asymmetric ECM deposition).Finally, the lack of requirement of lumen expansion for KV tissue remodeling and changes in cell volume is an interesting finding, but again this is not explored further.In summary, the paper provides interesting methodology and data, but in its current state has a lack of focus, which makes unclear its main contribution to our understanding of epithelial morphogenesis.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal her identity: Dany S Adams (Reviewer #1).Thank you for submitting your article "Asymmetric cell volume changes regulate epithelial morphogenesis in zebrafish Kupffer's vesicle" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.While the reviewers felt that the manuscript was much improved, there remain important issues that need to be addressed, as outlined below in the specific comments of reviewers 2 and 3. We hope you find their comments useful and look forward to receiving your revised manuscript.Reviewer #1:The clarity of the manuscript is greatly improved, and extra data that I requested has been collected, analyzed thoughtfully, and integrated. I believe it is a strong paper that makes a contribution to our understanding of KV development and, more generally, the interplay of different forces during morphogenesis. I also believe it makes a strong argument for the importance of addressing questions about morphogenetic forces and shape generation, and I think the structure of the project could serve as a useful model for how to conduct such studies. Specifically, the sequence they present, careful description of 3D morphology at multiple time points, followed by hypothesis generation (in their case using modeling), followed by experimental testing of those hypotheses, is a useful outline for these kinds of studies. I look forward to following this project, especially if these authors are able to expand their models into 3D the way they have expanded their descriptions. I recommend publication.Reviewer #2:The manuscript by Dasgupta et al. describes how asymmetric cell shape changes between the anterior and posterior cells of the zebrafish Kupffer's vesicle (KV). Using 3D analysis of single cells at defined single time points, the authors show that asymmetric cell shape changes in length, width and volume  occur between 4 and 6 somite-stages (ss) and that lumen expansion does not primarily contribute to asymmetric cell shape changes.Although the conclusion from the latter part is convincing, what drives asymmetric cell shape changes is largely unclear from the 3D analysis other than involvement of ion channels. The authors emphasize that live embryos are imaged at one stage to avoid potential photobleaching. This approach is good to initially identify the crucial parameters and duration. However, to identify the mechanisms underlying asymmetric cell shape changes requires the analysis of single cell behavior continuously during the critical time points (from 4ss to 6ss) based on time-lapse movies.There are several concerns to be clarified.1) It is confusing about the term "asymmetric cell volume changes", which reflects asymmetric cell shape changes in length and width but not in height . What is2) Despite the fact that the authors found out the crucial time point in this process between 4 ss and 6 ss , the maj3) I am uncertain about how informative the modeling part is . The ver4) There is inconsistency of the data upon ouabain treatment at 2ss . The for5) What is the biological significance of cell height changes at 2 ss in embryos treated with ion channel inhibitors or lgl2-morphant embryos? It is confusing, as cell height does not significantly change between anterior and posterior cells from 2ss to 8ss.Reviewer #3:The study by Dasgupta and colleagues show for the first time asymmetric volume changes in the KV and argue that this asymmetry contributes to the asymmetric cell shape changes contribute to KV cell shape changes. They also provide experiments to show that inhibiting lumen expansion does not affect cell shape changes associated with KV morphogenesis. The authors use a nice combination of experiments and modeling to assess this interesting issue. This study proposes a new process that could at work in the process of KV morphogenesis that promises to start exciting new avenues of investigation. Overall the article is a great piece of work and has been significantly improved since the last revision. We have a few comments.1) Inhibiting ion channels inhibits cell shape changes/asymmetric cell changes in KV are separable from lumen expansion.The statistical analysis. Ouabain treatment and cftr knock down lead to similar conclusion which is great. However, both experiments are based on the fact that the results are not significant in the treated embryos at 2SS and 8SS. For this type of analysis, the authors should provide a power analysis to demonstrate that the sample size is enough to conclude about not significant results. Similar comment for jupa and igl2 treatments.2) Mechanical properties of the surrounding cells. In simulations, asymmetric cell volume changes could account partially for cell shape changes, provided that the mechanical properties of the cells satisfy certain conditions.The issue about mechanical properties of cells is important. The mechanical properties of surrounding cells are important and need to be solid like at least anteriorly. Please provide reasonable explanation as to why anterior and posterior could be solid like and more generally, what are the arguments implying that mesodermal cells are solid like (this can be part of the Discussion). The 'pinning' hypothesis is interesting in that aspect and could be discussed. Lance Davidson’s work could be discussed as it seems that the notochord is a pretty rigid structure from his work. In addition, could the fact that fibronectin is enriched in the anterior side of the KV could increase the possibility that cells are solid like anteriorly?3) Cell volume changes and actomyosin. Given that their simulations show that cell volume changes alone could not account for cell shape changes, it is weird that in 4) The conclusion 'Asymmetric cell volume changes regulate epithelial morphogenesis'. It should be toned down providing that we do not know if the surrounding cells are solid like and that we do not know how the actomyosin network is compromised by the treatments used. The term 'regulate' seems too strong at this point. [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:[…] Weaknesses: the illustrations are not as clear as they need to be, and labels are inconsistent making it difficult to follow, particularly the first half, (up through the description of the model). The first half of the document is difficult to read in a way that I associate with an author who knows a subject so well that she or he forgets to provide the reader with the "obvious" background needed.Thank you for making this point. We have made significant revisions to the text and figures to improve clarity and consistency. Additional background has been added to the text and new 1) There is no discussion of total system volume. I wanted to know what is getting pushed where as the lumen increases in volume. I quickly counted the number of external cells in Our apologies that the manuscript did not make this clear. We do not change thenumber of cells in our simulations. Therefore, in the simulations, the increase in lumen area is accommodated by a  decrease in the preferred area for each of the external cells. The force-balanced states depicted in former 2) I think it is imperative that they add a description of the effect of the drugs and the MOs on the rest of the embryo, especially the external cells. That, plus the differences in the apparent extent of the KV in Thank you for making this suggestion. We have now examined the effect of thepharmacological inhibitor ouabain on the rest of the embryo and specifically on external cells surrounding the KV. We focused on ouabain treatments since it is already known that Cftr is expressed specifically in KV cells  and that loss of Cftr function does not alter tissues other than KV between 2 ss and 8 ss . This analysis is included in new “Since these treatments were global, we wanted to test whether blocking ion channels altered other tissues in the embryo, including cells surrounding KV that could have an impact on KV cell shapes. […] These results indicate that ouabain does not alter the geometry of cells surrounding KV and suggest that defects in KV cell shape changes result from altered ion flux in KV”.We did not repeat this analysis for Jup MO and Lgl2 MO treatments  since KV3) It was not clear to me why knocking out E-cadherin directly is lethal, but knocking out its accumulation is not. Is it just a dosage effect, or is something else going on?Yes, this is a dosage effect. Loss-of-function mutations in E-cadherin result in the arrest of epiboly movements of deep cells at the 70-80% epiboly stage development (8 hpf) prior to the appearance of KV (11 hpf) . With the MO dose we used in Jup knockdown embryos, E-cadherin enrichment at lateral KV membranes is only moderately reduced (~22% decrease) . These k4) The manuscript should be revised one more time, preferably by someone not intimately familiar with the project, who can identify where and why clarity is lacking. I've made some specific suggestions below, but it needs to be revised for consistency in terms  and illustrations .Thank you for these suggestions. We recruited two additional readers that have now critically evaluated our manuscript and figures and made helpful comments. We have made significant revisions to the text and figures to improve clarity and consistency. Additionally, in the schematics and in the graphs we have now consistently pseudo-colored KV-ant and KV-post cells associated with the middle plane of the organ as blue and red, respectively.The section describing the model, in particular, needs to include more information up front – even with long experience interpreting models, I had to go back and forth to the supplements many times before finding the background I needed to understand the model and its predictions.We have now extensively revised the modeling section and the correspondingsupplement. See below for details. We hope that clarity has significantly improved.Highly recommended: Since the authors have the 3D data, I was disappointed that the model was nevertheless simplified to 2D. I have to assume the authors are heading in that direction, so I was torn about whether to list this under required. I chose to recommend it highly on the basis that the model as is clearly provides useful testable predictions, which are cleverly explored experimentally in the latter half of the paper. But, it would be great if the model could be that much more representative of the actual geometry.This is an excellent point, which we carefully considered ourselves in writing thismanuscript. We are in fact headed in the direction of 3D models and we agree that it is important, but in order to use such a model for the KV, we first have to understand the simple, bulk mechanical properties of 3D models for tissues, which has not really been studied before.Some of us have a preprint that just appeared online , showing that there is indeed a fluid-solid transition in the 3D model. We next need to investigate whether adding additional interfacial tensions (as we’ve done here in 2D) are numerically stable, as there are some subtleties about numerical stability in 3D that we are actively working through now. Therefore, we didn’t want to use a model we do not yet fully understand here, and, as discussed below, we do not think it is necessary to explain the science.For us, the question one must always ask with modeling is whether the mechanism that one is proposing as biologically relevant can survive realistic perturbations to the model. In this case, the mechanism we propose is that solid-like behavior of the surrounding tissue can “pin” the interface between the anterior and posterior KV cells so that it remains at the mid-plane despite changes to cell volume and cross-sectional area, and this helps to generate the observed shape changes. We have added a new figure and text to highlight this mechanism.Now, we must ask whether this mechanism would survive a (major) perturbation to the model, which is going from 2D to 3D. We provide data that demonstrates that the 3rd dimension  contributes much less differential changes to cell volume than changes in the cross-sectional areas. We have added a table Reviewer #2:The study by Dasgupta and colleagues show for the first time asymmetric volume changes in the KV and argue that this asymmetry contributes to the asymmetric cell shape changes contribute to KV cell shape changes. They also provide experiments to show that inhibiting lumen expansion does not affect cell shape changes associated with KV morphogenesis. Although asymmetric volume change represents an interesting potential mechanism, the paper is severely hampered by the quality of the results, and specifically, the huge standard deviations for some experiments. I also have some doubts about the impact of the study considering that the described process lead to change in 20% of the size of the KV and that the Amack group showed that left-right patterning occurs normally even though KV size can change vary more than 30% between embryos .The sizeable standard deviations that can come with measuring KV cells are due to the wide variability in the size of KV from embryo to embryo. These standard deviations are important, as they accurately reflect the degree of variability in the system. We now report all raw cell volume data as an average + one standard deviation in the revised text (Results section). As noted, we recently reported a comprehensive study of the natural variation of KV size and number of ciliated cells in several wild-type and transgenic strains .Direct comparisons of KV size with left-right patterning (a readout for KV function) in individual embryos revealed that KV size only needed to exceed a certain  threshold to function normally. Thus, we agree with the reviewer that the amount that the KV cells change in size/volume, which varies from embryo to embryo, may not be that important. However, it is clear from our results that wild-type KV cells always change size in an asymmetric way along the anterior-posterior (AP) axis. Anterior KV cells always increase in size and posterior cells always decrease in size. These asymmetric cell size changes are linked to asymmetric cell shape changes along the AP axis that we know from previous studies are critical for KV function.We have modified the Results section and added a new paragraph to the Discussion section to clarify the point that it is the AP asymmetry rather than the absolute value of volume changes that are important for KV morphogenesis and function.First main point of paper: There are asymmetric cell volume changes that correlate with asymmetric cell shape changes.The presented data are not completely convincing. The reason is the approach used by the authors is not validated. For example the authors never made sure that the results are not simply due to photobleaching, changes in depth, etc. The authors should perform alternative cell volume measurements in KVs where cells are labeled in a mosaic with a membrane marker either by late mRNA injection, DNA injection or even cell transplantation and assess if the signal and therefore the cell volume measurements affected by the location of cells within the KV or by the stage of the embryos (the organ is deeper?) or by photo-bleaching (prolonged imaging). These points need to be addressed since the volume change reported here is only of about 20% between 2 and 8 ss.We apologize that there was confusion about our methodology. We have now substantially revised the text and added new figures and movies  to clariWe did not use an alternative labeling approach , since each approach would be subject to the same potential imaging artifacts (e.g. photobleaching or tissue depth). Instead, we took several precautions to avoid these artifacts. In our initial experiments, time-lapse imaging  was usedTo avoid differences in fluorescence signal due to differences in imaging depth, cells were not selected at random positions in the KV, but rather had to reside at the middle plane along the AP axis (defined as where the lumen diameter is largest) of the KV in all embryos analyzed. This was not made clear in our original manuscript and is now emphasized in the text. All images were also visualized in an YZ orientation  to deterOverall the best would be to provide a comprehensive analysis of cell surface or volume and present convincing results  of cell volume changes throughout the KV. At present the authors are dependent on mosaic experiments where the cells location is random and we are left with data presenting huge variability with scales on the plots that are not consistent and comparable between conditions .Mosaic labeling indeed randomly labeled KV cells. By selecting enough labeled cells from several embryos, we sampled KV cells from all positions along the middle plane of KV.This is now shown in new We also adjusted the scales on our graphs so that they are consistent and all data are comparable from figure to figure. We have emphasized that AP asymmetry, rather than the specific amount of volume change, is important for KV morphogenesis. This has been clarified in the text and added to the Discussion.Given that their simulations are in 2D, it might help to also perform a simpler experiment where they label cells exactly as they did in Wang 2012, and see if they see the expected differences in cross-sectional area between anterior and posterior cells. They could also see if their cross-sectional area measurements match up with their volume data .We did this analysis . Thus, interpreting these results in a systematic way was difficult and so we left this computation out of the manuscript.In addition, the authors need to clarify the following points:- Sample size and number of independent experiments;Thank you for pointing this out. This information has now been clarified in the figures and figure legends:jupa MO knockdown experiments were repeated four independent times and rescue experiments were performed three independent times. DFC specific knockdown of Jup was performed two independent times. Dominant negative Jup-naxos mRNA was also injected two separate times to validate MO knockdown phenotype. This is clarified in the figure legend. The number of embryos (N) analyzed for each experiment is indicated under the graphs.jupa MO knockdown experiments were repeated three independent times and lgl2 MO experiment was repeated two separate times at each stages (2 ss and 8 ss). The number of embryos (N) and cells analyzed are mentioned in the graphs and in the figure legend.jupa MO knockdown experiments were repeated two independent times and number of embryos analyzed (N) is denoted in the graph.- Experimental design; e.g. cell volume changes between 2 and 8 ss: did they follow the same cells overtime and therefore show single cell growth or shrinkage or simply measured the volume of different cells in embryos at different stages and compared average cell volumes?We used both approaches, but all data presented in the paper are averages thatcome from pooling results obtained from imaging different cells in different embryos at different stages. This method was selected to guard against potential imaging artifacts as discussed above. Our experimental design has now been clarified in the revised text:Tg embryos from the 2-somite stage (2 ss) when the lumen first forms to the 8 somite stage (8 ss) when the lumen is fully expanded . […] By analyzing enough embryos, we sampled KV cells from all positions along the middle plane of KV at different stages of development  of KV cell volumes at 2 ss and 8 ss instead of percentage volume changes . In wildThey write in the Discussion:"The finding that KV epithelial cells change volume during development is surprising and insightful since previous 2D analyses had not identified any statistically significant changes in KV cell size."Their previous study showed that cells reduced their cross-sectional area during morphogenesis (Wang 2012), suggesting that cells reduced their volume, but this study shows for first time that these volume changes are asymmetric. This point should be clarified.We have revised the Discussion to clarify this point:“The finding that KV epithelial cells change volume during development is insightful for thinking about mechanisms of KV morphogenesis since previous analyses  that were limited to 2D had not predicted any differences in KV cell size. […] It is therefore a striking that 3D analysis shows that KV cells do indeed change volume, and do so asymmetrically along the AP axis.”Second main point of paper: Inhibiting ion channels inhibits cell shape changes.Large error bars and inconsistent scales are a problem.As discussed above, the error bars represent the natural variation in KV observed in a population of embryos. Even with variability, sampling enough embryos allowed us to identify statistically significant differences. We have changed the scales in our graphs such that they are now consistent throughout the paper.Third main point of paper: In simulations, asymmetric cell volume changes could account partially for cell shape changes, provided that the mechanical properties of the cells satisfy certain conditions.The issue about mechanical properties of cells is important. It is unclear if the anterior and posterior mechanical properties of the KV environment are different and how this contributes to KV morphogenesis. If the mechanical properties of surrounding cells are important, could they simulate what happens when there are asymmetric differences in the environment .Thank you for this suggestion. We now additionally studied the influence ofasymmetries in the external cells. As may have been expected from our proposed mechanism (that solid-like cells “pin” the interface between the anterior and posterior cells at the middle plane during volume changes), we find that it does make a difference whether only anterior or only posterior external cells were solid or all external cells were solid.In particular, if only posterior external cells were solid , the AP In future work, we are planning to extensively study the fluidity, geometry, and mechanical properties of the external cells in more detail, in order to better constrain their effects on KV mechanics, but we believe this is beyond the scope of the current manuscript.Or when there are asymmetric differences in the KV cells .The case of solid anterior KV cells and fluid posterior KV cells is already included in our simulations. For small numbers of cells, the APA is also positive. We have added a brief discussion of this case to the main text and a more extensive discussion to a dedicated section in the supplement.Given that the effect of cell volume changes relies on the condition that surrounding cells are solid like, it would be important to perform an experiment to test that. It brings a different light to the Wang et al., 2012 paper, which used blebbistatin and MOs to disrupt mechanical properties of KV cells. Could some of the effects they saw be due to the changes in mechanical properties of surrounding cells rather than the KV cells themselves?This is an interesting point. Our previously reported blebbistatin treatments andglobal MO knockdowns of the Rho kinase Rock2b  likely impact the mechanical properties of both KV cells and surrounding cells. We previously did an experiment to test whether actomyosin contractility in KV cells vs. surrounding cells is involved in KV cell shape changes . To test this, MOs against Rock2b were injected into midblastula stage embryos for DFC/KV specific knockdown. KV cell shape changes failed to occur in embryos with Rock2b knocked down in KV cells, even though surrounding cells were normal. This indicates that cell-autonomous function of Rock2b and actomyosin activity is important for KV cell shape changes. Of course, this doesn’t rule out a role for surrounding cells, and future studies are needed to test this possibility. This point is now addressed in the Discussion.Given that their simulations show that cell volume changes alone could not account for cell shape changes, it is weird that in Is cell volume change somehow a prerequisite for cell shape changes via actomyosin contractility? They suggest there could be a link in the Discussion, referencing Saias et al., 2015.Considering the importance of actomyosin in the process of KV morphogenesis, it is important to show what happens to KV cell volume (anterior and posterior) when myosinII/rock2b is inhibited. It might be also good to check phosphorylated myosin via antibody staining (as they did in Wang 2012) to see if inhibiting ion channels somehow affects actomyosin contractility.We agree that is interesting and important to tease apart the relationships among ion flux, cell volume changes and cytoskeletal contractility. The suggested experiments are excellent and we plan to pursue this line of investigation in future work. As suggested, there are several possible outcomes. First, as mentioned in our Discussion section, contractile forces that generate cell shape changes may be directly linked to cell volume changes. In the KV system, it will be interesting to test whether AP asymmetric volume changes result in differential cytoskeletal contractility between KV-ant and KV-post cells. To do this, we are generating fluorescent reporters of actomyosin dynamics in KV cells to analyze this in real time.mypt1 mRNA) to perturb contractility and then assay volume changes. In addition, we are building genetic tools to address this possibility. Since we have established the sox17:CreERT2 transgenic line, we are using CRISPR to introduce loxP sites into cytoskeletal regulatory genes such as Rock2b. This system would allow us to delete Rock2b specifically in KV and then follow contractility, volume changes and shape changes.A second possibility is that actomyosin contractility is necessary for cells to change volume. Here, we can use treatments  or that the relationship between contractility and cell volume is complex (see possible outcomes discussed above).We feel that the experiments proposed to test the interplay between cell volume changes and cytoskeletal contractility represent a new line of investigation that goes beyond the scope of this paper and any new results would not change the conclusions of the present manuscript.Repeat experiments in As suggested, we used DFC/KV-specific knockdown to determine whether Jupfunctions cell-autonomously in KV cells to regulate KV lumen expansion. Similar to global Jup knockdowns, the DFC/KV-specific knockdowns resulted in a reduced KV lumen size without affecting the number of ciliated KV cells. These new data, presented in We did not repeat experiments in old Unfortunately, available lgl2 mutants are not useful for KV studies. As previously described  lgl2 mutants do not have phenotypes during early development (prior to 4 days post-fertilization) due to maternal contribution. Only MOs that target both maternal and zygotic expression develop KV phenotypes .Fourth main point: Lumen expansion does not affect KV cell shape change.This is A discussion of error bars and differences among control embryos appears aboveand is now included in the revised text.Fifth and concluding point: Inhibiting cell volume changes disrupts cell shape changes.Here key experiments are missing. The authors never demonstrate that asymmetric (anterior-posterior) cell volume changes are necessary or contribute KV morphogenesis.Our work with mosaic labeled cells has uncovered asymmetric volume changesalong the AP axis of KV that occur at the same developmental timing as AP asymmetric cell shape changes that were previously described. A previous study that used ouabain treatments in inhibit ion flux and lumen expansion  predicted that the forces associated with rapid KV lumen growth impacts asymmetric KV cell shape changes. Here, we show that in addition to blocking lumen expansion, inhibiting ion flux also blocked asymmetric changes in KV cell volume. Thus, the failure of cells to change shape when ion flux was altered could be due to blocked lumen expansion or blocked volume changes or both.To test this, we blocked lumen expansion by creating a leaky KV. In these embryos , asymmetFrom these experiments, we conclude that asymmetric cell shape changes that occur in KV depend on ion flux-mediated cell volume changes and do not depend on extrinsic mechanical forces associated with lumen expansion. We also used vertex models to test whether changes in cell volume  contribute to KV cell shape changes. This has been clarified in the Results section:“First, to investigate whether lumen expansion is necessary to create an asymmetry in KV cell elongation, we repeated the numerical simulations shown in Taken together, these results indicate asymmetric cell volume changes contribute to asymmetric cell shape changes in KV. These results have been emphasized for clarity in the revised manuscript.The authors should directly test the impact of cell volume change on KV morphogenesis and in particular the impact of asymmetric volume change between anterior and posterior. Photomorpholinos could provide temporal and spatial specificity. If they can get it to work, blocking cftr in anterior vs. posterior cells, and blocking cftr in left vs. right cells to see if an additional axis of asymmetry arise  would be necessary.Whether volume changes in anterior KV cells or posterior KV cells make equalcontributions to overall KV morphogenesis is very interesting question that we plan to address in future work. In addition to the suggested photo-MO experiments, we are currently generating genetic tools to test the contributions of asymmetric volume changes. We have identified a handful of genes that are asymmetrically expressed in KV– either in anterior cells or posterior cells – that we are using to generate transgenic lines that can overexpress or interfere with ion channels. As suggested, we plan to integrate results from these experiments with our models to fully test the contribution of asymmetric volume changes to KV form and function. We agree with the reviewer and predict that asymmetric ion channel activity is driving asymmetric volume changes. However, we feel that rigorously testing this hypothesis is beyond the scope of this paper.Finally, the authors need to rethink their final drawing because it is really misleading.We have significantly simplified model. In this model, cell volume changes  introduce cell shape changes along the AP axis of the KV in wild-type embryos. Perturbations of ion flux inhibit lumen expansion, prevent cell volume  changes and prevent KV cell shape changes. Weakening junction integrity inhibited normal lumen expansion, but asymmetric cell volume and cell shape changes occurred normally, albeit to a lesser extent compared to wild type.Reviewer #3:[…] My main concerns of the paper are:Methodology:a) The authors mix results and discussion of experimental data of cell shape (measured in 2D) with volume changes (in 3D). This is not correct and could lead to wrong interpretations.We have worked to ensure that we more clearly separate now between ourexperimental findings about the 3D KV and our 2D modeling of a cross section through the center of the KV along a plane that is perpendicular to the dorso-ventral (DV) axis. We also explain more clearly now that volume changes are correlated with cross-sectional area changes, and that our simulations show that asymmetric changes in the KV cell areas can induce asymmetric KV cell shape changes.For making a clearer connection between the two, parameters of cell shape should incorporate the 3 principal axes.Cell parameters for all 3 axes  were measured using 3D rendered cells and are reported in the paper. We chose to use a length to width ratio (LWR) to describe cell shapes for two reasons: 1) to make connections with our previous work (done in 2D) that used LWRs to describe cell shape changes  and 2) to make connections with simulations using vertex models. In our simulations, we quantify cell shape anisotropy based on a LWR, where the length corresponds to the apico-basal extension of the cells, and the width corresponds to the lateral extension perpendicular to the DV axis. Thus, the LWR quantified in the simulations corresponds to the LWR quantified in the experiments.Since the 2D simulations focus on a plane perpendicular to the DV axis, the cell extension along the DV axis  is not included in the modeling. Please see our response to reviewer 1 Point #6 for a detailed discussion of our justification for this choice. We have also edited the paper significantly to address this point.Also, the authors equal the term "epithelial remodeling" with the changes in cell shape in 2D. The authors should restrict the use of the term epithelial remodeling when addressing the epithelial changes in a broader manner, for example, in the introduction and discussion, and use "changes in cell shape" in the other cases.We agree and have reformulated. We now are explicit about ‘changes in cell shape’ and ‘changes in cell volume.’b) The authors developed a mathematical approach using a 2D vertex model to test the relationship between changes in cell shape and volume and lumen expansion. The model works well for testing the relationship between cell shape changes and lumen expansion in 2D. However, the authors extend the interpretation of the results to the changes in cell volume, assuming that changes in cell cross sectional areas (2D) are equivalent to the changes in cell volume (3D). However, this is not necessarily the case, and thus all the conclusions and predictions that involve changes in cell volume are not valid. The authors should be very careful and make more explicit the limitations of the model. Although the use of 2D models for 3D data is in many cases useful, it is not in this particular case, where the changes of cell volume are central for the mechanisms.We agree that one has to be careful when studying the effect of cell volume changes using a 2D model. However, we have explicitly measured the changes in cell volume and cell cross-sectional area in the plane perpendicular to the dorsal-ventral axis (e.g. where our 2D model resides) as well as the “height” of cells – their length along the DV axis. These are shown in the main text in Results:It remains unclear what the main conclusions of the paper are. The authors propose that changes in cell volume work upstream of the changes in tissue remodeling but there is no direct evidence of this, nor they explore the mechanisms that could mediate this process.In this work, we found experimental conditions that separate the process of lumen expansion  from the process that drives cell shape changes in KV. We conclude that ion flux and its associated asymmetric volume changes contribute to cell shape changes during epithelial morphogenesis in KV. We have explored possible mechanisms of how asymmetric KV cell volume changes create asymmetric KV cell shapes. However, we might not have explained this clearly enough.To improve our manuscript, we have modified the main text to more clearly explain our conclusions and the mechanism that is responsible for asymmetric KV cell shapes in the simulations when the external cells are solid. In addition, we created illustrations of these mechanisms for both presence and absence of lumen expansion . MoreoveAlso, it remains unexplored what makes the changes in cell volume to be AP asymmetric, and whether these changes are really "programmed autonomously" or if the mechanisms that control cell volume are symmetric but modulated in an asymmetric manner by non-autonomous forces (e.g. asymmetric ECM deposition).Finally, the lack of requirement of lumen expansion for KV tissue remodeling and changes in cell volume is an interesting finding, but again this is not explored further.As discussed above in response to reviewer #2, we are very interested indetermining how KV cell volumes change in an asymmetric way and how this asymmetry contributes to KV form and function. We are generating several genetic tools to perturb volume changes and test whether they are cell-autonomous. We will also perform experiments and simulations to test the role of external forces that include ECM and the mechanical properties of surrounding cells. Multiple approaches will be needed to tease apart contributions from cell autonomous and non-cell autonomous effects, which we feel is beyond the scope of this paper.In summary, the paper provides interesting methodology and data, but in its current state has a lack of focus, which makes unclear its main contribution to our understanding of epithelial morphogenesis.Thank you for this feedback. We have revised the manuscript text to more sharply focus on the goals and the main contributions of this study. We set out to develop new mathematical models and experimental methods that allow morphometric analysis of epithelial cells in the simple KV organ in order to investigate the interplay between intrinsic and extrinsic mechanisms that contribute to cell shape changes during epithelial morphogenesis. Using this combination of modeling and experimental approaches, we uncovered new mechanistic insights that contribute to our understanding of epithelial morphogenesis:1) Epithelial cells in KV undergo volume changes that are asymmetric along the AP axis during morphogenesis. KV-ant cells increase in volume and KV-post cells decrease in volume. These AP asymmetric volume changes coincide with AP asymmetric cell shape changes.2) Ion flux is an intrinsic mechanism that regulates asymmetric cell volume changes and cell shape changes in KV.3) Mathematical models indicate that mechanical properties of external cells surrounding the KV can impact cell shape changes in the KV. Models predicted that when external cells are solid like, asymmetric cell volume changes in KV cells contribute to cell shape changes even in the absence of lumen expansion. In particular, we have uncovered a possible mechanism for KV cell shape change: if the interface between KV-ant and KV-post cells was pinned, the volume changes directly affects the apico-basal dimension of the cells while the apical area would remain constant. Hence, asymmetric cell volume changes can induce asymmetric cell shape changes.4) Experiments determined that asymmetric cell volume and shape changes are separable from extrinsic biophysical forces associated with lumen expansion.Taken together, these results demonstrate ion flux serves as an intrinsic mechanism to regulate asymmetric epithelial morphogenesis in the KV organ and that changes cell morphology can be uncoupled from mechanical forces exerted during lumen expansion.These take-home points have been clarified throughout the revised text.[Editors' note: the author responses to the re-review follow.]Reviewer #2:[…] Although the conclusion from the latter part is convincing, what drives asymmetric cell shape changes is largely unclear from the 3D analysis other than involvement of ion channels. The authors emphasize that live embryos are imaged at one stage to avoid potential photo-bleaching. This approach is good to initially identify the crucial parameters and duration. However, to identify the mechanisms underlying asymmetric cell shape changes requires the analysis of single cell behavior continuously during the critical time points (from 4ss to 6ss) based on time-lapse movies.Thank you for your comments and this suggestion. We have now performed time-lapse imaging of mosaic labeled KVs between 4 ss and 6 ss and quantified volume dynamics of single cells at 5 minute intervals during this critical time point. This has allowed us to track and quantify changes in KV-ant and KV-post cell morphology in real time. Consistent with our results from analyzing ‘snap shots’ at specific developmental stages , we found that individual KV-anterior cells increase their volume between 4-6 ss and KV-posterior cells decrease their volume. This live imaging approach sheds light on KV cell behavior and how these cells change their morphology during morphogenesis. Our new results are presented in There are several concerns to be clarified.1) It is confusing about the term "asymmetric cell volume changes", which reflects asymmetric cell shape changes in length and width but not in height . What isWe use the term ‘asymmetric cell volume changes’ to describe regional changes in KV cell size along the anterior-posterior (AP) body axis: KV-anterior cells increase in volume over developmental time, whereas KV-posterior cells decrease in size. We apologize for the confusion, and have now clarified this in the Results section of the manuscript .Changes in cell volume are not necessarily tied to changes in cell shape: cells can change volume but remain the same shape or change shape without changing volume. Results presented in this paper indicate KV cell volume changes contribute to KV cell shape changes. Inhibitors of ion flux inhibit lumen expansion and AP asymmetric KV cell volume changes. This indicates ion flux, which drives fluid movements, is necessary for both lumen expansion (previously published) and KV cell volume changes (not surprising and consistent with a role for ion flux in cell volume changes in other systems). When we looked at KV cell shape changes by length and width (2D) in embryos treated with ion flux inhibitors, they did not occur. This indicates that KV cell shape changes depend on 1) lumen expansion, 2) KV cell volume changes or 3) both. Mathematical modeling suggested that KV cell shapes could change if KV cell volume changes occurred without lumen expansion. We next tested this experimentally. When we leave ion flux and asymmetric KV cell volume changes intact and only inhibit lumen expansion, KV cell shape changes (measured by length and width in 2D) occur normally. Taken together, this suggests that KV cell shape changes do not depend on full lumen expansion, but do depend on asymmetric KV cell volume changes. Thus, mechanistically, KV cell volume changes are required (drive) for cell shape changes. This has been clarified in the first paragraph of the Discussion section.As suggested, we have changed the title to “Cell volume changes contribute to epithelial morphogenesis in zebrafish Kupffer’s vesicle.”2) Despite the fact that the authors found out the crucial time point in this process between 4 ss and 6 ss , the majThank you for suggesting this experiment to test the effect of inhibiting ion channels during the critical stages of 4 to 6 ss. Our rationale for starting pharmacological treatments at the tailbud stage was to allow the compounds to penetrate inside the embryo and block ion channel function in KV throughout the lumen expansion process. We have now repeated the mosaic-labeling experiments with ouabain treatments that started at 4 ss and then imaged KVs at 6 ss. Over the course of several trials, we found, unfortunately, that treatments between 4-6 ss did not reduce KV lumen expansion  were not important in this process?We apologize for not making this clear. In wild-type embryos, KV lumen expansion involves extension of apical surfaces of both KV-ant and KV-post cells in dorsoventral axis  I am uncertain about how informative the modeling part is . The verThe purpose of our vertex model simulations was not a literal simulation of the precise time-dependent cell shape changes in vivo. As the referee correctly points out, we would be missing a lot of information for that . Rather, the purpose of our simulations was to identify potential mechanisms that could induce an asymmetric LWR in the KV cells. Since we do not know the values of the cell-cell interfacial tensions and the properties of the surrounding cells, we scanned the parameter space for many possible combinations of both, and we found there was a large regime where asymmetric cell volume change drives asymmetric KV cell shape change. Based on these simulations, we could identify a potential mechanism that translates volume asymmetry into cell shape asymmetry . Then we4) There is inconsistency of the data upon ouabain treatment at 2ss . The forThank you for pointing this out. We have now quantified the effect of ouabain on KV lumen expansion and present these results in 5) What is the biological significance of cell height changes at 2 ss in embryos treated with ion channel inhibitors or lgl2-morphant embryos? It is confusing, as cell height does not significantly change between anterior and posterior cells from 2ss to 8ss.Height changes reflect reduced lumen expansion as compared to controls . This difference is already significantly different at 2ss in ouabain, CFTR inh, CFTR MO treated embryos and becomes significant by 8ss in Lgl2 MO treated embryos. In wild-type and all treated embryos, height responds similarly in both anterior and posterior cells to lumen expansion (or lack thereof).Reviewer #3:[…] 1) Inhibiting ion channels inhibits cell shape changes/asymmetric cell changes in KV are separable from lumen expansion.The statistical analysis. Ouabain treatment and cftr knock down lead to similar conclusion which is great. However, both experiments are based on the fact that the results are not significant in the treated embryos at 2SS and 8SS. For this type of analysis, the authors should provide a power analysis to demonstrate that the sample size is enough to conclude about not significant results. Similar comment for jupa and igl2 treatments.cftr MO and lgl2 MO to increase the sample size of both KV-ant and KV-post cells. We have updated We have repeated the treatments with ouabain, 2) Mechanical properties of the surrounding cells. In simulations, asymmetric cell volume changes could account partially for cell shape changes, provided that the mechanical properties of the cells satisfy certain conditions.The issue about mechanical properties of cells is important. The mechanical properties of surrounding cells are important and need to be solid like at least anteriorly. Please provide reasonable explanation as to why anterior and posterior could be solid like and more generally, what are the arguments implying that mesodermal cells are solid like (this can be part of the Discussion). The 'pinning' hypothesis is interesting in that aspect and could be discussed. Lance Davidson’s work could be discussed as it seems that the notochord is a pretty rigid structure from his work. In addition, could the fact that fibronectin is enriched in the anterior side of the KV could increase the possibility that cells are solid like anteriorly?Our modeling results indicate that asymmetric cell shape changes in KV are more pronounced if only the anterior external cells have solid-like mechanical properties than if only the posterior external cells are solid-like, as the solid-like cells on the anterior side are able to "pin" the interface between the KV-ant cells and the KV-post cells. In order to determine general mechanisms, we have indeed kept the model very simple – for example, it does not take into account effects of additional tissues/structures (e.g. notochord) or extra-cellular matrix (ECM). It is possible that the notochord – which physically interfaces with KV-ant cells – may be a solid-like structure, and this may provide the pinning mechanism. Additionally, an AP gradient of ECM may help prevent neighbor exchanges and give rise to solid-like behavior only in anterior external cells. It is also possible that the solid-like ECM directly physically pins the KV-ant cells, so that the mechanism we have identified may operate even if the anterior cells beyond the ECM are fluid-like. These possibilities have been expanded upon in the discussion. In addition, we have provided new 3) Cell volume changes and actomyosin. Given that their simulations show that cell volume changes alone could not account for cell shape changes, it is weird that in Thank you for bringing up this important point. We propose that cell volume changes contribute to epithelial morphogenesis in KV and are likely linked to additional important factors  that are not currently in our model. We are quite intrigued about the relationship between cell volume changes and actomyosin contractility in KV epithelial morphogenesis. As suggested here, we used antibodies that detect phosphorylated myosin light chain (pMLC) to test whether treating embryos with ouabain alters non-muscle myosin II activity in KV cells. Our group  and the Heisenberg group  have previously used this pMLC antibody to qualitatively assess myosin II activity in KV. Over the course of several trials, we detected cortical pMLC staining in control and ouabain treated KV cells that localized to the cortex of KV cells marked by the tight junction protein ZO1 see . HoweverTg(actb2:myl12.1-MKATE2) embryos that express the fluorescent mKate2 protein fused to myosin light chain (Myl12.1). We and others  have found that the accumulation and intensity of Myl12.1-fusion proteins correlates with phosphorylated myosin II antibody staining intensity, and thereby serve as a good proxy for active myosin II. Embryos from homozygous Tg(actb2:myl12.1-MKATE2) parents were treated with ouabain starting at the tailbud stage and then KVs in live treated and control embryos were imaged at the 6 somite stage  The conclusion 'Asymmetric cell volume changes regulate epithelial morphogenesis'. It should be toned down providing that we do not know if the surrounding cells are solid like and that we do not know how the actomyosin network is compromised by the treatments used. The term 'regulate' seems too strong at this point.We have toned down the conclusions and changed the title to “Cell volume changes contribute to epithelial morphogenesis in zebrafish Kupffer’s vesicle.”


Despite persistent clinical pain in patients with painful diabetic neuropathy, longer pain duration was associated with more efficient CPM. Alleviation of pain, by either medical or surgical therapy, is accompanied by transition from less efficient, or pro-nociceptive, to efficient conditioned pain modulation (CPM). Spontaneous decrease or resolution of pain with disease progression is reported for some patients with painful diabetic neuropathy (PDN).To explore whether CPM changes similarly in parallel to spontaneous resolution of pain in PDN patients.In this cross-sectional study, thirty-three patients with PDN underwent psychophysical assessment of pain modulation on the forearm, remote from the clinical pain.P = 0.025, Spearman correlation). Patients with pain more than 2 years (median split) expressed efficient CPM that was not different from that of healthy controls. These patients also had lower temporal summation of pain than the short-pain duration patients group (P < 0.05). The 2 patient groups did not differ in clinical pain characteristics or use of analgesics.Pain duration was not correlated with neuropathic pain intensity, yet, it correlated with CPM efficiency; patients with longer pain duration had same pain level, but more efficient CPM than those with short-pain duration (ρ = −0.417; Pro-nociception, expressed by less efficient CPM and high temporal summation that usually accompanies clinical painful conditions, seems to “normalize” with chronicity of the pain syndrome. This is despite continuing pain, suggesting that pro-nociceptivity in pain syndromes is multifactorial. Because the pain modulation profile affects success of therapy, this suggests that different drugs might express different efficacy pending on duration of the pain in patients with PDN. Similar to other pain states, patients with painful diabetic neuropathy (PDN) demonstrate enhanced activity of facilitatory pain pathways40 Studies on surgical and medication treatments for pain have shown that the pain alleviation is accompanied by improvement of pro-nociceptivity, with transition from less efficient to efficient conditioned pain modulation (CPM).49 However, it is unknown whether the spontaneous alleviation of neuropathic pain along with PDN duration is similarly associated with reversal of pro-nociceptivity.An interesting feature of the neuropathic pain of diabetic origin relates to the reports of spontaneous pain improvement or resolution with disease progression.The aim of this study was to evaluate pain modulation in PDN of different pain duration and its association with pain levels. We hypothesized that longer neuropathic pain duration would be associated with lower clinical pain levels and reversal of the pro-nociceptive characteristics of pain modulatory system.49). Patients met the following inclusion criteria: (1) lower limb distal neuropathic pain for more than 3 months and (2) mean pain severity during the last month of ≥40 on a 0  to 100 (“most painful imaginable sensation”) numerical pain scale (NPS). The exclusion criteria were as follows: (1) intake of a monoamine oxidase inhibitor (MAO-I) within the last 14 days, (2) narrow-angle glaucoma, (3) inability to undergo psychophysical testing, (4) pain complaints at the upper limbs, and (5) chemotherapy for cancer and known vitamin B12 deficiency. Patients who were on regular use of serotonin–noradrenaline reuptake inhibitors (SNRIs) were tested after 1 month drug washout.Thirty-five patients with PDN participated in this study. The diagnosis of PDN was confirmed by a qualified neurologist or endocrinologist with special training in diabetes care. This article reports the results of baseline (pretreatment) assessment of the patients cohort who then underwent treatment with duloxetine  collected under same psychophysical protocol as for patients with PDN served as the control group. The inclusion criteria for the control group were as follows: (1) absence of chronic pain history; (2) no use of analgesic or psychiatric medication on a regular basis; (3) age between 40 and 80. All participants were able to communicate and understand the instructions of the study and were asked to refrain from any pain relief medications 24 hours before the experimental trial. The study was approved by the local ethics committee, and informed consent was obtained from all participants.All experiments were conducted in the same setting by the same experimenter (H.N.-A.) at the Laboratory of Clinical Neurophysiology in Rambam Health Care Campus, Haifa, Israel. Subjects sat comfortably in a quiet room with an ambient room temperature of 22°C to 23°C.Patients were asked to report duration of (1) diabetes, (2) sensory neuropathic symptoms such as, (3) mean and maximal neuropathic pain intensity during the last week, and pain duration. In addition, the information regarding current or past pain-relieving drugs consumption was collected.2 contact thermode according to the method of limits.45 The 32°C baseline temperature decreased or increased at a rate of 1.0°C/s and was stopped by pressing the response button at the moment that warm or cold sensation was perceived. Warm detection threshold and CDT were calculated as the mean values of the 3 measurements. The mechanical detection thresholds (MDT) were measured using von Frey filaments . The weight was increased using consecutive filaments until the patient reported that sensation was perceived. Each filament was applied 3 times until the filament pressure was detected in at least 2 of the 3 trials. The first filament to be detected at ≥2/3 times was determined as the MDT.As part of a clinical examination, sensory detection thresholds were measured at the dorsum of the foot to assess neuropathy severity. Warm detection thresholds (WDT) and cold detection thresholds (CDT) were measured 3 times each using the thermal sensory analyzer (TSA-II)  with a Peltier 30 × 30 mmAll participants, the patients with PDN and the control subjects, underwent CPM and temporal summation (TS) assessments to assess pain modulation. All tests were delivered to the volar aspect of the forearm. This site is less commonly affected by the neuropathy, allowing normal or near-normal peripheral sensory function. This is because genuine assessment of the central effect depends on intactness of the peripheral conduction of neural signals.Mechanical temporal summation of pain (TS)—the stimuli were delivered with an 180gr (# 6.45) von Frey filament applied to the volar forearm. Subjects were exposed to a series of 10 stimuli with an interstimulus interval of 1 sec, applied within an area of 1 cm in diameter. Subjects were asked to rate the level of pinprick pain intensity using verbal NPS for the first and the tenth stimuli. The difference between the tenth and the first pain scores served as a score of the TS.10 The baseline temperature was 32°C, which increased at a rate of 2°C/s to the destination temperature, which was held for 30 seconds, and decreased back to baseline at the same rate. Subjects were asked to rate the intensity of the test stimulus along the stimulus at the 10th, 20th, and 30th second. The mean pain score served as the pain level of “test stimulus.” After a 15-minute break, subjects were exposed to the conditioning stimulus by immersion of the nondominant hand into a hot water bath at 46.5°C  up to the wrist for 1 minute. Water-induced pain was rated every 10 seconds during the first 30 seconds of immersion; the mean score of the 3 ratings of the immersed hand served as the pain level of the “conditioning stimulus.” During the last 30 seconds of the conditioning stimulation, an identical test stimulus was repeated, and pain of the test stimulus was rated again every 10 seconds. The CPM effect was calculated as the difference between 2 test-stimuli applications: the conditioned test stimulus minus the stand-alone test stimulus, for each single individual pair of stimuli, obtained at 10, 20, and 30 seconds of test stimuli , and as CPMmean for averaged 3 CPM values. More negative values indicated more efficient CPM. This paradigm of conditioned pain stimulation is routinely used in our laboratory.43Conditioned pain modulation was assessed using the parallel paradigm in which 2 identical test stimuli were given; once before and then simultaneously with a noxious conditioning stimulus. The test stimulus was a tonic noxious contact heat stimulus applied to the volar aspect of the dominant forearm using TSA. The intensity of the test stimulus was predetermined individually based on the psychophysical parameter of Pain60 temperature. This method was based on delivery of several triplets of 7-sec long stimuli of various intensities; the closest temperature that induced pain at a level of 60 on a 0-100 NPS was considered as the Pain60 temperature.35 and anxiety levels .34 The patients with PDN also reported their self-perceived health by filling out a Hebrew version of the short-form 36 (SF-36) Health Survey.20 SF-36 contains the following subscales: vitality, physical functioning, bodily pain, general health perceptions, physical role functioning, emotional role functioning, and social role functioning. Higher scores on each summary scale agree with better general health.At the beginning of the experimental session, all participants completed several pain-related psychometric questionnaires for the assessment of pain catastrophizing P < 0.05. The data are presented as mean (SE) if not indicated otherwise. Statistical analyses were performed using Microsoft Excel  and JMP .The primary interest of this study is to characterize the relationship between pain duration and pain psychophysics. Preliminary examination of data indicated that skewed pain duration data distributions, with some extreme values, would result in problematic Pearson correlations, with extreme points having unacceptably high leverage. Therefore, nonparametric Spearman (rank-order) correlations were performed to test overall monotonicity of relationships between neuropathic pain level and duration and psychophysical measures. To further examine the effect of pain duration on pain modulation profile (PMP) characteristics, additional analyses were conducted by dividing the patients into 2 groups based on the median score of pain duration. The results of 2 subgroups of patients were compared with the control subjects with the addition of age as a covariate, in a regression model. The post hoc analysis was performed to confirm the group differences. The level of significance was set at P = 0.004); therefore, subjects' age was added as a covariate into the regression models.Two patients did not provide full information about their neuropathic pain duration, so the final sample of patients with PDN consisted of N = 33 . Demographic and clinical parameters as well as the values of PCS and STAI of the patients with PDN as well as the results of sensory quantitative sensory assessment are presented in Table P < 0.001); (2) higher severity of the neuropathy as measured by higher CDT  and warm detection threshold , all tested on feet. Because no consistent responses were obtained to the assessment of mechanical detection threshold, these data are not reported. It is important that the pain duration did not correlate with neuropathic pain intensity  or age .Patients with longer pain duration had (1) higher duration of neuropathy  or its temporal segments; CPM10 , and CPM20  or CPM30 . In line, no correlation was found between pain intensity and TS . However, looking at the relationships between pain duration and pain modulation capabilities, we found that patients with longer pain duration had (1) increased efficiency of CPMmean  and CPM20  but not for CPM30 , (2) a trend toward lower TS magnitude .No significant correlations were found between the neuropathic pain intensity and CPMmean  and 20 seconds (CPM20) of the CPM assessment, as compared with short-pain duration patients, and were not different of those from the healthy controls.Patients were divided into 2 groups based on the median split of the pain duration. Patients with pain duration of 2 years or less  were considered as short-pain duration group, whereas patients with pain duration longer than 2 years  were considered as long-pain duration group. The results of 3 group comparisons for psychophysical variables are presented in Table r = 0.055; P = 0.784), anxiety state , or PCS . Comparing the long- and short-pain duration PDN patients with the healthy subjects, no significant group differences were observed for the scores for anxiety state or trait; the PCS scores were significantly higher in the patients with PDN, with no differences between the 2 patient groups , or for the neuropathy severity in terms of foot warm  or cold sensation threshold  . In addition, the short- and long-pain duration patients were not different for any component of the SF-36 Health Survey . In addition, the number of used drug families was similar between short- and long-pain duration patients with PDN .Among the 33 patients, 17 (52%) were currently using pain medications . The proportion of patients on pain medications was not different between the short (10 patients with and 10 patients without medications) and long  pain duration groups , confirming a methodological advantage of shorter CPM assessment.11 Second, less-efficient CPM at early stages of painful neuropathy may suggest higher efficacy of mechanism-based analgesic choice (such as serotonin-norepinephrine reuptake inhibitors) during the first years of the neuropathic pain, whereas other treatment choices may be preferable for the long-time pain sufferers. This approach, however, should be confirmed from a study on a large group of patients with PDN.Our results shed light on 2 additional aspects of potential clinical implication of PMP; first, the significant relationship between pain duration and CPM was restricted to first 20 seconds of dual stimulation (CPM14 However, this issue is far from being conclusive because of contrary findings.38 The other limitation is that this is a cross-sectional study; the ideal design for a study that relates to durations is a longitudinal study. Because no prospective follow-up was designed, we rely on patients' memory as far the durations of both neuropathy and pain, rather than on documented data.Our main study limitation is the quite small sample, which is a general problem in clinical research. In addition, the middle-aged control subjects were significantly younger than the patients with PDN. Although age was included as a covariate in all group comparisons, the younger age of control subjects can be a potential bias for the results on CPM because of several reports on more-efficient CPM at younger ages.To conclude, this is a first report on the association between duration of neuropathic pain and pain modulation. Pain modulation seems to return to normal for longer duration of pain, whereas clinical pain levels seem to persist for a long time, at least in our cohort of patients with PDN. Our findings on the difference of antinociceptive PMP in PDN patients with longer pain duration highlight the significant role of the “time window” in which the assessment of PMP has clinical meaning and should be considered as a factor in treatment choice and evaluation of treatment efficacy.The authors have no conflicts of interest to declare.This study was sponsored by an IIT grant from Eli Lilly Inc.Y. Granovsky contributed to logistics of this study, data analysis, and the manuscript preparation. H. Nahman-Averbuch contributed to data collection. M. Khamaisi contributed to the patients recruitment and consenting. M. Granot contributed to data analysis and the manuscript preparation. All the authors approved the final draft of the manuscript.