Soluble Notch agonist enables human ameloblast maturation and enamel-like tissue formation for tooth regeneration

Abstract

Enamel, the hardest mineralized material in the human body, protects the underlying living tissues, the dentin and pulp of the tooth. However, over 90% of adults have lost or damaged enamel and cannot regenerate the protective structure due to lack of enamel-producing cells, ameloblasts. iPSC-derived secretory Ameloblasts (isAM) have promise in future regenerative dentistry. Today, it is not known why iAM maturation requires intimate contact with the dentin-producing cell type, odontoblast. Here, we reveal that one of the critical signaling ligands emanating from odontoblasts for ameloblast maturation is Delta, the ligand for Notch receptor. We showed that our designed, soluble Notch agonist can induce iAM organoid maturation in an unprecedented manner, without interactions with odontoblast layer. Notably, soluble Notch agonist induces the iAM maturation to a novel, WDR72-positive mature secretory AM stage (ismAM) in our ameloblast organoid model. When transplanted under the kidney capsule of NOD-SCID mice, these ismAM organoids generated enamel-like calcified material, as confirmed by microCT analysis, marking the first demonstration that Notch-activated iAM organoids can form such tissue in vivo. This novel maturation procedure enabled us to analyze the specific requirements of DLX3 function in ameloblasts, independent of its known function in odontoblasts. We now show that DLX3, a gene associated with Amelogenesis Imperfecta, is required on a cell-autonomous manner in human ameloblasts for the expression of Enamelin, MMP20, and WDR72, a role not previously demonstrated in mouse models.

References

[1] Crawford PJ, Aldred M, Bloch-Zupan A. Amelogenesis imperfecta. Orphanet J. Rare Dis.. 2007, 2. 17

[2] Duverger Oet al. . Neural crest deletion of Dlx3 leads to major dentin defects through down-regulation of Dspp. J. Biol. Chem.. 2012, 287: 12230-12240.

[3] Zhang Zet al. . Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis. PLoS ONE. 2015, 10: e0121288.

[4] Ohta M, Nishimura H, Asada Y. Association of DLX3 gene polymorphism and dental caries susceptibility in Japanese children. Arch. Oral. Biol.. 2015, 6055-61.

[5] Li F, Xu S. Association between the risk of dental caries and DLX3 gene polymorphisms in Chinese children. Oral. Health Prev. Dent.. 2024, 22: 503-510

[6] Alotaibi RNet al. . Genome-Wide Association Study (GWAS) of dental caries in diverse populations. BMC Oral. Health. 2021, 21. 377

[7] Alghadeer Aet al. . Single-cell census of human tooth development enables generation of human enamel. Dev. Cell. 2023, 58: 2163-2180.e9.

[8] Kim THet al. . Osterix regulates tooth root formation in a site-specific manner. J. Dent. Res.. 2015, 94: 430-438.

[9] Chen Wet al. . Cbfβ deletion in mice recapitulates cleidocranial dysplasia and reveals multiple functions of Cbfβ required for skeletal development. Proc. Natl. Acad. Sci. USA. 2014, 111: 8482-8487.

[10] Feng Jet al. . Abnormalities in the enamel in bmp2-deficient mice. Cells Tissues Organs. 2011, 194: 216-221.

[11] Qiu Wet al. . MACF1 deficiency suppresses tooth mineralization through IGF1 mediated crosstalk between odontoblasts and ameloblasts. Genes Dis.. 2024, 11: 101103.

[12] Chan Let al. . Loss of Stat3 in Osterix+ cells impairs dental hard tissues development. Cell Biosci.. 2023, 1375.

[13] Guo Fet al. . Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression. J. Cell Physiol.. 2015, 2301871-1882.

[14] Ono W, Sakagami N, Nishimori S, Ono N, Kronenberg HM. Parathyroid hormone receptor signalling in osterix-expressing mesenchymal progenitors is essential for tooth root formation. Nat. Commun.. 2016, 7. 11277

[15] Omi, M. et al. BMP-Smad signaling regulates postnatal crown dentinogenesis in mouse molar. JBMR Plus4, https://doi.org/10.1002/JBM4.10249 (2019).

[16] Bray, S. J. Notch signalling in context. https://pubmed.ncbi.nlm.nih.gov/27507209/ (2016).

[17] Aster JC, Pear WS, Blacklow SC. The varied roles of Notch in cancer. Annu Rev. Pathol.. 2017, 12: 245-275.

[18] Mitsiadis TA, Graf D, Luder H, Gridley T, Bluteau G. BMPs and FGFs target Notch signalling via jagged 2 to regulate tooth morphogenesis and cytodifferentiation. Development. 2010, 137: 3025-3035.

[19] S. BORKOSKY Set al. . Sequential expressions of Notch1, Jagged2 and Math1 in molar tooth germ of mouse. Biocell. 2008, 32: 251-257.

[20] Jungers KA, Le Goff C, Somerville RPT, Apte SS. Adamts9 is widely expressed during mouse embryo development. Gene Expr. Patterns. 2005, 5: 609-617.

[21] Alotaibi RNet al. . Multivariate GWAS of structural dental anomalies and dental caries in a multi-ethnic cohort. Front. Dent. Med.. 2022, 2. 771116

[22] Alghadeer Aet al. . Protocol for generating three-dimensional induced early ameloblasts using serum-free media and growth factors. STAR Protoc.. 2024, 5: 103100.

[23] Mout Ret al. . Design of soluble Notch agonists that drive T cell development and boost immunity. Cell. 2025, 1885980-5994.e28.

[24] Duverger O, Morasso MI. Pleiotropic function of DLX3 in amelogenesis: from regulating pH and keratin expression to controlling enamel rod decussation. Connect Tissue Res.. 2018, 59: 30-34.

[25] Duverger O, Ohara T, Bible PW, Zah A, Morasso MI. DLX3-dependent regulation of ion transporters and carbonic anhydrases is crucial for enamel mineralization. J. Bone Miner. Res.. 2017, 32: 641-653.

[26] Hanson-Drury, S. et al. Single cell RNA sequencing reveals human tooth type identity and guides in vitro hiPSC derived odontoblast differentiation (iOB). Front. Dent. Med.4, https://doi.org/10.3389/FDMED.2023.1209503 (2023).

[27] Wang, Y. talklr uncovers ligand-receptor mediated intercellular crosstalk. Preprint at bioRxivhttps://www.biorxiv.org/content/10.1101/2020.02.01.930602v2 (2020).

[28] Hermans, F. et al. Establishment of inclusive single-cell transcriptome atlases from mouse and human tooth as powerful resource for dental research. Front. Cell Dev. Biol.10, https://doi.org/10.3389/FCELL.2022.1021459 (2022).

[29] Edman NIet al. . Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies. Cell. 2024, 187: 3726-3740.e43.

[30] Morohashi, Y. et al. C-terminal fragment of presenilin is the molecular target of a dipeptidic γ-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L- alanyl]-S-phenylglycine t-butyl ester). J. Biol. Chem. 281, 14670–14676 (2006). .

[31] Felszeghy S, Suomalainen M, Thesleff I. Notch signalling is required for the survival of epithelial stem cells in the continuously growing mouse incisor. Differentiation. 2010, 80: 241-248.

[32] Harada Het al. . Stratum intermedium lineage diverges from ameloblast lineage via Notch signaling. Biochem. Biophys. Res. Commun.. 2006, 340: 611-616.

[33] Kim HYet al. . Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral. Int. J. Oral. Sci.. 2023, 15: 55.

[34] Luca VCet al. . Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity. Science. 2017, 3551320-1324.

[35] Kuintzle, R., Santat, L. A. & Elowitz, M. B. Diversity in Notch ligand-receptor signaling interactions. eLife12, https://doi.org/10.7554/ELIFE.91422.2 (2024).

[36] Wu Ket al. . De novo design of modular peptide-binding proteins by superhelical matching. Nature. 2023, 616: 581-589.

[37] Watson JLet al. . De novo design of protein structure and function with RFdiffusion. Nature. 2023, 620: 1089-1100.

[38] Dauparas Jet al. . Atomic context-conditioned protein sequence design using LigandMPNN. Nat. Methods. 2025, 22: 717-723.

[39] Divine, R. et al. Designed proteins assemble antibodies into modular nanocages. Science372, https://doi.org/10.1126/science.abd9994 (2021).

[40] Fallas JAet al. . Computational design of self-assembling cyclic protein homo-oligomers. Nat. Chem.. 2017, 9: 353-360.

[41] Zhao, Y. T. et al. F-domain valency determines outcome of signaling through the angiopoietin pathway. EMBO Rep.22, https://doi.org/10.15252/embr.202153471 (2021).

[42] Klein ODet al. . Meeting report: a hard look at the state of enamel research. Int J. Oral. Sci.. 2017, 9: e3-e3.

[43] Kim YJet al. . Unexpected identification of a recurrent mutation in the DLX3 gene causing amelogenesis imperfecta. Oral. Dis.. 2016, 22: 297-302.

[44] Whitehouse LLEet al. . Novel DLX3 variants in amelogenesis imperfecta with attenuated tricho-dento-osseous syndrome. Oral. Dis.. 2019, 25: 182-191.

[45] Zhang, Z. et al. Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis. PLoS ONE10, https://doi.org/10.1371/journal.pone.0121288 (2015).

[46] Katsura Ket al. . WDR72 regulates vesicle trafficking in ameloblasts. Sci. Rep.. 2022, 12: 2820.

[47] Mitsiadis TA, Hirsinger E, Lendahl U, Goridis C. Delta-Notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation. Dev. Biol.. 1998, 204420-431.

[48] Mitsiadis TAet al. . Adam10-dependent Notch signaling establishes dental epithelial cell boundaries required for enamel formation. iScience. 2022, 25: 105154.

[49] Malecki MJet al. . Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes. Mol. Cell Biol.. 2006, 264642-4651.

[50] Sprinzak Det al. . Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature. 2010, 465: 86-90.

[51] Miao Z, Deng K, Wang X, Zhang X. DEsingle for detecting three types of differential expression in single-cell RNA-seq data. Bioinformatics. 2018, 34: 3223-3224.

[52] Cheng J, Zhang J, Wu Z, Sun X. Inferring microenvironmental regulation of gene expression from single-cell RNA sequencing data using scMLnet with an application to COVID-19. Brief. Bioinform.. 2021, 22: 988-1005.

[53] Qiu Cet al. . A single-cell time-lapse of mouse prenatal development from gastrula to birth. Nature. 2024, 626: 1084-1093.

[54] Establishment of scRNA-seq atlases from mouse and human tooth - Mendeley Data https://data.mendeley.com/datasets/2kskdknngb/1.

[55] Bouxsein MLet al. . Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Miner. Res.. 2010, 25: 1468-1486.

[56] Ausk BJ, Gross TS, Bain SD. Botulinum toxin-induced muscle paralysis inhibits heterotopic bone formation. Clin. Orthop. Relat. Res.. 2015, 473: 2825-2830.

Funding

This work is supported by ISCRM Fellows Program (Anjali Patni) and grants from the National Institutes of Health DE033016 (J.M., R.A.C. and H.R-B.), 1P01GM081619, R01GM097372, R01GM083867, NHLBI Progenitor Cell Biology Consortium (U01HL099997; UO1HL099993) SCGE COF220919 (H.R-B), Molecule (J.M. and H.R-B.), and AHA 19IPLOI34760143, Brotman Baty Institute (BBI), DOD PR203328 W81XWH-21-1-0006 and Stem Cell Gift Funds for H.R-B. The Birth Defects Research Laboratory was supported by NIH award number 5R24HD000836, to IAG and DD, from the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

RIGHTS & PERMISSIONS

The Author(s)