Single Cell RNA Sequencing Reveals Critical Functions of Mkx in Periodontal Ligament Homeostasis

Kaho Takada; Tomoki Chiba; Takayuki Miyazaki; Lisa Yagasaki; Ryo Nakamichi; Takanori Iwata; Keiji Moriyama; Hiroyuki Harada; Hiroshi Asahara

doi:10.3389/fcell.2022.795441

Single Cell RNA Sequencing Reveals Critical Functions of Mkx in Periodontal Ligament Homeostasis

Front Cell Dev Biol. 2022 Feb 4:10:795441. doi: 10.3389/fcell.2022.795441. eCollection 2022.

Authors

Kaho Takada^{1

2}, Tomoki Chiba¹, Takayuki Miyazaki^{1

3}, Lisa Yagasaki^{1

4}, Ryo Nakamichi⁵, Takanori Iwata⁴, Keiji Moriyama³, Hiroyuki Harada², Hiroshi Asahara^{1

5}

Affiliations

¹ Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Japan.
² Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Japan.
³ Department of Maxillofacial Orthognathics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Japan.
⁴ Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Japan.
⁵ Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, United States.

Abstract

The periodontal ligament (PDL) comprises a fibrous tissue that connects teeth to alveolar bone and is essential for periodontal function. The transcription factor mohawk homeobox (Mkx) is expressed in the PDL where it plays an important role in the development and maintenance of the PDL. However, the precise and critical functions of Mkx in the cell populations comprising PDL have not yet been elucidated. The present study aimed to clarify the effects of a Mkx deficiency on PDL cellular heterogeneity and differences between gene expression in PDL tissues from wild-type (WT) (Mkx ^+/+ ) and Mkx knockout (Mkx ^-/- ) rats using single-cell RNA sequencing. We identified 12 cell clusters comprising mesenchymal cells and macrophages. The expression of Mkx and scleraxis (Scx; another key transcription factor of PDL), was mutually exclusive, and partitioned mesenchymal cell clusters into Mkx and Scx types that dominantly expressed proteoglycans and elastic fibers, and type 1 and 3 collagen, respectively. Ossification-related genes were upregulated in mesenchymal cell and osteoblast clusters with more Mkx ^-/- than Mkx ^+/+ PDLs. Increased number of cells and inflammatory mediators were observed in macrophage clusters of Mkx ^-/- PDL. These results suggested that Mkx plays an important role in maintaining PDL homeostasis by regulating specific cell populations and gene expression.

Keywords: MKX; SCX; collagen; extracellular matrix (ECM); inflammation; ossification; periodontal ligament; single cell RNA-sequencing (scRNA-seq).