Inclusive, Exclusive and Hierarchical Atlas of NFATc1⁺/PDGFR-α⁺ Cells in Dental and Periodontal Mesenchyme

Reviewer #1 (Public Review):

In this study, Yang et al. investigated the locations and hierarchies of NFATc1+ and PDGFRα+ cells in dental and periodontal mesenchyme. By combining intersectional and exclusive reporters, they attempted to distinguish among NFATc1+PDGFRα+, NFATc1+PDGFRα-, and NFATc1- PDGFRα+ cells. Using tissue clearing and serial section-based 3D reconstruction, they mapped the distribution atlas of these cell populations. Through DTA-induced ablation of PDGFRα+ cells, they demonstrated the crucial role of PDGFRα+ cells in the formation of the odontoblast cell layer and periodontal components.

Main issues:

(1) The authors did not quantify the contribution of PDGFRα+ cells or NFATc1+ cells to dental and periodontal lineages in PDGFRαCreER; Nfatc1DreER;LGRT mice. Zsgreen+ cells represented PDGFRα+ cells and their lineages. Tomato+ cells represented NFATc1+ cells and their lineages. Tomato+Zsgreen+ cells represented NFATc1+PDGFRα+ cells and their lineages. Conducting immunostaining experiments with lineage markers is essential to determine the physiological contributions of these cells to dental and periodontal homeostasis.

(2) The authors attempted to use PDGFRαCreER; Nfatc1DreER;IR1 mice to illustrate the hierarchies of NFATc1+ and PDGFRα+ cells. According to the principle of the IR1 reporter, it requires sequential induction of PDGFRα-CreER and Nfatc1-DreER to investigate their genetic relationship. Upon induction by tamoxifen, NFATc1+PDGFRα- cells and NFATc1-PDGFRα+ cells were labeled by Tomato and Zsgreen, respectively. However, the reporter expression of NFATc1+PDGFRα+ cells was uncertain, most likely random. Therefore, the hierarchical relationship of NFATc1+ and PDGFRα+ cells cannot be reliably determined from PDGFRαCreER; Nfatc1DreER; IR1 mice.

Reviewer #2 (Public Review):

Summary:

Yang et al. present an article investigating the spatiotemporal atlas of NFATc1+ and PDGFR-α+ cells within the dental and periodontal mesenchyme. The study explores their capacity for progeny cell generation and their relationships - both inclusive and hierarchical - under homeostatic conditions. Utilizing the Cre/loxP-Dre/Rox system to construct tool mice, combined with tissue transparency and continuous tissue slicing for 3D reconstruction, the researchers effectively mapped the distribution of NFATc1+ and PDGFR-α+ cells. Additionally, in conjunction with DTA mice, the study provides preliminary validation of the impact of PDGFR-α+ cells on dental pulp and periodontal tissues. Primarily, this study offers an in-situ distribution atlas for NFATc1+ and PDGFR-α+ cells but provides limited information regarding their origin, fate differentiation, and functionality.

Strengths:

(1) Tissue transparency techniques and continuous tissue slicing for 3D reconstruction, combined with transgenic mice, provide high-quality images and rich, reliable data.
(2) The Cre/loxP and Dre/Rox systems used by the researchers are powerful and innovative.
(3) The IR1 lineage tracing model is significantly important for investigating cellular differentiation pathways.
(4) This study provides effective spatial distribution information of NFATc1+/PDGFR-α+ cell populations in the dental and periodontal tissues of adult mice.

Weaknesses:

(1) In the functional experiment section, the investigation into the role of NFATc1+/PDGFR-α+ cell populations is somewhat lacking.

(2) The author mentions that 3D reconstruction of consecutive tissue slices can provide more detailed information on cell distribution, so what is the significance of using tissue-clearing techniques in this article?

(3) After reading the entire article, it is confusing whether the purpose of the article is to explore the distribution and function of NFATc1+/PDGFR-α+ cells in teeth and periodontal tissues, or to compare the differences between tissue clearing techniques and 3D reconstruction of continuous histological slices using NFATc1+/PDGFR-α+ cells?

(4) The researchers did not provide a clear definition of the cell types of NFATc1+/PDGFR-α+ cells in teeth and periodontal tissues.

(5) In studies related to long bones, the author defines the NFATc1+/PDGFR-α+ cell population as SSCs, which as a stem cell group should play an important role in tooth development or injury repair. However, the distribution patterns and functions of the NFATc1+/PDGFR-α+ cell population in these two conditions have not been discussed in this study.

Reviewer #3 (Public Review):

Summary:

This groundbreaking study provided the most advanced transgenic lineage tracing and advanced imaging techniques in deciphering dental/periodontal mesenchyme cells. In this study, authors utilized CRISPR/Cas9-mediated transgenic lineage tracing techniques to concurrently demonstrate the inclusive, exclusive, and hierarchical distributions of NFATc1+ and PDGFR-α+ cells and their lineage commitment in dental and periodontal mesenchyme.

Strengths:

In cooperating with tissue clearing-based advanced imaging and three-dimensional slices reconstruction, the distribution and hierarchical relationship of NFATc1+ and PDGFR-α+ cells and progeny cells plainly emerged, which undoubtedly broadens our understanding of their in vivo fate trajectories in craniomaxillofacial tissue. Also, the experiment design is comprehensive and well-executed, and the results are convincing and compelling.

Weaknesses:

Minor modifications could be made to the paper, including more details on the advantages of the methodology used by the authors in this study, compared to other studies.