Plap-1 lineage tracing and single-cell transcriptomics reveal cellular dynamics in the periodontal ligament

  1. Tomoaki Iwayama
  2. Mizuho Iwashita
  3. Kazuya Miyashita
  4. Hiromi Sakashita
  5. Shuji Matsumoto
  6. Kiwako Tomita
  7. Phan Bhongsatiern
  8. Tomomi Kitayama
  9. Kentaro Ikegami
  10. Takashi Shimbo
  11. Katsuto Tamai
  12. Masanori A. Murayama
  13. Shuhei Ogawa
  14. Yoichiro Iwakura
  15. Satoru Yamada
  16. Lorin E. Olson
  17. Masahide Takedachi
  18. Shinya Murakami

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Abstract

Periodontal tissue supports teeth in the alveolar bone socket via fibrous attachment of the periodontal ligament (PDL). The PDL contains periodontal fibroblasts and stem/progenitor cells, collectively known as PDL cells (PDLCs), on top of osteoblasts and cementoblasts on the surface of alveolar bone and cementum, respectively. However, the characteristics and lineage hierarchy of each cell type remain poorly defined. This study identified periodontal ligament associated protein-1 (Plap-1) as a PDL-specific extracellular matrix protein. We generated knock-in mice expressing CreERT2 and GFP specifically in Plap-1-positive PDLCs. Genetic lineage tracing confirmed the long-standing hypothesis that PDLCs differentiate into osteoblasts and cementoblasts. A PDL single-cell atlas defined cementoblasts and osteoblasts as Plap-1−Ibsp+Sparcl1+ and Plap-1−Ibsp+Col11a2+, respectively. Other populations, such as Nes+ mural cells, S100B+ Schwann cells, and other non-stromal cells, were also identified. RNA velocity analysis suggested that a Plap-1highLy6a+ cell population was the source of PDLCs. Lineage tracing of Plap-1+ PDLCs during periodontal injury showed periodontal tissue regeneration by PDLCs. Our study defines diverse cell populations in PDL and clarifies the role of PDLCs in periodontal tissue homeostasis and repair.

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  1. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    This study aims to gain a better understanding of PDLCs and their associated cementum and alveolar bone. The study provides a very clear results for differential expression of Plap-1 and IBSP the periodontal fibroblast and associated cementoblasts and osteoblasts.

    The most infesting is the generation of reporter mice for identification of Plap-1+ cells. The generation of this mice lined allowed then to gain insight to the regeneration of periodontium as well as heterogeneity in of Plap-1+ cells.

    Minor issues:

    1. Many abbreviation in the papers have to be better defined. (Spp1, Bgn, Sparc, Col1a. also DN and DP in legends to Figure 3.
    2. Legends to all figure can be written more clearly.
    3. Statement in the result (line 27 and 28) cement oblasts and osteoblasts were aligned ..... should be eliminated as the figure 1A does not allow appreciation of such features. Also, the statement does not add anything to the manuscript and its results.
    4. The statement on Page 5 (line 1, 2) the protein distribution of Plap-1 needs to be described.
    5. Line 14 and 15 on page 5. It should be noted that very few/if any cells are co-expressing Ibsp and td-tomato. The number is so few that brings questions to the conclusion.

    Major/important issues to be addressed:

    1. The authors have very nicely and clearly shown that Ibsp is expressed by cementoblasts and osteoblasts but not by PDL fibroblasts. Therefore, the lineage tracing experiments after PDL injury should be followed by examination of Ibsp in cementoblasts and osteoblasts originating from the Plap-1+ cells.
    2. It is also important to know what is the percentage of Plap-1+/Ly6a+ cells.
    3. The author should include a stronger statement for the possible role of Plap-1+/Ly6a+ cells (not Plap-1+ alone) as a source pf progenitors for periodontium.

    Significance

    by providing new markers and new transgenic animal model, the paper makes an important and significant contribution to the field

  2. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    • The authors describe in a richly illustrated manuscript periodontal ligament associated protein-1 Plap-1 as a periodontal fibroblast (PDLC) associated molecule that has the possibility to differentiate both into cementoblasts lining the tooth surface and into osteoblasts lining the alveolar bone.
    • In the introduction, please not only refer to higher expression of Plap-1 in certain tissues, but also refer to the function, as revealed by Sakashita et al (also on the periodontium/susceptibility to periodontitis). Apart from the fact that it is a 43 kDa ECM protein, subtype of the leucine-rich etc., it is also important to briefly sum-up -if scientific data allow - the function of the protein.

    • Page 5, line 2: have the authors investigated Plap-1 in tissues other than the periodontal ligament? These experiments seem essential to demonstrate the uniqueness of Plap-1, possibly as a confirmation of the Sakashita et al. paper of 2021. It is always a good habit to confirm previous work in a next study.

    • Page 5 on lineage tracing with Tomato: please spend a few lines on the essence of the experiment, either on page 5 or in the legend of Fig. 2, or both. You will thus keep the readers involved who are not familiar with lineage tracing.

    • Page 6 and 7: the description of the protocol is very valuable. It is also important the cell numbers of the various cell types were described in great detail (Fig 3). So, authors have now used cells derived from extracted teeth, which is world-wide a sample of convenience. However, after extraction, half of the PDLCs are likely attached to the alveolar bone of the tooth socket. Have the authors ever considered to harvest these cells? In principle, and biologically, PDLC cells at the site of the alveolar bone could be the more osteogenic cells. The PDLC that are attached to tooth could in principle be quite different, being anti-osteogenic and anti-osteoclastogenesis-stimulating.

    • Page 6, line 8: indicate what CD51+ cells are. In corresponding figure 3, explain the abbreviations in the X-axis in the legend.

    • Page 7 line 1: The proerythroblasts in the PDL are a surprise to me! I assumed that the bone marrow would be the natural niche. Authors are also encouraged to highlight plasma cell specific RNAs in their atlas, since these are quite abundant in periodontitis lesions.

    • In figure 4, its seems to me that the stromal cells in 4C are scattered more or less in 3 domains. This idea is strengthened when interpreting 4E Plap-1 and lbsp. Could the authors specify these domains?

    • On Page 7: again for the not-so-informed reader: briefly, in the first sentence, describe the phenomenon of RNA velocity.

    • Page 7, line 20: delete "were".

    • In figure 5A it could be helpful to put the numbers in the figure as well.

    • In figure 6G, it seems like that some osteocytes are positive, which means that they were derived from the TomatoRed cells within 7 days. That is quite remarkable and should gain some attention. Probably use a white arrow and specific mentioning in the legend.

    • In the discussion, I miss a clear link and comparison with human periodontal ligament. Is all this mouse specific or are some of these aspects also present in the human periodontal ligament? One study comes to mind that has actually studied gene expression of Plap-1 etc. in PDLC and in alveolar bone derived cells: Loo-Kirana R, et al., Frontiers in Cell and Developmental Biology, 2021: DOI: 10.3389/fcell.2021.709408. But there is bound to be other studies as well. A brief mirroring of these findings with other studies would be in place.

    CROSS-CONSULTATION COMMENTS

    I have read and seen the comments of the other reviewers. They are more or less in line with mine, and I have nothing to add.

    Significance

    Authors identify Flap-1 postive cells as key cells contributing to stem cell ness of the periodontium. With advanced techniques using GFP and Tomato Rd mice they are able to show a kind of hierarchy in cell differentiation. They also describe the presence of all kind of cells in the peridoontal ligament as well as the capacity of the Plap-1 positive cells to contribute to regeneration. It is a very valuable addition to existing literature.

    Audience: those, basic scientist but also dentists in general for whom the biology of the periodontal ligament is crucial.

    My expertise: periodontal ligament specialist, but more the human part. I use PDL to study osteogenesis and osteoclastogenesis, in presence of bacterial products, inflammatory and anti-inflammatory reagents.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    Comments:

    • In the presented study the authors attempt to perform an in vivo characterisation of the cellular hierarchies and cellular dynamics of the periodontal ligament (PDL), a largely unknown compartment of the periodontal tissue which function is to support mammalian teeth. Periodontal diseases are one of the major causes of adult tooth loss, hence understanding the cellular dynamics of this compartment is of particular interest. To do this, the authors develop a novel traceable mouse line based on the Plap-1 marker, which they identify specifically labels periodontal fibroblasts. The developed Plap-1-GFP-2A-CreER mice are then crossed onto an inducible Rosa26-tdTomato to enable in vivo tracing of Plap-1 PDL fibroblasts. This tool is of significant relevance as it allows for the first time to analyse the cellular fate of the elusive populations that constitute PDL under normal homeostatic and regenerative conditions. This mouse model also enables the authors to sort the cells of interest (as marked by GFP) to perform single-cell RNA sequencing analysis, providing further knowledge on the cellular heterogeneity of PDL cells.

    • The work presented in this article is of interest for the periodontal stem cell field and more generally the mesenchymal stem cell field. In particular, through the development of new tools, including a novel lineage traceable mouse line amenable for lineage tracing studies, the authors provide knew knowledge advancing our understanding on the populations and hierarchies that constitute the PDL. Having said this, I find this study rather descriptive and, in certain cases, the significance of the results are somewhat overinterpreted. For instance, lineage tracing studies are rather vague... based on the colocalization of a widely induced traceable fluorophore and markers present in the relevant cell populations, or even just histological positioning of cells; something that entails numerous technical implications and potential artefacts. It would be more convincing to titrate down the tamoxifen levels used to induce Plap-1 traceable mice, in order to track how single-cell derived clones actually contribute to the formation of other PDL populations, and validate this using the relevant markers at critical time points. Quantification of clonal distribution would also provide a deeper understanding of the process.

    • Another rather technical, but critical, aspect is the need for further validation of their new mouse model. Particularly, in order to interpret any prospective data on clonal dynamics, it is first important to know whether their new Cre system is tightly regulated, or whether there is leakage in the absence of Tamoxifen induction. Imaging of aged un-induced animals would help clarify this point.

    • Finally, the scRNA-seq is rather superficial, a more in-depth analysis would be required to support the statements based on hierarchies and trajectories proposed by the authors.

    • Despite all this, I believe the authors have the tools and data to address most of the aspects discussed below, which would make the study sound and result of advancement in the relevant field.

    Significance

    See above

  4. Version published to 10.1242/dev.201203
  5. Version published to 10.1101/2022.07.16.500314 on bioRxiv