Runx2 Regulates Mouse Tooth Root Development Via Activation of WNT Inhibitor NOTUM

Abstract

Progenitor cells are crucial in controlling organ morphogenesis. Tooth development is a well-established model for investigating the molecular and cellular mechanisms that regulate organogenesis. Despite advances in our understanding of how tooth crown formation is regulated, we have limited understanding of tooth root development. Runt-related transcription factor 2 (RUNX2) is a well-known transcription factor in osteogenic differentiation and early tooth development. However, the function of RUNX2 during tooth root formation remains unknown. We revealed in this study that RUNX2 is expressed in a subpopulation of GLI1+ root progenitor cells, and that loss of Runx2 in these GLI1+ progenitor cells and their progeny results in root developmental defects. Our results provide in vivo evidence that Runx2 plays a crucial role in tooth root development and in regulating the differentiation of root progenitor cells. Furthermore, we identified that Gli1, Pcp4, NOTUM, and Sfrp2 are downstream targets of Runx2 by integrating bulk and single-cell RNA sequencing analyses. Specifically, ablation of Runx2 results in downregulation of WNT inhibitor NOTUM and upregulation of canonical WNT signaling in the odontoblastic site, which disturbs normal odontoblastic differentiation. Significantly, exogenous NOTUM partially rescues the impaired root development in Runx2 mutant molars. Collectively, our studies elucidate how Runx2 achieves functional specificity in regulating the development of diverse organs and yields new insights into the network that regulates tooth root development. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Keywords: Gli1; NOTUM; ROOT DEVELOPMENT; Runx2; WNT/β-CATENIN.

Fig 1

Colocalization of RUNX2 and GLI1+ MSCs and their progeny in developing roots. (A–D) RUNX2 (green) and GLI1 (stained by β‐gal in red) co‐immunofluorescence of sagittal sections of mandibular molars from PN3.5 heterozygous Gli1‐LacZ mice. (E–L) RUNX2 immunofluorescence (green) and visualization of tdT (red) of sagittal sections of mandibular molars from Gli1‐Cre ^ERT2 ;tdTomato ^fl/+ mice at PN7.5 (E–H) and PN21.5 (I–L) after induction at PN3.5. The progeny of the GLI1+ lineage are presented in red. Boxes in A, E, and I are enlarged in B–D, F–H, and J–L, respectively. White dashed lines outline HERS; arrows indicate colocalization. Scale bars = 100 μm. β‐gal = β‐galactosidase; tdT = tdTomato.

Fig 2

Loss of Runx2 in GLI1+ MSCs results in root development defects. (A–D) μCT images of Runx2 ^fl/fl control (A,B) and Gli1‐Cre ^ERT2 ;Runx2 ^fl/fl (C,D) mandibular molars at PN21.5. (E–J) H&E staining of Runx2 ^fl/fl control (E–G) and Gli1‐Cre ^ERT2 ;Runx2 ^fl/fl (H–J) mandibular molars at PN21.5. Boxed areas in E and H are shown magnified in F–G and I–J, respectively. Yellow arrowheads indicate the absence of cementoblasts and periodontal ligament. (K–P) Dspp in situ hybridization (red) at PN21.5 (K,N), and Ki67 immunofluorescence (red) indicating proliferating cells at PN7.5 (L,M,O,P) in sagittal sections of mandibular molars in Runx2 ^fl/fl control and Gli1‐Cre ^ERT2 ;Runx2 ^fl/fl mice. Arrows in K, M, and P indicate positive signals; arrowheads in N indicate absence of signal. Boxes in L and O are enlarged in M and P, respectively. Dashed white lines outline HERS. Scale bars in A–D = 400 μm; scale bars in all others = 100 μm. AB = alveolar bone; C = cementoblast; D = dentin; DP = dental pulp; OD = odontoblast; PDL = periodontal ligament.

Fig 3

Integrated analysis of bulk RNA‐seq and scRNA‐seq reveals specific downstream targets of Runx2. (A) Bulk RNA‐seq revealed that 219 genes were upregulated and 208 genes were downregulated with >1.8‐fold change (p < .05) upon Runx2 deletion, represented here by volcano plot and heat map. (B) UMAP plots showed 19 clusters within PN7.5 molars after integration of the Runx2 ^fl/fl control and Gli1‐Cre ^ERT2 ;Runx2 ^fl/fl scRNA sequencing data with Seurat v3. Different clusters represent different cell types in the mouse molar, defined by expression of known marker genes. Dashed lines outline clusters representing the same cell type. Clusters 0, 1, 3, 4, and 15: dental papilla cells and odontoblasts. Cluster 2: dental follicle cells. The feature plot of the first gene in the list is shown. (C,D) Cluster 1 maps to the apical region of dental mesenchyme by two marker genes, Dio3 (C) and Itga4 (D). Dashed white lines outline tooth, arrows indicate positive signals. Scale bars = 100 μm. (E) Feature plots and box plots of four differentially expressed genes mapping to cluster 1. They were identified as potential downstream targets of Runx2. The differences in expression levels were consistent between feature plots of scRNA‐seq and box plots of bulk RNA‐seq.

Fig 4

In vivo validation of putative downstream targets upon deletion of Runx2 in the dental mesenchyme. RUNX2 immunofluorescence (A–D) and RNAscope in situ hybridization (red) of Gli1 (E–H), Pcp4 (I–L), NOTUM (M–P), and Sfrp2 (Q–T) of sagittal sections of mandibular molars from PN7.5 Runx2 ^fl/fl control and Gli1‐Cre ^ERT2 ;Runx2 ^fl/fl mice. The boxed area is enlarged on the right. Dashed lines outline HERS. Arrowhead indicates positive signals; asterisks indicate altered staining in targeted region of mutant samples. n = 3 sections were examined from multiple littermate mice per group. Scale bars = 100 μm.

Fig 5

NOTUM is a direct target of RUNX2 and activates the expression of odontoblast marker Dspp in vitro. (A) Genome browser snapshots representing the peak of ATAC‐seq from PN7.5 control mouse molars colocalized with the RUNX2 binding site at the promoter region of NOTUM. (B) ChIP analysis revealed the binding of endogenous RUNX2 to the genomic loci of NOTUM. DNA before immunoprecipitation (input) and after immunoprecipitation with an anti‐RUNX2 or rabbit IgG was amplified by qPCR using primers that amplify the regions containing RUNX2‐binding motifs in the NOTUM promoter. The value of input was defined as 1, and relative levels are shown. (C–J) RNAscope in situ hybridization (red) and qPCR of Dspp in cultured dental pulp cells treated with CM, OM, and OM + NOTUM protein (C–F), as well as OM + control siRNA, OM + Runx2 siRNA, and OM + Runx2 siRNA + NOTUM protein (G–J), insets in C–E and G–I are enlarged images of the cells pointed to by arrows in the same image. Scale bars = 25 μm. CM = control growth media; OM = odontoblastic media.

Fig 6

Ectopic NOTUM partially rescues the root defect in Gli1‐Cre ^ERT2 ;Runx2 ^fl/fl mice. H&E staining (A–I) and RNAscope in situ hybridization of Dspp (J–O) of sagittal sections of tooth germs from Runx2 ^fl/fl control and Gli1‐Cre ^ERT2 ;Runx2 ^fl/fl mice cultured for 3 weeks under kidney capsules with BSA or NOTUM beads. The control explants developed well; two roots with columnar odontoblasts, thick dentin, and predentin are identifiable (A,D,G). In Gli1‐Cre ^ERT2 ;Runx2 ^fl/fl molars treated with BSA beads (B,E,H), the root dentin is irregular, and predentin is unseen, arrowheads indicate there are few odontoblast‐like cells along with the dentin, some cells are embedded into the dentin. After treatment with NOTUM beads (C,F,I), the root dentin became more regular with detectable predentin, and many odontoblast‐like cells accumulated at the surface of the dentin (indicated by black arrows). Dspp expression is strong in control samples (J,M), whereas in Gli1‐Cre ^ERT2 ;Runx2 ^fl/fl molars treated with BSA beads (K,N), there are only a few positive signals. Following treatment with NOTUM beads, Dspp is detectable in the apical region (L) and furcation region (O). Insets in J, K, and L are lower magnification images of the same sample. White arrows indicate positive signal; asterisks indicate absence of signal. B = bead; D = dentin; PD = predentin; DP = dental pulp; OD = odontoblast. n = 4 samples were collected and analyzed for each group. Scale bars = 100 μm.