Cellular basis of accelerated whole-tooth regeneration

Reviewer #3 (Public review):

Summary:

This is an interesting paper. The process of tooth exfoliation and replacement in vertebrates remains an intriguing and fascinating subject of inquiry. As the scientists noted, there are no mammalian models that can be used to examine signaling pathways in real time.

Strengths:

This work integrates in vivo and high-resolution transcriptomics. The study confirms previous findings and emphasizes the need for additional research into the processes that drive the restoration of missing teeth for future therapeutic uses.

Weaknesses:

I disagree with the use of the phrase "plucking". Instead, the authors use tooth extraction or tooth removal, which is clinically more correct for the procedure they are doing.

The title is rather broad and appears to be more appropriate for a review than an original research work. I would advise specifying the species under research and/or the sort of damage model used in the transcriptome analysis.

It's uncertain whether the findings are exclusively based on regeneration. The presence of tooth remnants, as well as unintended harm to surrounding tissues, may have triggered repair mechanisms, thereby biasing the current data. How did the authors handle this issue? The oral cavity was under severe manipulation, increasing the inflammatory stimuli, a situation that does not take place in physiological exfoliation.

The authors indicated the use of microCT analysis; however, no such information appears in the main text. In fact, this manuscript lacks anatomical information. It is required to conduct histological examinations of the regenerated teeth at various time points.

Although the current findings confirm previously found and verified signaling pathways, the absence of functional data lends uniqueness to this work.

Reviewer #1 (Public review):

Summary:

The authors used single-nucleus RNA sequencing (snRNA-seq) to investigate accelerated tooth replacement following tooth plucking in cichlid fish. They analyzed four stages of regeneration using elegant and well-designed approaches to characterize cellular trajectories and interactions within the dental epithelium and mesenchyme during the accelerated replacement process. Their analyses identified cell-type-specific gene expression profiles and intercellular signaling interactions associated with whole-tooth regeneration.

Strengths:

This is a highly interesting and thoughtfully executed study that provides compelling and convincing insights into the mechanisms underlying accelerated tooth regeneration.

Weaknesses:

The manuscript currently lacks experimental validation of the single-nucleus RNA-seq data.

Reviewer #2 (Public review):

Summary:

Mubeen and colleagues studied the cellular basis of tooth regeneration in cichlid fish. Using an elegant tooth plunking strategy followed by single-nucleus RNA-sequencing, the authors were hoping to achieve an atlas of cellular and transcriptional changes that occur within and between cells during whole tooth replacement.

Strengths:

The major strengths of the methods and results are high novelty in the approach in a vertebrate with continuous tooth replacement, the temporal analysis of analyzing at plucking and three later time points, the thorough and sophisticated analysis of the snRNA-seq data, including the inference of trajectories and signaling events, and the robust signal of transcriptional differences induced by tooth plucking.

Weaknesses:

The major weaknesses of the methods and results are no validation of any of the inferred cell types, no functional tests of whether any of the changes in signaling pathways affect the plucking-induced tooth replacement process, and perhaps no clear takeaway message for biologists not necessarily interested in tooth replacement.

Conclusion:

The authors achieved their aims of identifying the changes in gene expression and cellular composition that occur during whole tooth replacement accelerated by plucking. Overall, the results support their conclusions, although some slight semantic qualifiers should probably be added (e.g., referring to "cell types" as "putative cell types").

The work should have a high impact in the field of tooth and organ regeneration, and the novel methodological paradigm established here of accelerating tooth replacement three-fold by plucking has great promise for future follow-up studies to further study this process. The work could also have a strong impact through the computational methods used here to infer trajectories and signaling interactions. Specific pathways, genes, and cell types could be tested in other fish, such as zebrafish, to test function during tooth replacement.

The work is unique and interdisciplinary, and also has significance by establishing that robust phenotypically plastic accelerations in regeneration rates occur upon tooth removal. There are very few studies like this one that combine genetic and environmental studies of regeneration. The result that three different species of cichlid fish that normally have very different tooth patterns all accelerate tooth replacement threefold upon tooth plucking also has significance in revealing a highly conserved plucking response.

Author response:

Many thanks to the three reviewers and the editors for their comments and review. These are fair, consistent (across positives and negatives), and largely expected comments. On behalf of my coauthors, I use this letter as a provisional response to indicate what we can and intend to change in a revised manuscript.

(1) A major comment from all three referees is that our single-nucleus RNA-seq data should be validated. The reviewers differ in the detail of exactly what they think should be validated, but they refer, individually, to (1) the discovery of ‘cell types’ themselves, (2) pathways inferred from trajectory analysis, (3) differentially expressed genes in plucked vs control condition at four time points and/or (4) inferred ligand-receptor pairs from cell-cell communication analysis, across the same time course. 

I think we’re actually on pretty good footing for 1-3, because of work we’ve published in the cichlid fish model.

I tally that in references cited in the manuscript, and highlighted below (References 1, 10, 11, 29, 30, 31), we present 29 figures with 273 individual figure panels of histology, in situ hybridization and immunohistochemistry featuring genes expressed across stages of tooth development and replacement. These genes are markers of dental competency and regenerative potential.

In addition, in multiple of these papers, we use pharmacology to manipulate the role of key pathways (Hh, BMP, Wnt, Notch) in cichlid tooth development and replacement. Identification and validation of cell types make use of these published data in cichlids (for markers matched to mouse), as well as an unbiased computational approach (SAMap) that draws homology between cichlid and mouse dental cell types, based on shared global patterns of gene expression.

In short, experiments to validate cell types, gene expression and pathways active in cichlid teeth are published and referenced herein. I noticed that these references (some of which include Gareth Fraser as an author, when he was a postdoc in my group; for Reviewer 2) were cited in the Introduction and not the Rationale/Methods or Results section (such that reviewers may have missed them). We will be clearer about this in the revision. 

We have not validated nor analyzed functionally the ligand-receptor pairs inferred from cell-cell communication analysis, across four times points of accelerated replacement. This work is beyond the scope of the current paper, and we will include a statement that these computational inferences represent hypotheses to be tested (although many of these ligand-receptor pairs have been noted in other ‘tooth’ publications that we cite).

(2) The biggest weakness of our manuscript, noted by referees, is that we do not provide serial histology to accompany our snRNA-seq time course after plucking. We describe this as a limitation in the “Study limitations and future direction” section of the Discussion, but we can and will be stronger about why this is a weakness (e.g., we do not explicitly know for instance, the degree of damage done to tissue in the plucking paradigm). We do know that the jaw recovers quickly, but we do not know how different the plucked side is from the control side (which is also undergoing active replacement and remodeling). Uniting reviewer comments 1 and 2 here, the best future approach is a spatial transcriptomics reference at distinct stages of the plucking<>recovery paradigm, as we framed in the Discussion section, because this addresses simultaneously the state of dental/jaw tissue and the in situ expression of thousands of genes.

(3) Reviewers asked about the presence of stromal cells in our snRNA-seq data. Because of this and another comment on the posted preprint version of our manuscript, we will take another look at the mesenchymal compartment of the snRNA-seq data and trajectories built from it.

(4) Multiple (minor) suggestions for clarification in text and figures will be adopted. 

Generally, I don’t think we’ll require reviewer re-engagement on the revision; editor review should be sufficient.

References cited in the manuscript, highlighted here:

(1) Fraser, G. J. et al. An Ancient Gene Network Is Co-opted for Teeth on Old and New Jaws. PLoS Biol. 7, e1000031 (2009).

(10) Fraser, G. J., Bloomquist, R. F. & Streelman, J. T. Common developmental pathways link tooth shape to regeneration. Dev. Biol. 377, 399–414 (2013).

(11) Bloomquist, R. F. et al. Developmental plasticity of epithelial stem cells in tooth and taste bud renewal. Proc. Natl. Acad. Sci. 116, 17858–17866 (2019).

(29) Streelman, J. T., Webb, J. F., Albertson, R. C. & Kocher, T. D. The cusp of evolution and development: a model of cichlid tooth shape diversity. Evol. Dev. 5, 600–608 (2003).

(30) Fraser, G. J., Bloomquist, R. F. & Streelman, J. T. A periodic pattern generator for dental diversity. BMC Biol. 6, 32 (2008).

(31) Bloomquist, R. F. et al. Coevolutionary patterning of teeth and taste buds. Proc. Natl. Acad. Sci. 112, (2015).