Organ-Founder Stem Cells Mediate Post-Embryonic Neuromast Formation In Medaka

  1. Karen Gross
  2. Tuğçe Raif
  3. Ali Seleit
  4. Jasmin Onistschenko
  5. Isabel Krämer
  6. Lazaro Centanin

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Abstract

Mammals display a species-specific number, size and location of organs exclusively built during embryogenesis. In fish and amphibians, however, organs must adapt to life-long growth either by expanding in size and/or increasing in number. Here we use neuromasts, small sensory organs that increase in number as fish grow in size, to explore organogenesis during post-embryonic stages. Using iterative imaging, we reveal that post-embryonic organogenesis in the medaka caudal-neuromast-cluster (CNC) is mediated by organ-founder stem cells that delaminate from a functional neuromast. Organ-founder stem cells undergo epithelial-to-mesenchymal (EMT) transition as shown by molecular markers and cellular rearrangements. Chemokine signaling controls the dynamics of organ-founder stem cell delamination, which occurs at a stereotypic position that endures experimental and genetic perturbations. 2-photon laser ablation experiments reveal that organ-founder stem cells are rapidly reconstituted and suggest that these do not constitute a pre-defined population but are rather specified in situ . Our findings contribute to better understanding physiological stem-cell mediated organogenesis, a growth strategy present in life-long growing vertebrates. We speculate that a similar strategy could operate in vertebrates with determined-size as a template for pathological conditions like metastasis, where cells detach from their original organ and expand remotely.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors ask the important question whether post-embryonic organ formation follows the same mechanisms as during embryonic development. They focus on neuromasts of the lateral line system in the caudal fin of medaka. They used live imaging to find that the post-embryonic caudal-neuromast-cluster develops from organ-founder neural stem cells as a bud (and not as individual migrating stem cells as in zebrafish) that detaches and migrates from the founding neuromast (P0). They show that the formation of post-embryonic neuromasts does not require the lateral line nerve, which establishes another difference to the process in zebrafish. Artificial reduction in Cxcr4b chemokine signalling slows down stem cell delamination, which invariably occurs at the anterior aspect of the P0-neuromast. They then show (via changes in cadherin-type gene expression and cellular imaging) that stem cell delamination from the P0 neuromast involves an epithelial-to-mesenchymal transition. Forcing this process in the entire neuromast accelerates new organ formation, but directionality is maintained. Finally they ask whether the stem cells required for this process are pre-specified or are generated from the neuromast by ablating the pre-bud stem cells. They find that other stem cells within the same organ rearrange and re-establish organ founder cells. The authors liken this new mechanism of organ formation to pathological metastases in humans and name it metastatic-like organogenesis.

    Major comments

    • In Fig. 2D both P0- and PE1-neuromasts appear with fewer hair cells, the reasons for this should be explained. Did the ablation also damage the primordium or is the pLL nerve required for complete neuromasts to form?
    • p. 6: The reasoning behind generating a cxcr4b l-o-f mutant does not become clear, since a mutant already existed with a non-migratory primordium. Why did the authors expect their mutant would have a different phenotype?
    • p. 7: The authors state that K15::cxcr7 larvae lack secondary embryonic neuromasts, but it seems from Figs. 3B,D that they might simply be delayed (note that the last one is missing in 3B). This delay may have been taken over from the delay in embryonic primordium formation of P0 and this result (as shown in Fig. 3) would not contradict the assumption that PE1 can form in the absence of cxcr4b signalling. I suggest that the authors actually show and quantify that K15::cxcr7 adults have fewer CNC neuromast numbers, because this seems to be the definitive proof that overexpressing the "sink" may be enough to reduce cxcr4b signaling to a level where its requirement for the formation of the PE1-neuromast can be assayed.
    • Fig. 6A': The scheme is at least ambiguous and interpretation of it requires more supporting information: In A the stippled lines represent position within the neuromast, but what do they represent in A', numbers of BrdU+ cells? So what does e.g. the grey peak at the anterior mean - position or number of cells? Is the area inside the coloured lines important or the edge-points? Stage III is the only distribution with a clear left-bias, the others are centered (with a left-extreme for stage II), so what in the figure is the anterior proliferation peak? These are just some questions this reviewer had. Maybe the problem lies in the octagonal lines, meaning different things in both images? It is further unclear how the means given in the text can be derived from the figure. Maybe it would be best to try to represent the data with a heat map-overlay of the image in A, one for each stage?
    • The authors propose two competing models regarding the origin of founder stem cells (p. 9, first sentence: early determination vs. in situ generation). In the third sentence again two scenarios are presented as to why experimentally prompting EMT does not trigger organ founder cell migration. This paragraph would benefit from stating more precisely which of these questions is addressed by the BrdU- and ablation experiments, together with a clearer statement at the end of that section as to which hypothesis in each case (origin and migration) is preferred.

    Minor comments

    • K15+ cells are described as neuromast stem cells and Fig. 1 suggests that these are the mantle cells: Please comment on the question whether all mantle cells are stem cells.
    • p. 5: The reference (Seleit, Krämer et al., 2017) is ambiguous, as there are 2 references listed that fit the abbreviation.
    • Fig.1B-F': Even though, as the authors state, there is variation in the timing of the budding process, it would be helpful to add an exemplay time frame to stages I-V.
    • Fig. 4A'-D': The cell bodies of the support cells should have a distinct colour, otherwise they are easily confused with the nuclei of the other cell types. This would make it easier to understand the schematic at first sight.
    • p. 9: T2A and H2A should be explained.
    • Nuclei are shown protruding posteriorly in wildtype neuromasts (Fig. 5A-A'), while P0 neuromasts stem cells protrude anteriorly. Please explain the significance of the difference.
    • Fig. 5 legend: Quantification is "E"; "and increased" should probably read "an increased"?
    • p. 10: Unconventionally, Fig 7 is mentioned prior to Fig. 6B-C, I suggest combining both figures into one.

    Significance

    The manuscript describes a new mechanism of post-embryonic organ formation. Investigating how accesory neuromasts are formed during growth of juvenile medaka, the authors find that stem cells from a founder neuromast undergo epithelial-mesenchymal transition and migrate away directionally to form a complete new organ. This new mechanism is likened to that of cancer metastases.

    Importantly, and different from zebrafish, this process is not dependent on innervation of the neuromast and is not a budding process, but relies on neural stem cells leaving the organ.

    The interesting question posed by the authors, and answered here positively for accessory neuromasts in juvenile fish, is whether the mechanisms of organ formation differ between embryonic and post-embryonic development. The reported findings should be of interest to the stem cell communities and to researchers interested in post-embryonic development.

    This review was written by a developmental biologist.

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    Referee #2

    Evidence, reproducibility and clarity

    Authors study how new sensory organs of the lateral line form post-embryonically in medaka fish.

    Evidence, reproducibility, and clarity

    Overall, I find the study very preliminary, since hardly any conclusions are sufficiently backed by quantifications and statistical analysis as detailed below.

    Major comments:

    Figure 1: Authors describe events as "detached cells migrate" in the results text and in the legend as "detached stem cells...move". They should clarify how these images were exactly generated, since this has implication on whether they can conclude that cells migrate. Are these frames from movies in which the entire process was seen to occur in individual larvae? Are they derived from repeated imaging of the same larvae? Or are they images from different larvae and authors assume that the sequence of events occurs as presented? While it's likely that cell migration occurs, a wave of de novo transgene expression could also travel through the tissue. Thus, authors can only conclude that it's migration if they have seen cells move relative to their surroundings using live imaging or via lineage-tracing. e.g. using a photoconvertible fluorophore.

    Page 5: "The process of post-embryonic neuromast formation in the CNC displays variability in timing". Authors should add information on the actual timing. This relates to issue 1, maybe they don't know, but if they did live imaging or repeated imaging of the same larvae they should have information on how long it takes from delamination of cells to new neuromast formation.

    Figure 2A: where is the data showing that the laser ablation actually worked and supporting the statement "All ablated fish showed escaping cells from the P0-neuromast (N=7/7 fish)"?

    Figure 2B-D: I find it difficult to see how these data support the conclusions the authors draw. First of all, it would be helpful to show the structures labeled by the individual transgenes for the non-initiated readers, so readers can judge themselves which of the signals are derived from the kremen and which from the eya1 transgene. Second, in B' readers are supposed to "Notice the uninjured primordium that continued migration and embryonic neuromast formation", yet where is the uninjured primordium? There are a lot of signals in the image. And how do we know that it "continued migration?" Relative to what? Finally in D, the label "PE1 neuromast" is printed in between a structure that is weakly and one that is strongly labeled. Do the authors suggest that there are 2 PE1 neuromasts here? Both the P0 and the P1 neuromast(s?) on the experimental side seem to be much smaller that on the control side, but authors do not mention this. Does this show that neuron proliferation or differentiation are nerve dependent?

    Cxcr4 mutant: it's unclear why the authors mention the new mutant, albeit they don't use it. The only reason to justify this would be if the community were to benefit from this new allele. However, this requires that authors clarify how it differs from the previously existing one (e.g by showing the predicted protein sequence) and whether it has any advantages, e.g. is it more likely to be null? Further, where are the data supporting this statement? "cxcr4bD625 larvae display the same phenotype as the previously published cxcr4b mutants".

    Figure 3: authors should validate the k15:cxcr7 line by showing cxcr7 (over) expression in k15+ cells using in situ hybridization.

    Figure 3B: how do the authors distinguish single and double transgenics? If they cannot and only assume that those that have a phenotype are the double transgenic they need to confirm this by genotyping embryos post imaging.

    Figure 3D: sample size is missing. Significance should be tested.

    "Adult Tg(K15::Cxcr7) fish display significantly lower CNC neuromast numbers compared to wild type fish (N= 5 WT fish, 3.8 organs per CNC; N= 4 Tg(K15:Cxcr7), 2.2 organs per CNC". Which statistical test has been used to support the use of the term "significantly" in this statement? Images should be shown to support this conclusion.

    Fig. 4C: authors should show YFP and CFP channels to allow readers to see why authors have false-colored 2 cells. What do they mean by the term "repolarization" that they put in the figure label?

    Fig. 4D: "Subsequently, the organ-founder stem cells start elongating lamellipodia-like processes in the anterior direction". How do the authors know that this is "subsequently"? These are obviously not frames from a movie, so how do they know that the cells the point their arrow at delaminated from the neuromast considering that there are also quite a few other YFP+ cells in this frame?

    Fig. 4C, D: These observations must be supported by a least rudimentary information on reproducibility. How many neuromasts have the authors analyzed? How often did they see this?

    Fig. 4F, G: These conclusions must be supported by quantifications and statistics (e.g. E- and N-cadherin staining intensities).

    Fig. 5: is the number of neuromasts with protruding cells significantly different between the control and experimental group? % should be rounded to significant figures.

    Fig. 5D: these data are not convincing since it is unclear how authors identify the neuromast, considering that almost all cells seem to contain H2B-GFP.

    Fig. 5E: is there a significant difference?

    Fig. 6A: Exemplatory images of BrdU+ cells should be shown.

    Fig. 6A: "This approach revealed a proliferation peak at stage I-II (mean: 6 and 5.4 BrdU+ cells, respectively". This statement does not support that this is a peak in the absence of information on how much proliferation there is at other stages. Why don't the authors show a graph plotting this with variation and statistical analyses to support that the numbers differ at different stages.

    Fig.6A: also the conclusion that more proliferation happens in anterior positions must be backed by statistics.

    Fig. 6: ablation data. Text mentions that PE1 formed in 7/8 ablated larvae, but images show differences between the control side, where PE1 has not yet formed and the ablated side where it has... Is this a non-representative image? Authors should clarify.

    Fig. 7: Supplementary Movie 2 does not work, so I could not review it.

    Fig. 7: Conclusions need to be supported by quantification and statistics, it's not sufficient to show only frames from one movie.

    Fig. 7: Where is the data supporting this statement "(mean: 0.05 BrdU+ stem cells; N=8 P0-neuromasts)"? How was the BrdU incorporation experiment performed?

    This statement needs to be supported by quantifications and statistical analysis: "We noticed that in most cases, the ablated P0-founder neuromast was considerably smaller than the non-ablated founder organ in the contralateral side (Fig.6 B',C)(N=4/7 larvae)"

    This statement needs to be supported by quantifications and statistical analysis: The PE1-neuromast, however, reached the regular size in all cases, regardless of the status of the P0-founder neuromast.

    Minor:

    I think the author pitch the fact that they are examining "organ-founder stem cells" a bit too aggressively. It would be more appropriate to stick to the term "mantle" cells in all figures that describe data and reserve the "organ-founder stem cells" term to text where they interpret their results. It's particularly strange that Fig. 4C is labelled with such an interpretation.

    Significance

    There are many interesting open questions about post-embryonic development and in particular about how novel structures/organs form in those species where this happens. Thus, the overall research topic of this study is very interesting. The model is also well suited to derive novel mechanistic insight into these questions. The problem with the study in its current form is that I find it quite anecdotal, since hardly any conclusions are sufficiently backed by thorough data, in particular there is a rather shocking lack of quantifications and statistical analyses throughout. However, if the authors back their conclusions with such data, it'll certainly make a very interesting paper.

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    Referee #1

    Evidence, reproducibility and clarity

    Groß and colleagues explore the mechanisms by which post-embryonic organogenesis occurs. To do so, they use medaka caudal fin neuromasts as a model, in which they follow the genesis of a new neuromast, PE1, from an existing neuromast, P0. Previously the group has established that ablation of the P0 neuromast results in the absence of PE1 neuromast formation, and lineage tracing confirmed that the P0 neuromast gives rise to all the neuromasts in the caudal-neuromast-cluster.

    To dynamically assess PE1 formation, the authors use a broad lateral line transgene reporter, labeling keratin 15-expressing cells (K15+). Through this method, they observe that anterior K15+ cells of the P0 neuromast detach from the organ, migrate anteriorly, and give rise to the PE1 neuromast. By ablating the posterior lateral line (pLL) nerve prior to PE1 formation, which innervates the P0 neuromast initially, the authors report that the pLL is not necessary for proper PE1 formation, as it still develops in the absence of this nerve. Further, the authors explore chemokine signals which may underlie this process. The authors discuss a previous finding that the Cxcr4b/Cxcr7/Cxcl12a signaling pathway has been shown to regulate primordium migration, which is an organ required for neuromast formation. Therefore, the authors modulated the signaling pathway by first generating a missense cxcr4b mutant, only to find that this resulted in the absence of P0 formation. To circumvent this issue, the authors instead overexpressed Cxcr7, a non-signaling receptor that competes with Cxcr4b for the Cxcl12a ligand, in K15+ cells. Results from these experiments displayed a role for the Cxcr4b/Cxcr7/Cxcl12a signaling pathway in the temporal regulation of post embryonic neuromast formation. At the developmental time that wild type fish develop post embryonic neuromasts, Tg(K15:Cxcr7) animals have reduced numbers of post embryonic neuromasts. However, the authors find that post embryonic neuromasts do form in these animals at a later stage of development. Finally, the authors show throughout the report that the organ founder stem cells, which go on to form the PE1 neuromast, arise exclusively from the anterior side of the P0 neuromast. They questioned whether some cells were pre-specified to become organ-founder stem cells or if all stem cells have the same capacity to generate a new organ. To understand this, the authors used laser ablation on the anterior side of the P0 neuromast and found that the neuromast cells were capable of rearranging and initiating migration from the anterior side.

    In all, this report highlights a potentially novel cellular mechanism by which a post embryonic neuromast is generated from a pre-established neuromast. By using longitudinal imaging, the authors were able to observe this process in developing animals and begin to probe its molecular mechanism. Several aspects of the manuscript should be strengthened prior to publication to more rigorously support the authors' model.

    Major Comments:

    A key aspect of the current manuscript is whether the morphogenetic process that they report is fundamentally different from the "budding" mechanism previously observed in zebrafish during neuromast stitch formation (Wada et al., 2010; Wada et al., 2013) or simply represents a different description of an analogous process in medaka. Inspection of the previous work in zebrafish reveals several similarities at the cellular level that leave this reviewer unconvinced that this is really a different process, rather than differences due to reporters used (membrane and nuclear here vs cytoplasmic previously). Specifically, Figures 2 and 3 in Wada et al., (2010) and Figure 2 in Wada et al., (2013) look quite similar to the morphogenetic process herein. Wada et al., (2013) describe stitch formation as: "a budding process that begins when a few cells elongate away from the founder neuromast". Here, on pg 4 Groß and colleagues describe a process that "is executed by a few organ-founder neural stem cells that detach from their original organ and migrate to generate a new organ remotely." How exactly are these two processes different? One key mechanistic difference is potentially the nerve dependence, but there are concerns with the interpretation of this experiment (see comment below). Unless the authors can definitively demonstrate that what they describe is a distinct process, they might be better served by reframing their results in light of previous findings by Wada and colleagues.

    A second important thrust of the paper is that migrating K15+ cells act as "organ founder stem cells." However, the authors do not show conclusive evidence of K15+ cells acting as stem cells during PE1 formation. To support their claim, the authors should more rigorously define the K15+ organ-founder cells as stem cells. This could be done by several approaches (e.g., via lineage tracing, molecular analysis, or live-cell imaging). Importantly, it has previously been shown that zebrafish hair cell regeneration is predominantly driven by surrounding support cell proliferation (reviewed by ​​Lush and Piotrowski, 2014 doi: 10.1002/dvdy.24167). Therefore, it is important for the authors to determine whether their organ-founder cells are in fact stem cells or, alternatively, migrate along with K15- support cells from the P0 neuromast.

    The rates of post-embryonic development in teleost fish, including medaka (Iwamatsu et al., 2003 doi: 10.2108/zsj.20.607), depend on several housing conditions (e.g., rearing density and feeding). It was surprising to this reviewer, therefore, that the authors performed all of their staging by days post-fertilization rather than standard length. Results from most experiments, especially those in Figures 1 and 3, would be more easily interpretable (and reproducible across different labs) if authors were to report standard length of the animals being used. For example, are Tg(K15:Cxcr7) animals smaller than their wildtype counterparts? If so, could this explain why PE1 neuromast formation is delayed? This goes for the interpretation of Figure 5E as well.

    The authors should include statistical analyses of all their quantification. For example, it would be appropriate to use a statistical test to compare groups in Figures 3D and 5E.

    The authors use the transgene Eya1:mCFP to visualize and laser ablate the pLL nerve, stating that Eya1 labels the nerve. However, a few sentences after introducing this transgene, the authors now use the transgene Kremen:mYFP to label the pLL nerve, and in double transgenic Kremen:mYFP; Eya1:mCFP fish, the Eya1 transgene is used to "assess the differentiation state of the putative newly formed PE1 neuromast". In the figure legend, the authors explain that Tg(Eya1:mCFP) labels the pLL in Figure 2A, but in Figure 2B it is used to visualize the primordium. The primordium is not the pLL nerve, and if the Eya1 transgene is labeling the primordium, and not the pLL (the text suggests it fulfills both roles in different scenarios) then the authors should repeat this experiment using the Kremen transgene to appropriately label the pLL nerve for accurate ablation. If, however, the Eya1 transgene labels both the primordium and the pLL (it is hard to tell from the double labeled images, as they are in grayscale), then this should be made explicitly clear.

    Consider tempering the interpretation of the pLL nerve ablation experiment. Since the neurons and associated Schwann cells are still present following the severing of the nerve, albeit at a distance, could the nerve not still signal via a diffusible paracrine molecule? Alternatively, the authors could ablate the pLL ganglion. Since pLL ablation is performed 18 days prior to imaging, it may be possible that there is nerve regeneration occurring. To make the authors' findings more convincing, use of a secondary reporter, or antibody staining with a pan-neuronal antibody to confirm the absence of neuromast innervation at 21 dpf should be considered. Additionally, in Wada et al., (2013), neuromasts still extend cellular processes after nerve ablation. Since Wada et al. uses a membrane reporter, and here we see the use of a nuclear reporter, is it possible that we are seeing the same results as Wada et al.? To enhance the strength of this figure, authors should also consider using a membrane reporter or performing IHC similar to that in Figure 4D, so that readers can visualize there is no cellular process connecting the P0 and PE1 neuromasts in this condition.

    Supplemental Figure 1 is a colorimetric in-situ hybridization image which depicts the localization of cxcl12 transcripts. The authors state that there is transcript expression in the vicinity of the P0 neuromast, however, the image is of poor quality and the expression does not appear spatially restricted to the anterior portion of the neuromast. Consider either discussing how a uniformly localized chemokine cue fits with their model and/or providing more detailed evidence of the expression pattern, e.g., by co-staining with a neuromast-specific marker and performing high-resolution imaging. Ideally the authors could quantify cxcl12 expression relative to the A/P axes of the P0 neuromast.

    In Figure 4, the authors provide evidence that: 1) K15+ cells in the anterior of the P0 neuromast change shape and extend invasive, lamellipodia-like protrusions, and 2) that a cluster of cells to the anterior of the P0 neuromast show decreased E-cadherin and increased N-cadherin staining. These results are consistent with a subset of K15+ cells undergoing a MET. However, the authors could strengthen this portion of the manuscript in several ways. First, is it possible to live-image delamination from the neuromast? This would provide unambiguous evidence in support of their model. Second, is it possible to combine E- and N-cadherin staining with visualization of the K15+ population? The authors state that "N-cadherin is clearly upregulated in migrating organ-founder stem cells", but at the moment the evidence is circumstantial that K15+ cells switch cadherin expression. Co-visualization of the K15+ population would strengthen this point. Ideally, the authors could quantify E- and N-cadherin levels in K15+ cells relative to neighboring cells to support their claim. Minor point: have the E- and N-cadherin antibodies previously been validated in medaka? If so, the authors should cite the relevant work. If not, the authors should provide evidence of antibody specificity.

    In Figure 5, the authors use Tg(K15:snail1b-T2A-H2A-mCherry) to address whether snail1b expression is sufficient to drive ectopic exit of cells from the P0 neuromast. The argument that snail1b is functional is an increase in neuromasts with protruding cells, but it is not demonstrated that any cells actually undergo EMT. A simple explanation for the observed lack of ectopic delamination is that snail1b is not expressed at sufficient levels from the transgene. With the current data, this reviewer would suggest tempering the claims related to interpretation of this experiment. On a related point, is snail1b normally expressed in delaminating cells? If so, this would provide further evidence to support the authors' EMT model in Figure 4. Minor point: the authors state that "not a single case of ectopic cell migration was observed when we analyzed the P0-neuromast by live imaging" - please clarify: what was the imaging time window and at what stage was the imaging performed?

    Minor Comments:

    Abstract, first sentence. This sentence is confusing since mammalian organs certainly grow at post-embryonic stages.

    On page 6 of the manuscript, in the "pLL nerve is dispensable for organ-founder stem cell migration and PE1-neuromast formation" section, the authors describe the transgenic animals used in their pLL ablation studies as, "double Tg(Eya1:mCFP) - to label the pLL nerve - (K15:H2B-RFP) - to visualize migrating neuromast stem cells". The phrasing of the transgenic description is cluttered and confusing to read. Authors should consider rewriting this description as something such as, "double Tg(Eya1:mCFP); (K15:H2B-RFP) animals were used to visualize the pLL nerve as well as migrating neuromast stem cells, respectively".

    On page 6 of the manuscript, in the "pLL nerve is dispensable for organ-founder stem cell migration and PE1-neuromast formation" section, the authors use the term escaping point. This is the first and only time the term is used, and it is not well defined. Presumably this refers to the anterior side of the P0 neuromast. This should be rewritten to more clearly articulate the meaning of this term.

    Could the authors refer the reader to Figure 1A in the corresponding section of the introduction? It might also be helpful to the reader to label the primary vs secondary neuromasts on the schematic diagram.

    In figure 2, the authors conclude that PE1 neuromast formation is not hindered by pLL ablation, however, in Figure 2C and D, it is apparent that the resulting PE1 neuromast post-pLL ablation is significantly smaller in size. The authors should address this, especially since they refer to the new PE1 neuromast in this condition as "mature". Is an organ mature if it is substantially smaller than in control conditions? Are the other resident cell types present in the proper proportions? Does the size of this organ grow to become wild-type further along in development?

    On page 6, in the results section for Figure 2C and D, Eya1:GFP is being used to visualize "post-mitotic neurons" in the P0 and PE1 neuromasts, however, Figures 2C and 2D look more representative of the K15 reporter. Also this reviewer is not aware of post-mitotic neurons within neuromasts. The corresponding figure legend states that Tg(Eya1:EGFP) labels neuromast hair cells. Could the authors please clarify what is being labeled?

    "Although specific for neural stem cells in the mature neuromast, the K15 promoter drives expression at earlier stages, after primary neuromasts were deposited by the primordium". This statement leads to confusion about the specificity of the K15 promoter, indicating it may be more broadly expressed than the authors state. The K15 promoter should be more rigorously described in the text, and evidence for its specificity should be clearly cited/provided.

    The last sentence of the first paragraph of the Discussion is unnecessary and possibly overstating the findings within the report, as no evidence for "hijacking" of this post embryonic neuromast formation process was assayed.

    The authors might consider discussing similarities and differences between their work and anchor cell invasion in C. elegans, which also involves post-embryonic organ remodeling by an invasive cellular behavior.

    Methods transgene construction - please provide concentrations of nucleotides and proteins injected.

    Methods Live-imaging section - "tranquilized" should probably read "anesthetized". More details on the imaging are needed. e.g., at what temperature was the imaging performed? What objective(s) was used?

    Methods BrdU section - how were animals fixed? Please also describe the antigen retrieval step in detail.

    It would be helpful for the supplemental movies to have labels for the transgenes, axes, and timestamps (as appropriate).

    Supplemental Movie 2 shows significant xy movement between timepoints. Perhaps registration of the timepoints would help eliminate this and make the movie easier to interpret?

    Significance

    Groß and colleagues present an intriguing new model for post-embryonic morphogenesis of neuromasts in medaka. However, in its current state it is unclear whether these findings truly represent a new model for organ morphogenesis, rather than an alternative description of a previously described process. If it is the latter, the manuscript still has new cellular and molecular insights, but should be reframed. This work is likely to appeal to basic scientists, e.g., developmental biologists interested in organogenesis and neurobiologists interested in cell-cell interactions. This reviewer has expertise in teleost development and organogenesis.

  5. Version published to 10.1101/2022.12.16.520711v1 on bioRxiv