Spatiotemporal dynamics of sensory neuron and Merkel-cell remodeling are decoupled during epidermal homeostasis

Reviewer #1 (Public Review):

We conclude that this descriptive study has some strengths but additionally, we propose several ways in which to increase its potential impact and to strengthen some of the claims. This study describes the remodeling of Merkel cells and their innervating sensory axons in the skin. By using transgenic mouse lines in which these cells were genetically fluorescently labeled, the authors performed a series of analyses mostly focusing on the number and location of Merkel cells and the sensory axons that innervate them.

One of the major strengths of the study is the establishment of intravital imaging techniques to investigate the dynamic simultaneous behavior of Merkel cells and their innervation during homeostasis and hair regeneration. However, how the findings integrate into the existing knowledge of skin development is unfortunately only partially addressed.

To the best of our understanding, a few technical limitations of the study define its major weaknesses: First, Merkel cell loss is dramatic and it's unclear whether this reduction is part of a developmentally controlled reduction in cell number, or whether additional cells are expected to be integrated into the system. Longer windows of imaging might help here. Second, the depilation agent might be too aggressive and lead to cell death and thus better controls might be suggested. Similarly, ablating Merkal cells throughout development might cause developmental issues that might mask the proposed homeostasis analyses. A controlled adult specific ablation might be suggested. Finally, the TrkC based transgenic mouse is expected to be heterozygous - could that be an issue? Either better controls, or a textual addressing of this topic are advised.

All in all, we think this study has the potential to establish a high resolution description of Merkel cells - sensory axon dynamic interactions. We hope that the authors will be encouraged to improve the paper based on our comments, something that will likely improve its potential significance and impact.

Reviewer #2 (Public Review):

Clary et al. utilized 2-photon intravital imaging techniques to investigate the dynamic behavior of Merkel cells and their innervation during homeostasis and hair regeneration. The authors demonstrated that both Merkel cells (Atoh1-GFP) and the branched axons (TrkC) innervating them undergo significant plasticity and remodeling during homeostasis. Merkel cells were added, removed, and relocated, while axons showed growth and regression. By taking advantage of live imaging, the authors identified two different ways in which Merkel cells interact with axons: creating the stable kylikes and the previously undescribed dynamic Bouton structure. Using live imaging and extensive quantification tools, the authors thoroughly characterized Merkel cell and axon plasticity. They found that Merkel cell plasticity is associated with the hair cycle, while axon plasticity is not. Moreover, newly generated Merkel cells have a short lifespan. By comparing the survival of afferents associated with Merkel cells to empty ones and analyzing Atoh1 cKO, the authors concluded that Merkel cells have a stabilizing effect on axon terminals.

Strengths:

The authors developed an intravital imaging system that enables the simultaneous tracking of both Merkel cells and axon branches. Live imaging, combined with numerous quantification tools, enabled an in-depth characterization of the different behaviors and dynamic nature of Merkel cells, axon branches, and their interaction. The authors' approach has the particular strength of allowing for the comparison of the dynamic behavior of axons associated with Merkel cells to those not innervating Merkel cells within the same touch dome, as well as describing a Bouton structure as a novel morphology mediating Merkel cell and nerve interaction.

Weaknesses:

Although the authors provide an in-depth analysis of Merkel cell dynamics and its association with hair growth, these concepts have been previously reported by the authors and others. Therefore, the extent of novel concepts and scientific advances should be better explained.

The authors suggest that Merkel cell association is a stabilizing factor on innervated axon branches by comparing branch plasticity between branches connected to Merkel cells and empty ones and using Atoh cKO. While the first set of experiments are compelling and provide interesting observations, additional experimental models, such as Merkel cell ablation in adults, may better strengthen the authors' claims. The authors currently use K14-Cre;Atoh1 cKO to support their observations. However, the absence of Merkel cell development in this model, might also lead to developmental defects in nerve patterning (absence of target organ) leading to the phenotype observed by the authors.

Finally, the authors use intravital imaging to describe the Bouton structure and dynamic. Though very interesting there is not enough data to support authors claim for interaction between axon and Merkel cells through the Bouton structure. The paper can benefit from additional functional analysis of this structure.

Reviewer #3 (Public Review):

This study documents the dynamics of Merkel cells and their axonal afferents during the hair growth cycle. Methodologically, the study is impressive-using two transgenic lines and repeated 2-photon imaging allowed the researchers to monitor Merkel cells and afferent axons over the course of weeks. These exciting tools and methods will enable future studies of these cell interactions. The manuscript is well written, the figures are clear and appealing, the statistical analyses are rigorous and appropriate, and potentially confounding issues (e.g., damage caused by 2-photon imaging or hair removal) were thoughtfully considered and controlled for. The clear and rigorously analyzed findings make the conclusions well justified. The impact of this study could be enhanced with further experiments that provide more functional characterization of boutons and kylikes, and that characterize axonal dynamics in Atoh mutants lacking Merkel cells.

Reviewer #4 (Public Review):

In this manuscript, Clary and colleagues use two-photon imaging to visualize the dynamics of Merkel cells and their innervating sensory axons using a combination of transgenic lines, where these parts of the mechanosensory organs of the skin are labelled with distinct fluorescent proteins. It is noteworthy that this study does not stand alone, but should be compared to prior published work cited by the authors, such as Wright et al., Developmental Biology 422 (2017) 4-13.

The study demonstrates a comparably high degree of remodelling, with a large fraction of Merkel cells (50% in three weeks) and a similar fraction of elaborated (cup-like) axons endings disappearing. It appears by timing and correlation that changes in Merkel cells can clearly drive axonal remodelling, while axons can still remodel even if the Merkel cells remain stable by the parameters measured here. Moreover, changes in Merkel cells partially relate to the hair growth cycle.

The imaging approach chosen is straightforward and clearly suited in principle to reveal the dynamism of the studied cellular structures. To co-visualize two synaptic partners in a vertebrate sensory organ in vivo - while not unprecedented - certainly remains quite challenging, and represents a strength of the paper. Similarly, understanding how stable structures in the nervous system are under homeostatic (rather than developmental) conditions, remains an understudied topic. I also found some of the correlative analysis in the later parts of the study quite interesting, albeit not always straightforward to interpret.

My central concern is the very high disappearance rate of Merkel cells. This, in my view is not compatible with a steady state situation in an adult animal - and not with the prior literature (especially the similar study by Wright et al. cited above). Obviously, if this rate were to continue, Merkel cells would all be lost in early adulthood in mice. Whether this is the case in the specific anatomical location was not examined in the study - but it would also imply that the study really addresses a dynamic developmental remodelling situation and should be written up accordingly. I am more suspicious of the depilation agent (plus the shaving). As Wright et al. already show that shaving causes some changes in Merkel cell dynamics (but, as far as I can tell, did not chemically depilate), I would not be surprised that we see an artificially high remodelling rate. Such skin treatment-related biology is probably less relevant in the context of neurobiology (albeit probably quite interesting to other audiences). So, my recommendation to the authors would be to invest some energy to find out, what causes the swift Merkel cell loss.

Another technical point that warrants discussion is the axonal labelling - first, I do not find the innervation patterns always easy to discern in the images provided, so I am not always sure how reliable this part is. Any artefact here creates the impression of dynamics, as during in vivo imaging stability is more reassuring than change. There are many ways not to see or recognize something, while there are few options to explain by an artefact why something did not change. Additionally, it might be good to explicitly mention that the TrkC mice are knock-in/knock-out (this is how I understood the JAX entry) - so the observations were made under reduced TrkC expression. It would help to explain, why this cannot affect axonal dynamics or Merkel cell-axon interactions.

Overall, while I feel that the authors performed an interesting in vivo imaging study, I think technical aspects make it difficult to conclude with confidence, whether we are watching a normal and physiological process here or dynamics that are induced by specific interventions. While these interventions might represent conditions that can occur also outside the laboratory, it would be important to clarify how the reader should contextualize this study.