Single-Cell Virtual Perturbation Screening Identifies STAT3 as a Key Regulator of Dentinogenesis

Abstract

Dentine formation constitutes a physiological process precisely regulated by signal transduction modules governing odontoblast differentiation and mineralisation. First, by constructing a single-cell transcriptional landscape of odontogenic tissue, we defined EFNB2+ mesenchymal cells as a primary progenitor cluster, marking the origin of the odontogenic lineage. Integrating CellRank-based fate mapping and SCENIC-based regulon specificity analysis, we identified signal transducer and activator of transcription 3 (STAT3) as a pivotal transcriptional regulator of the odontoblast lineage. Subsequently, in silico perturbations using CellOracle predicted that STAT3 ablation disrupted the developmental vector field, redirecting the fate of mesenchymal precursors away from the odontoblast lineage. To substantiate these bioinformatic predictions, functional validation using shRNA-mediated silencing and pharmacological modulation demonstrated that STAT3 was essential for the proliferation and differentiation capacity of dental mesenchymal cells. Furthermore, we generated conditional knockout mice targeting Stat3 in Osterix-expressing odontoblast progenitors, which consequently exhibited significant dentine dysplasia. Mechanistically, RNA-seq and chromatin immunoprecipitation (ChIP) assays revealed that STAT3 directly bound to the WNT2B promoter, transcriptionally activating the Wnt/β-catenin signalling pathway in dental mesenchymal cells. Overexpression of WNT2B partially rescued the odontogenic defects induced by STAT3 inactivation. This ‘prediction to verification’ study establishes STAT3 as a critical regulator of dentinogenesis and provides potential therapeutic targets for the treatment of dentine developmental disorders and the advancement of dentine regeneration.

Keywords

dentine development / dentinogenesis / in silico perturbation / odontoblast differentiation / STAT3 / Wnt signalling pathway / WNT2B

References

[1] X. Xu, L. Zhang, X. Gong, et al., “Primary Cilia Length Stability Is Essential for Dentinogenesis,” Journal of Bone and Mineral Research 41, no. 1 (2025): 73–84.

[2] X. Gong, H. Zhang, X. Xu, et al., “Tracing PRX1(+) Cells During Molar Formation and Periodontal Ligament Reconstruction,” International Journal of Oral Science 14, no. 1 (2022): 5.

[3] W. Zhang and P. C. Yelick, “Tooth Repair and Regeneration: Potential of Dental Stem Cells,” Trends in Molecular Medicine 27, no. 5 (2021): 501–511.

[4] S. Wang, F. Song, S. Liang, C. Huang, and H. Yang, “Mechanobiology of Collagen Synthesis, Assembly, and Mineralization,” Journal of Dental Research 104, no. 13 (2025): 1432–1442.

[5] H. Ritchie, “The Functional Significance of Dentin Sialoprotein-Phosphophoryn and Dentin Sialoprotein,” International Journal of Oral Science 10, no. 4 (2018): 31.

[6] C. Kornsuthisopon, N. Nowwarote, T. Srisuwan, W. L. Dissanayaka, and T. Osathanon, “Regulatory Pathways Governing Odonto/Osteogenic Differentiation in Dental Pulp Stem Cells,” International Endodontic Journal 58 (2025): 449–466.

[7] M. C. Riou, S. Touraivane, A. Berdal, et al., “Physiology of Dentinogenesis and Pathophysiology of Dentinogenesis Imperfecta: How Does It Affect Dentin Structure and Biomechanics?,” Acta Biomaterialia 207 (2025): 131–141.

[8] E. J. Lira Dos Santos, K. Nakajima, J. Po, et al., “Dental Impact of Anti-Fibroblast Growth Factor 23 Therapy in X-Linked Hypophosphatemia,” International Journal of Oral Science 15, no. 1 (2023): 53.

[9] L. Zhou, S. Zhao, and X. Xing, “Effects of Different Signaling Pathways on Odontogenic Differentiation of Dental Pulp Stem Cells: A Review,” Frontiers in Physiology 14 (2023): 1272764.

[10] H. Pan, Y. Yang, H. Xu, et al., “The Odontoblastic Differentiation of Dental Mesenchymal Stem Cells: Molecular Regulation Mechanism and Related Genetic Syndromes,” Frontiers in Cell and Development Biology 11 (2023): 1174579.

[11] A. V. Villarino, Y. Kanno, J. R. Ferdinand, and J. J. O'Shea, “Mechanisms of Jak/STAT Signaling in Immunity and Disease,” Journal of Immunology 194, no. 1 (2015): 21–27.

[12] P. S. Yadav, S. Feng, Q. Cong, H. Kim, Y. Liu, and Y. Yang, “Stat3 Loss in Mesenchymal Progenitors Causes Job Syndrome-Like Skeletal Defects by Reducing Wnt/Beta-Catenin Signaling,” Proceedings of the National Academy of Sciences of the United States of America 118, no. 26 (2021): e2020100118.

[13] X. Huang, Y. Zhu, S. Sun, et al., “Exercise Maintains Bone Homeostasis by Promoting Osteogenesis Through STAT3,” International Journal of Biological Sciences 19, no. 7 (2023): 2021–2033.

[14] S. Zhou, Q. Dai, X. Huang, et al., “STAT3 Is Critical for Skeletal Development and Bone Homeostasis by Regulating Osteogenesis,” Nature Communications 12, no. 1 (2021): 6891.

[15] J. Krivanek, R. A. Soldatov, M. E. Kastriti, et al., “Dental Cell Type Atlas Reveals Stem and Differentiated Cell Types in Mouse and Human Teeth,” Nature Communications 11, no. 1 (2020): 4816.

[16] F. A. Wolf, P. Angerer, and F. J. Theis, “SCANPY: Large-Scale Single-Cell Gene Expression Data Analysis,” Genome Biology 19, no. 1 (2018): 15.

[17] R. Lopez, J. Regier, M. B. Cole, M. I. Jordan, and N. Yosef, “Deep Generative Modeling for Single-Cell Transcriptomics,” Nature Methods 15, no. 12 (2018): 1053–1058.

[18] K. Street, D. Risso, R. B. Fletcher, et al., “Slingshot: Cell Lineage and Pseudotime Inference for Single-Cell Transcriptomics,” BMC Genomics 19, no. 1 (2018): 477.

[19] G. S. Gulati, S. S. Sikandar, D. J. Wesche, et al., “Single-Cell Transcriptional Diversity Is a Hallmark of Developmental Potential,” Science 367, no. 6476 (2020): 405–411.

[20] P. Weiler, M. Lange, M. Klein, D. Pe'er, and F. Theis, “CellRank 2: Unified Fate Mapping in Multiview Single-Cell Data,” Nature Methods 21, no. 7 (2024): 1196–1205.

[21] B. Van de Sande, C. Flerin, K. Davie, et al., “A Scalable SCENIC Workflow for Single-Cell Gene Regulatory Network Analysis,” Nature Protocols 15, no. 7 (2020): 2247–2276.

[22] K. Kamimoto, B. Stringa, C. M. Hoffmann, K. Jindal, L. Solnica-Krezel, and S. A. Morris, “Dissecting Cell Identity via Network Inference and In Silico Gene Perturbation,” Nature 614, no. 7949 (2023): 742–751.

[23] D. Osorio, Y. Zhong, G. Li, et al., “scTenifoldKnk: An Efficient Virtual Knockout Tool for Gene Function Predictions via Single-Cell Gene Regulatory Network Perturbation,” Patterns (N Y) 3, no. 3 (2022): 100434.

[24] T. Qu, C. Miao, Z. Zhang, et al., “Prognostic Signature of Endoplasmic Reticulum Stress-Related Long Noncoding RNAs in Head and Neck Squamous Cell Carcinoma: Association With Somatic Mutation and Tumor Immune Microenvironment,” Journal of Dental Sciences 18, no. 2 (2023): 541–550.

[25] G. Li, M. Liu, S. Zhang, et al., “Essential Role of IFT140 in Promoting Dentinogenesis,” Journal of Dental Research 97, no. 4 (2018): 423–431.

[26] R. Sheng, Y. Wang, Y. Wu, et al., “METTL3-Mediated m(6) A mRNA Methylation Modulates Tooth Root Formation by Affecting NFIC Translation,” Journal of Bone and Mineral Research 36, no. 2 (2021): 412–423.

[27] F. Liu and S. E. Millar, “Wnt/Beta-Catenin Signaling in Oral Tissue Development and Disease,” Journal of Dental Research 89, no. 4 (2010): 318–330.

[28] I. J. de Souza Araujo, R. S. Perkins, W. Zhang, S. A. Krum, and G. T. Huang, “Canonical and Noncanonical WNT-Loaded Hydrogel for Dentin-Pulp Regeneration,” Journal of Dental Research 105 (2025): 450–462.

[29] T. Tanaka, A. Takahashi, Y. Kobayashi, et al., “Foxf2 Represses Bone Formation via Wnt2b/Beta-Catenin Signaling,” Experimental & Molecular Medicine 54, no. 6 (2022): 753–764.

[30] Y. Chai, X. Jiang, Y. Ito, et al., “Fate of the Mammalian Cranial Neural Crest During Tooth and Mandibular Morphogenesis,” Development 127, no. 8 (2000): 1671–1679.

[31] S. Alves-Costa, M. Romandini, and G. G. Nascimento, “Lip and Oral Cancer, Caries and Other Oral Conditions: Estimates From the 2021 Global Burden of Disease Study and Projections up to 2050,” Journal of Periodontal Research 60, no. 6 (2025): 544–558.

[32] W. Yin, Y. M. Yang, H. Chen, et al., “Oral Health Status in Sichuan Province: Findings From the Oral Health Survey of Sichuan, 2015-2016,” International Journal of Oral Science 9, no. 1 (2017): 10–15.

[33] M. J. Barron, S. T. McDonnell, I. Mackie, and M. J. Dixon, “Hereditary Dentine Disorders: Dentinogenesis Imperfecta and Dentine Dysplasia,” Orphanet Journal of Rare Diseases 3 (2008): 31.

[34] T. Liang, H. Zhang, Q. Xu, S. Wang, C. Qin, and Y. Lu, “Mutant Dentin Sialophosphoprotein Causes Dentinogenesis Imperfecta,” Journal of Dental Research 98, no. 8 (2019): 912–919.

[35] Q. Gao, Z. Deng, L. Yang, K. Liu, J. Yang, and Q. Du, “A Novel DSPP Mutation in Dentinogenesis Imperfecta Shields Type II: Clinical, Genetic and Stem Cell Perspectives,” International Dental Journal 75, no. 5 (2025): 100937.

[36] P. M. Yamaguti, M. de La Dure-Molla, S. Monnot, et al., “Unequal Impact of COL1A1 and COL1A2 Variants on Dentinogenesis Imperfecta,” Journal of Dental Research 102, no. 6 (2023): 616–625.

[37] X. Ni, Y. Gong, Y. Jiang, et al., “The First Compound Heterozygous Mutations of DMP1 Causing Rare Autosomal Recessive Hypophosphatemic Rickets Type 1,” Journal of Clinical Endocrinology and Metabolism 108, no. 4 (2023): 791–801.

[38] D. E. Johnson, R. A. O'Keefe, and J. R. Grandis, “Targeting the IL-6/JAK/STAT3 Signalling Axis in Cancer,” Nature Reviews. Clinical Oncology 15, no. 4 (2018): 234–248.

[39] J. J. O'Shea, S. M. Holland, and L. M. Staudt, “JAKs and STATs in Immunity, Immunodeficiency, and Cancer,” New England Journal of Medicine 368, no. 2 (2013): 161–170.

[40] W. Ono, N. Sakagami, S. Nishimori, N. Ono, and H. M. Kronenberg, “Parathyroid Hormone Receptor Signalling in Osterix-Expressing Mesenchymal Progenitors Is Essential for Tooth Root Formation,” Nature Communications 7 (2016): 11277.

[41] T. H. Kim, C. H. Bae, J. C. Lee, et al., “Osterix Regulates Tooth Root Formation in a Site-Specific Manner,” Journal of Dental Research 94, no. 3 (2015): 430–438.

[42] D. Chen, X. Li, F. Lu, Y. Wang, F. Xiong, and Q. Li, “Dentin Dysplasia Type I-A Dental Disease With Genetic Heterogeneity,” Oral Diseases 25, no. 2 (2019): 439–446.

[43] A. Takahashi, M. Nagata, A. Gupta, et al., “Autocrine Regulation of Mesenchymal Progenitor Cell Fates Orchestrates Tooth Eruption,” Proceedings of the National Academy of Sciences of the United States of America 116, no. 2 (2019): 575–580.

[44] H. Choi, J. K. Jeong, D. Adasooriya, S. W. Cho, and E. S. Cho, “Notum as a Crucial Regulator of Matrix Integrity in Dentinogenesis,” Journal of Cellular Physiology 240, no. 7 (2025): e70070.

[45] M. Amir, L. Jeevithan, M. Barkat, et al., “Advances in Regenerative Dentistry: A Systematic Review of Harnessing Wnt/Beta-Catenin in Dentin-Pulp Regeneration,” Cells 13, no. 13 (2024): 1153.

[46] C. H. Bae, T. H. Kim, S. O. Ko, J. C. Lee, X. Yang, and E. S. Cho, “Wntless Regulates Dentin Apposition and Root Elongation in the Mandibular Molar,” Journal of Dental Research 94, no. 3 (2015): 439–445.

[47] Y. Cheng, S. Xiao, L. Lan, et al., “WNT2B High-Expressed Fibroblasts Induce the Fibrosis of IBD by Promoting NK Cells Secreting IL-33,” Journal of Molecular Medicine (Berlin, Germany) 102, no. 10 (2024): 1199–1215.

[48] M. Purwaningrum, C. M. Giachelli, T. Osathanon, S. Rattanapuchpong, and C. Sawangmake, “Dissecting Specific Wnt Components Governing Osteogenic Differentiation Potential by Human Periodontal Ligament Stem Cells Through Interleukin-6,” Scientific Reports 13, no. 1 (2023): 9055.

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