Dendritic atoh1a+ cells serve as transient intermediates during zebrafish Merkel cell development and regeneration

  1. Evan W. Craig
  2. Erik C. Black
  3. Camille E.A. Goo
  4. Avery Angell Swearer
  5. Nathaniel G. Yee
  6. Jeffrey P. Rasmussen

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Abstract

Sensory cells often adopt specific morphologies that aid in the detection of external stimuli. Merkel cells encode gentle touch stimuli in vertebrate skin and adopt a reproducible shape characterized by spiky, actin-rich microvilli that emanate from the cell surface. The mechanism by which Merkel cells acquire this stereotyped morphology from basal keratinocyte progenitors is unknown. Here, we establish that dendritic Merkel cells (dMCs) express atonal homolog 1a (atoh1a) , extend dynamic filopodial processes, and arise in transient waves during zebrafish skin development and regeneration. We find that dMCs share molecular similarities with both basal keratinocytes and Merkel cells, yet display mesenchymal-like behaviors, including local cell motility and proliferation within the epidermis. Furthermore, dMCs can directly adopt the mature, microvilliated Merkel cell morphology through substantial remodeling of the actin cytoskeleton. Loss of Ectodysplasin A signaling alters the morphology of dMCs and Merkel cells within specific skin regions. Our results show that dMCs represent an intermediate state in the Merkel cell maturation program and identify Ectodysplasin A signaling as a key regulator of Merkel cell morphology.

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    Reply to the reviewers

    'The authors do not wish to provide a response at this time.'

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript from Craig et al., (2023) leverages a previously reported atoh1a reporter to drive expression of lifeact-egfp in Merkel cells (MC) to assess MC morphology during both scale development and regeneration, in the optically tractable zebrafish. Using a combination of live-imaging approaches and genetic perturbations, the authors show that MCs arise from a more immature population of dendritic Merkel cells (dMC) and that dMCs themselves derive from basal keratinocytes. The authors show that following injury, dMCs are the major cell type to infiltrate the regenerating scale region, with MCs becoming the predominant cell type at later stages of regeneration (presumably as the dMCs mature). The authors present evidence suggesting that dMCs are molecularly similar to both keratinocytes and MCs and argue that dMCs may represent an intermediate cell type. Data in the manuscript suggests MC and dMC protrusions are differently polarized, and that MC and dMC dynamics are also different. The authors provide direct evidence that dMCs mature into MCs morphologically and suggest that the reverse may also occur. Finally, the authors show that MC microvilli morphology is impaired in eda-/- mutants, suggesting a role for eda in the normal morphology of MCs, more specifically in the trunk.

    Major comments:

    1. The discovery and characterization of dMCs in this study relies entirely on their labeling by an atoh1a-lifeact transgenic reporter. Given the striking similarity of dMCs to melanocytes, it is important to confirm the atoh1a reporter labels dMCs and MCs specifically, and not melanocytes. For example, it would be useful to see confirmation of cell type by double labelling of dMCs, e.g. with atoh1a:lifeact-egfp together with an antibody for atoh1a or preferably, another MC/dMC marker. dMCs look morphologically similar to melanocytes, which also display many of the behaviors noted in this manuscript. According to RNA-seq data (see https://hair-gel.net/), atoh1 is expressed in melanocytes in embryonic mouse skin and hair follicle stem cell precursors in post-natal skin. We recommend that the authors mine a similar dataset for zebrafish to ascertain whether atho1a is also expressed in pigment cells (e.g. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=GSE190115). We would also recommend that the authors run a stain for a melanocyte marker such as Mitf/Tyr/Dct to show this is not expressed in dMCs.
    2. A major conclusion of the paper is that dMCs display molecular properties that overlap with both MCs and basal keratinocytes based on expression of three markers. I feel this conclusion is a little strong given the evidence presented; global transcriptomic analysis of these cells (RNA-seq) would better define where along a differentiation trajectory dMCs lie.
    3. More data regarding the function of the dMC intermediate cell type would greatly strengthen the significance of the study. The characterization of dMCs forms the core of the report, yet little is shown/discussed regarding the function of this cell population. For example, why is this intermediary even required? Presumably this is to facilitate the migration of MCs from the basal layer into the upper strata and their dispersion upon arrival. In this case, one could argue that the morphology of the dMC is directly related to its migratory function, as the authors suggest dMCs arise from basal keratinocytes, then migrate upwards towards the more superficial strata, where mature MCs are located. However, very little evidence in support of this upward migration is presented - most of the migratory data are related to lateral movement. Experiments to alter the migratory properties of dMCs, for example using inhibitors of Arp2/3, would address whether migration is the key function of dMCs. Finally, there is insufficient evidence to suggest contact-inhibition is occurring, and in the cell division movie 5, it doesn't appear to happen (or the movie isn't long enough to show it). More examples are required or this observation should be reworded accordingly.
    4. Eda is shown to be important for MC morphology, especially in MCs located in the trunk. More discussion of how eda may function would be helpful to the reader. For example, in what cells are Eda and Edar expressed? Do the authors think Edar signaling is cell autonomous within the MCs? Or does the loss of Eda indirectly affect MC morphology as a result of impaired scale formation? Additionally, the authors state that corneal MCs in both WT and eda-/- have similar microvilli morphologies. The figure, however, shows that corneal MCs from these genotypes do look different, with eda-/- corneal MCs having a more evenly distributed microvilli than the polarized microvilli of their WT counterparts. The metric '% of MCs with microvilli' does not capture this aspect of their morphology.
    5. In several places, the number of biological replicates is unclear. A major concern is that data presented as 'number of cells' may only have been collated from n=1 animal. The authors should specify the number of both biological and technical replicates per experiment and consider displaying the data in superplots. Where stats are undertaken, particularly on percentages, it should be made clear whether the stats test was perfomed on raw numbers or the % (particularly true for Chi square). Examples of this issue can be found in figures 3C-H, 4F-H, 5B-C and supplemental.

    Minor comments:

    • Line 124. Why did the authors choose developmental stages 11mm and 28mm for the quantification? The images in Figure 1 show 8, 10 and 12mm but not 11mm.
    • Line 126. It is unclear what the difference is between circularity and roundness.
    • Line 645 and Fig 1I. 'Cells manually classified as MC or dMC'. Please provide further clarification on this categorization (e.g. number of protrusions/roundness value etc.)
    • Line 141 and Fig 1O. The authors comment on the mosaic nature of DsRed expression, but it seems particularly sparse in the image. Similarly, there are numerous GFP+ cells that do not express DsRed, and the ones that do are found at a distance from the DsRed+ basal keratinocytes. Further explanation is required here. For example, if MCs ultimately arise from dMCs, why are so few of them labelled? It would be useful to know the % of cre-recombination that is actually occurring (i.e. how efficient the cre driver is in keratinocytes by DsRed+/total number) to put these data in context.
    • Line 170 and 179. The authors do not comment on the possibility of de/trans-differentiation of mature MCs as an explanation of how dMCs and 'new' MCs arise on regenerating scales.
    • Line 176. Can the authors comment on how quickly the nls-Eos protein turns over? This is pertinent given it is possible that by 7 dpp all the red nls-Eos could potentially have been replaced by green nls-Eos in an 'existing' atoh1a+ cell.
    • Figure 2M-P. Both channels (green and magenta) should be shown here. Cells will express both and it is unclear from the image panel what this looks like.
    • Line 186, 200 and 206. 'regenerating dMCs' this is confusing. Perhaps reword to 'dMCs associated with regenerating scales'.
    • Line 186. Why did the authors focus on 5dpp, particularly given that at 3 dpp the proportion of dMCs:MCs is more evenly spread?
    • Figure 3A-B. An additional panel with DAPI is needed here to enable Tp63 negative nuclei to be visualized. Also, what is the cell in the top right of 3B? It has a red nucleus but is not marked by an asterisk.
    • Figure 3D-E. This data panel also needs to show a dMC that is negative for SV2.
    • Figure 4D-E and line 235. It is intuitive that dMCs will not have basal facing processes if they are already in the basal layer of keratinocytes - there simply isn't the physical space (unless they penetrate the scales/basement membrane which presumably they don't). Also, the authors need to comment on, and quantify dMC protrusions in relation to the directionality of dMC migration in the main text. This is referred to in line 762 as part of the figure legend (Fig 5) and Movie 3 legend (line 809), but this is not quantified anywhere.
    • Line 258. How do these unipolar protrusions correlate with directionality?
    • Line 287 and Figure 5G. There is insufficient evidence to conclude that MCs can revert back to dMCs, particularly given that MCs are considered post-mitotic. N=2 (cells/fish?) is not sufficient without further evidence, and the MC depicted in Figure 5G doesn't resemble a bona fide MC at the start of imaging. Suggest removing this conclusion and data or increasing n and providing further evidence.
    • Line 394. 'These protrusions extended from lateral-facing membranes and interdigitated between basal and suprabasal keratinocytes'. Did the authors specifically show this? It is not clear from the data.
    • Line 430. The reference to Merkel Cell carcinoma needs more commentary with regards to the relevance of the authors' findings.
    • Line 491. Denoise.ai was used on images as stated. Can the authors confirm that any image quantification was done on raw images prior to using the Denoise.ai function?
    • Line 528. Include details of the tp63 antibody here.

    Significance

    Overall, the data are novel and of interest to researchers in several fields, including development, skin biology and MC carcinoma. This work provides an important step forward in our understanding of how basal keratinocytes give rise to MCs in zebrafish - via a dMC intermediary cell type. The imaging presented therein is of a high quality, and the movies are beautiful; capturing the cellular behaviors very clearly. This paper does not however, comment on the molecular mechanisms regulating this transition, nor on the cellular mechanisms resulting in the altered morphology and migration of dMCs and maturation into MCs. Inclusion of data as described above in the major comments section would increase the significance and impact of this work. Notwithstanding, the observations made in this work describe, for the first time to my knowledge, a morphologically distinct cell type in zebrafish (dMCs) similar to that having been described in other vertebrates and provide the ground work for future investigation.

    Reviewer expertise: skin biology, live-imaging, zebrafish, mouse, developmental biology.

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    Referee #2

    Evidence, reproducibility and clarity

    This work by Craig et al., defines intermediate steps in Merkel cell (MC) differentiation during development and regeneration in the zebrafish model system. Using live imaging, the authors describe a number of previously unappreciated steps that lead to the MCs differentiation from basal keratinocytes through a dendritic MC (dMC) intermediate. Live imaging of MSs' microvilli as well as dMSc show a previously unrecognized dynamics of dMSs, including the presence of long actin-based protrusions and their dynamics. The authors also carefully analyzed dMCs migration, dynamics of dMC-dMC contacts and their division. Moreover, lineage tracing identified basal keratinocytes as dMC precursors, showing that basal keratinocytes give rise to this intermediate cell population. Their marker expression analysis provides further evidence that dMCs indeed represent a transitional state between basal keratinocytes and MCs. They also look at the MCs renewal during skin regeneration and show that MCs in regenerated epidermis form predominantly de novo. Although the Eda requirement for MCs differentiation is not novel, they show that microvilli are absent in mutant cells. This adds some mechanistic insight into the MC protrusion formation. I found the study rigorous, well-controlled and their conclusions supported by the presented data. It clearly adds to our basic understanding of this important cell type. I only have a few general and minor comments.

    Major comments:

    One burning question is what controls the transition of dMCs into MCs? An obvious candidate is innervation. If the authors can demonstrate that, it would certainly take their work to another level.

    What happens to the MC regeneration in eda mutants? Is it already known? If not, it would help to address its role in the MC differentiation process.

    In their discussion they talk about directionality of MCs' protrusions in other species. Can they resolve MCs in 3D to address special orientation of their protrusions in zebrafish?

    Minor comments:

    The authors should comment on the eda expression; is it present in dMSs and MCs?

    The difference between corneal and trunk dMCs and MCs in eda mutants is striking. The authors should comment on this in their discussion. Can they speculate on the basis of these differences?

    Referees cross-commenting

    Reviewer 3 made an important point about atoh1a expression and the reporter line. I agree that the authors should confirm their atoh1a reporter indeed marks dMCs and MCs.

    Significance

    The strength of this work is the ability to follow MCs' differentiation in a live animal over time. One of its limitations is that the work is mostly descriptive. The main advance is showing that dMCs are the MCs intermediate population derived from basal keratinocytes. The study will be of an interest to sensory neuroscientists as well as those studying various aspects of skin development and regeneration.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, the authors use confocal imaging techniques to morphologically characterize Merkel cells during their maturation process in the zebrafish skin. Using an F-actin reporter, they identify two morphologically distinct populations of atoh1a+ cells: 1) Mature Merkel cells (MCs), which had previously been described in zebrafish, and 2) a transient population sharing morphological characteristics with so called dendritic Merkel Cells (dMCs), that were described in mice and humans but not previously identified in zebrafish. It was unknown whether dMCs represent a developmentally immature MC state or a functionally distinct subpopulation of neuroendocrine cells. The authors go on to show that dMCs represent the primary atoh1a+ cell type during skin regeneration and share features of both basal keratinocytes and Merkel cells, leading them to speculate that they could be MC precursors. Confocal time lapse imaging further showed that MCs and dMCs differ in the polarity of their protrusions. In some of the lapses, dMC can be seen maturing into MCs, providing evidence that they could be precursor cells. MC to dMC reversion events are also observed, albeit less often. Finally, the authors show that loss Ectodysplasin A (Eda) signaling disrupts MC microvilli formation, identifying this pathway as a potential regulator of MC morphology.

    Major comments:

    • The authors conclude that dMCs represent an intermediate state in the MC maturation program. This is based on the observation that the percentage of dMCs decreases over time and the fact that they share characteristics of both keratinocytes and MCs. In addition, dMCs are observed to mature into MCs in time lapses. However, these findings do not completely rule out the possibility that dMCs represent a transient, functionally distinct population of MCs. The authors should discuss this possibility. Additionally, some clarifications on the data could help strengthen their conclusion:
      • Figure 1 I-K: The interpretation of the simultaneous increase of dMCs and MCs is not clear. Shouldn't the percent of dMCs be highest at 8-9mm and then go down, when MCs first start to appear?
      • Fig. 2K: These results could also mean that dMCs numbers stay the same and only MCs increase in number. Does not imply lineage as stated in line 182 where the authors say that dMCs are a transient population. Please also report the total number of dMCs.
      • Figure 5 F and G: In these time lapses, "a small subset of dMCs (n>10)" is observed to adopt MC morphology. Does this mean 10 cells, and if so, out of how many? The authors should clarify how many time lapses were taken, and quantify the percentage of dMCs undergoing this process. The same goes for the reciprocal process, MC to dMC conversion, which happens only "in rare instances (n=2)".
    • Use photoconversion of single cells to establish lineage relationship. The 2 time lapses shown are not statistically significant and the identity of MCs in these movies is solely based on morphology.
    • In the last part of the paper, the authors show that trunk dMCs and MCs adopt abnormal morphologies in the absence of Eda signaling. However, this phenotype is not seen in the corneal epidermis, which is not squamated. Since Eda mutants do not develop scales, could the altered morphology in the trunk be due to the absence of scales? If possible, the authors should inhibit Eda signaling after the formation of scales or tone down their conclusions.
    • Line 264: The authors write: 'Consistent with this notion, dMC-dMC or dMC-MC contacts resulted in lateral dMC movement away from the contact (Movie 4). Together these observations suggest that MCs are immotile, epithelial-like cells, whereas dMCs are motile, mesenchymal-like cells that undergo contact inhibition upon encountering another atoh1a+ cell'. The lateral movement of dMCs after contacting MCs needs to be quantified before it can be interpreted as contact inhibition.

    Minor comments:

    • 'Defects in the morphogenesis of actin-based protrusions are linked to a variety of diseases, including colorectal cancer and deafness'. Please provide refs.
    • Line 145: this experiment does not show motility. Just that basal keratinocytes give rise to them.
    • Line 165. Cells increase by 14dpp and do not seem to plateau at 7dpp. Please discuss.
    • Line 190. Does Figure 3A not show basal keratinocytes? Only Figure 3B is cited.
    • Figure 3: Within individual cells, is there a negative correlation between SV2 staining and tp63 staining in dMCs? Or between sphericity and tp63 staining?
    • If dMCs are immature, are they already innervated by somatosensory axons?
    • Line 284: Indeed, during our live-imaging of juvenile and regenerating adult skin, we observed a small subset of dMCs (n>10) withdraw their long protrusions, round up their cell body, and rapidly extend microvilli reminiscent of the mature "mace-like" MC morphology (Figure 5F; Movies 6,7). I do not think movie 7 shows that. If it does, please indicate which of the cells shows this behavior.

    Optional:

    Published scRNASeq of the zebrafish skin exists and I am wondering if the authors could have searched for dMC and MC genes in these data which then could be used to generate lineage tracing tools or perform a pseudotime analysis that could indicate lineage relationships.

    Significance

    The aim of the study was to test if motile, dividing dMCs are precursors of immotile, post-mitotic MCs or a functionally distinct subpopulation of neuroendocrine cells. The manuscript is largely descriptive, well written and the findings are supported by beautiful imaging. The authors performed a series of experiments that strongly support the interpretation that dMCs are immature MCs. The findings will be of interest to developmental and stem cell biologists who study cell specification and differentiation. The most direct evidence that dMCs and MCs share a lineage relationship are the observations of a few dMCs that acquire the morphology of MCs in time lapse analyses. The other results support this interpretation but are correlative and do not exclude the possibility that dMCs are a functionally distinct cell type. To substantiate their interpretation the authors could take advantage of their photoconvertible line and photoconvert individual dMCs to determine if they differentiate into MCs.

  5. Version published to 10.1101/2023.09.14.557830v2 on bioRxiv
  6. Version published to 10.1101/2023.09.14.557830v1 on bioRxiv