Abstract
BackgroundDevelopment of dental tissue is regulated by extensive cell crosstalk based on various signaling molecules, such as bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) pathways. However, an intact network of the intercellular regulation is still lacking.ResultTo gain an unbiased and comprehensive view of this dental cell interactome, we applied single-cell RNA-seq on immature human tooth germ of the growing third molar, discovered refined cell subtypes, and applied multiple network analysis to identify the central signaling pathways. We found that immune cells made up over 80% of all tooth germ cells, which exhibited profound regulation on dental cells via Transforming growth factor-β, Tumor necrosis factor (TNF) and Interleukin-1. During osteoblast differentiation, expression of genes related to extracellular matrix and mineralization was continuously elevated by signals from BMP and FGF family. As for the self-renewal of apical papilla stem cell, BMP-FGFR1-MSX1 pathway directly regulated the G0-to-S cell cycle transition. We also confirmed that Colony Stimulating Factor 1 secreted from pericyte and TNF Superfamily Member 11 secreted from osteoblast regulated a large proportion of genes related to osteoclast transformation from macrophage and monocyte.ConclusionsWe constructed the intercellular signaling networks that regulated the essential developmental process of human tooth, which served as a foundation for future dental regeneration engineering and the understanding of oral pathology.
Highlights
Development of dental tissue is regulated by extensive cell crosstalk based on various signaling molecules, such as bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) pathways
A series of genes and signaling pathways play an important role in regulation, including Sonic hedgehog (Shh) pathway [5], Wingless-related integration (Wnt) pathway [6], fibroblast growth factors (FGF) pathway [7], Transforming growth factor-beta (TGF-β) [8] pathway and bone morphogenesis proteins (BMPs) family [9,10,11]
By Gene Ontology (GO) analysis, we found that genes showing decreasing expression in the cascade mainly took part in Wnt signaling pathway, mesenchymal development (GO P = 1.91 × 10− 8, OR = 5.08), BMP signaling pathway (GO P = 1.16 × 10− 7, OR = 6.12, Fig. 4B and Additional file 2: Table S2)
Summary
Single‐cell composition of human tooth germ We isolated tooth germ tissue from two patients with different developmental statuses of left mandibular third molars (Fig. 1A and Additional file 1: Figure S1). By combination of ligand-receptor-TF-target regulation results, we identified several important pathways such as (See figure on page.) Fig. 3 Non-T immune cell subtypes and their cellular interaction patterns. At the significance threshold of false discovery rate (FDR)-adjusted P < 0.01 and log fold change > 1, we found 111 genes that were elevated during monocyte-to-osteoblast transformation and 149 genes that were elevated during macrophage-toosteoblast transformation (Additional file 2: Tables S8, S9) We merged these two gene lists into 183 unique transformation-related genes and applied nichenetr [39] to discover the upstream signaling pathways that regulated them (Fig. 6B). For TCF4 targets, we did not observe significant functional enrichment
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