IntroductionPulp infection, necrosis, or periapical inflammation due to trauma or caries frequently affect immature permanent teeth, hindering root development. These teeth have short roots and thin dentin walls, lacking an apical stop, which increases the risk of root fracture.1,2,3 Thus, promoting root development in teeth with pulp necrosis is a desirable treatment approach. It is essential to examine and enhance the signaling molecules and mechanisms regulating tooth root development.Root development follows crown formation, initiated by Hertwig’s epithelial root sheath.4,5 Mesenchymal stem cells in the tooth germ receive developmental signals from the sheath, possessing multi-directional differentiation potential. These cells can differentiate into odontoblasts, dental pulp cells, cementoblasts, and periodontal ligament cells, all crucial for root development.6,7Stem cells from Apical Papilla (SCAP), derived from the apical papilla tissue of immature permanent teeth, were first isolated and cultured by Sonoyama et al.8 SCAP are considered the precursor to odontoblasts, elongating and polarizing under the influence of the epithelial root sheath to secrete and mineralize dentin.9 Due to their significant proliferation ability and differentiation potential, SCAP are frequently used as an ideal cell model in laboratory studies focused on tooth development and odontoblast differentiation.10,11As structural proteins, extracellular matrix proteins interact with receptors and integrins on cell membranes, regulating growth factor and protease activities, which are crucial for dentin formation and mineralization.12,13 Recently, SLIT3, a member of the extracellular matrix protein SLIT family, has been implicated in numerous biological processes, such as angiogenesis, tumorigenesis, inflammation, and bone metabolic balance.14,15,16 SLIT3 acts as a novel bone coupling factor, promoting bone formation and inhibiting resorption in bone metabolic homeostasis.17 Although bone and dentin are distinct mineralized tissues, they share similarities in mineralization processes. However, research on SLIT3’s role in tooth development remains limited.The mechanisms driving SLIT3’s potential role in odontogenesis are largely unexplored. A key candidate pathway is the Akt/GSK3β/β-catenin axis, a positive regulator of SCAP proliferation and odontogenic differentiation.18,19 This pathway, in turn, is classically regulated by the kinase Akt, which can phosphorylate and inactivate GSK3β, thereby preventing β-catenin degradation.20 Interestingly, SLIT/ROBO signaling, particularly through SLIT2, a homolog of SLIT3, directly influences Akt activity. This suggests a possible SLIT3-Akt-GSK3β-β-catenin signaling cascade in SCAP. Consequently, this study aims to investigate SLIT3’s effects on SCAP odontogenic differentiation and evaluate the specific hypothesis that SLIT3 activates the Akt/GSK3β/β-catenin signaling pathway.The objective of this study was to examine SLIT3’s spatiotemporal expression during tooth development in vivo and in vitro, assessing its impact on SCAP proliferation and odontogenic differentiation. The role of Akt/GSK3β/β-catenin signaling pathway in the effect of SLIT3 on odontogenic differentiation of SCAP were also evaluated.ResultsDefining dental mesenchyme subgroups at different developmental stages in the integrated single-cell transcriptomic atlasData cleaning and clustering analyses were conducted on GSE189381 in a manner similar to that described in a previously published study on cranial neural crest cells.21 The single-cell RNA sequencing data underwent preprocessing, including quality control, integration, principal component analysis, and dimensionality reduction. UMAP plots (Fig. 1a) visualized single-cell data from E13.5 to PN7.5 in dental and surrounding tissues. Subclustering analyses, alongside known markers for dental mesenchyme, identified dental mesenchyme subclusters marked by dotted frames (Fig. 1a, b).Fig. 1Full size imageSLIT3 displays dynamic expression patterns across embryonic to postnatal tooth development. a UMAP plots illustrating single-cell data from embryos to postnatal stages in the tooth and surrounding tissues; b Expression of SLIT3 at different embryonic and postnatal stages, with dotted lines indicating mesenchymal cell subpopulationsAt E13.5, early tooth development stages revealed two primary clusters: dental mesenchyme and epithelial cell clusters. By E14.5, distinct clusters of dental follicle and dental papilla cells emerged, indicating lineage segregation. At E16.5, the dental follicle differentiated into lateral and apical follicles, while dental papilla split into coronal and apical papillae. By PN3.5, approaching root development onset, follicle differentiation mirrored patterns at E16.5, and papilla subclusters comprised coronal, middle, apical papillae, and odontoblast clusters. At PN7.5, cell populations resembled those at PN3.5, suggesting completion of tooth root development. Notably, Slit3 mRNA maintained consistent expression in dental mesenchyme subclusters with minimal to no expression in epithelial cell subclusters (Fig. 1b).SLIT3 is continuously expressed in odontoblasts of developing mandibular first molar in miceImmunohistochemical staining of paraffin sections from the first mandibular molar tissues of PN1 mice showed SLIT3 expression in odontoblasts, ameloblasts, and the middle layer (Fig. 2a). On PN7, SLIT3 expression was observed in the dental papilla, odontoblasts, ameloblasts, and middle layer (Fig. 2b). In PN14 mice, SLIT3 was expressed in the odontoblast layer (Fig. 2c) but not elsewhere. By PN21, SLIT3 was expressed in both crown and root odontoblasts, with stronger expression in root odontoblasts (Fig. 2c, d). These findings suggest SLIT3’s significant role in odontoblast differentiation and maturation, and hard tissue formation. The strong protein signal in epithelial-derived ameloblasts contrasts with scRNA-seq and microarray data, which localize Slit3 mRNA expression to the dental mesenchyme, indicating a paracrine action of the SLIT3 protein.Fig. 2Full size imageSLIT3 expression progressively increases during in vivo odontoblast maturation and in vitro odontogenic differentiation of SCAP. a Immunohistochemical staining results of mandibular first molar sections from PN1 mouse (20×, scale bar: 100 μm), and magnified immunohistochemical staining results of mandibular first molar sections from PN1 mouse (40×, scale bar: 50 μm); b Immunohistochemical staining results of mandibular first molar sections from PN7 mouse (20×, scale bar: 100 μm), and magnified immunohistochemical staining results of mandibular first molar sections from PN7 mouse (40×, scale bar: 50 μm); c Immunohistochemical staining results of mandibular first molar sections from PN14 mouse (20×, scale bar: 100 μm), and magnified immunohistochemical staining results of mandibular first molar sections from PN14 mouse (40×, scale bar: 50 μm); d Immunohistochemical staining results of mandibular first molar sections from PN21 mouse(20×, scale bar: 100 μm), and magnified immunohistochemical staining results of crown of mandibular first molar sections from PN21 mouse (40×, scale bar: 50 μm), and magnified immunohistochemical staining results of root of mandibular first molar sections from PN21 mouse (40×, scale bars: 50 μm); e Result of RT-PCR showed that the mRNA expression of SLIT3 was increased in the SCAP differentiated into odontoblasts. *P