Sfrp4 Is Required for Proper Dental Formation and Stem Cell Regulation

Abstract

Our studies have established that loss-of-function mutations in the Wnt signaling antagonist SFRP4 (Secreted Frizzled Related Protein 4) cause Pyle disease, a rare skeletal disease characterized by limb deformity. Pyle disease can also present with a number of dental conditions, including tooth decay and taurodontism. Aside from the sparse clinical descriptions of the tooth anomalies seen in Pyle disease, the role of Sfrp4 in teeth has not been investigated. Here we show that in adult mouse incisors, Sfrp4 is expressed in the mesenchymal and epithelial compartments and their derivatives as well in the developing apex of the molar roots, pulp, odontoblasts, and periodontal ligament. We report that Sfrp4 deletion in mice leads to markedly shorter incisors, with reduced dentin mineral apposition and enamel volume. In addition, we find that Sfrp4 deletion leads to a reduction in the root length and bifurcation height of the developing molars, both features of taurodontism. Rodent incisors grow continuously during the animal’s lifetime, thanks to the maintenance and interactions of epithelial (EpSCs) and mesenchymal stem cells (MSCs) at their apex. As such, the mouse incisor is a powerful tool for studying adult stem cells, their interactions, and their regulation. Using this model, we found that Sfrp4 deletion leads to a significant decrease in Gli1⁺ MSCs and in Ki67 levels in mesenchymal transient amplifying cells and preameloblasts. Sfrp4-null incisors have reduced type I collagen levels and altered amelogenin and Mmp20 secretion accompanied by changes in the enamel maturation stages. When incisor growth is accelerated by preventing occlusion through tooth clipping, Sfrp4-null incisors grow significantly slower than wt incisors do. Our study suggests a key role for Sfrp4 in the fast-growing teeth (incisors) and developing molars, where there is a need to balance the maintenance and differentiation of the stem cell niche.

Introduction

Mammalian teeth develop through interactions between the dental epithelium and mesenchyme, which give rise to enamel-depositing ameloblasts and dentin-secreting odontoblasts, respectively (Balic and Thesleff 2015). The proper deposition of dentin and enamel contributes to resistance to caries in childhood and adult life (Balic and Thesleff 2015; Krivanek et al 2017). The role of Wnt signaling in dental stem cells is well established, and mutations in its components have been associated with both syndromic and nonsyndromic tooth anomalies (Zhang et al 2013; Tamura and Nemoto 2016; Vogel et al 2016; Fan et al 2018; Jarvinen et al 2018; Xiong et al 2019; Jing et al 2021; Lav et al 2023). Given the critical clinical relevance of Wnt signaling, the diverse and sometimes conflicting outcomes associated with its dysregulation underscore the need for a more comprehensive understanding of the mechanisms by which Wnt signaling governs tooth development. Members of the Wnt signaling include several Wnt ligands, receptors, co-receptors, agonists, and antagonists (Gori and Baron 2021). Among the latter group is Sfrp4 (Secreted Frizzled Related Protein 4), which functions as a decoy receptor for Wnt ligands and thereby can block both canonical and noncanonical Wnt cascades (Simsek Kiper et al 2016; Gori and Baron 2021). We were the first to report that, in humans, loss-of-function mutations in SFRP4 cause Pyle disease, a rare skeletal disease with fewer than 40 reported cases characterized by limb deformity and fragility fractures (OMIM#265900) (Simsek Kiper et al 2016). Using Sfrp4^−/− mice, which mirror the Pyle disease skeletal phenotype, we showed that Sfrp4 maintains cortical bone homeostasis by regulating both periosteal bone formation and endosteal remodeling (Simsek Kiper et al 2016). Sfrp4 regulation of endosteal remodeling occurs mainly via local regulation of the Wnt5a/Ror2 Wnt cascade (Chen et al 2019a). With regard to the periosteal formation, Sfrp4 plays a key role in the maintenance, differentiation, and function of cathepsin K–lineage periosteal stem cells (Chen et al 2023).

Pyle disease can present also with several dental conditions, including tooth decay that might result from altered mineralization caused by defects in odontoblast and ameloblast function, and taurodontism, a developmental tooth anomaly in which the molar pulp chamber is enlarged at the expense of the roots (Beighton 1987; Narayananan et al 2006; Simsek Kiper et al 2016; Bhattacharjee et al 2018; Chetty et al 2022). Therefore, investigations using Sfrp4-deficient mice offer translational insight into the molecular mechanisms underlying similar pathologies in human teeth. While aware of the difference between human and murine dentition, studies in mouse incisors are largely used to investigate the molecular mechanisms controlling tooth development. Rodent incisors grow continuously during the animal’s lifetime, thanks to the maintenance of dental epithelial (EpSCs) and mesenchymal stem cells (MSCs) at their apex (Sharpe 2016; Hermans et al 2021; Jing et al 2022). As the front of the incisors becomes shorter due to abrasion, apical stem cells exit their quiescent status, become proliferating epithelial (eTACs) or mesenchymal transit amplifying cells (mTACs), and migrate along the proximal–distal axis of the incisor while differentiating into ameloblasts or odontoblasts, which deposit new enamel and dentin, respectively (Krivanek et al 2017). Therefore, functional analysis of the incisor niche environment is key to advance our understanding of the signaling networks that regulate dental stem cells and mineralization, with major implications for regenerative medicine (Volponi and Sharpe 2013; Zhang and Yelick 2021).

Sfrp4 is expressed in the mouse incisors, and recent single-cell RNA seq data have identified Sfrp4 as a marker for incisors (Leimeister et al 1998; Krivanek et al 2020). However, the role of Sfrp4 in tooth development, growth, and eruption has not been investigated. Given the well-established importance of Wnt signaling in tooth development, together with reports of dental anomalies in individuals with Pyle disease, we sought to use our Sfrp4^−/− mice to investigate the role of Sfrp4 in the regulatory mechanisms involved in these processes.

Materials and Methods

Animals

Sfrp4^−/− mice have been previously reported (Simsek Kiper et al 2016; Chen et al 2019a; Chen et al 2023). Animal studies were conducted as outlined in the Appendix. The study complied with the ARRIVE 2.0 guidelines.

Micro–Computed X-Ray Tomography, Histomorphometry Analysis, and Scanning Electron Microscopy

A high-resolution, bench-top micro-tomographic imaging system X (Scanco μCT40, Scanco Medical AG) was used to scan the male and female skulls as described in the Appendix. Dynamic histomorphometric analysis of the dentin apposition rate was performed as outlined in the Appendix. For scanning electron microscopy–energy-dispersive spectroscopy, the upper incisors were prepared as outlined in the Appendix.

Immunoflourescence and BrdU Pulse Chase

Immunofluorescence analysis and BrdU pulse chase were performed on paraffin-embedded sections as detailed in the Appendix.

Clipping Cxperiments

Clipping experiments and growth rate were assessed as outlined in the Appendix.

Isolation of Dental Mesenchymal Cells, Quantitative Real-Time Polymerase Chain Reaction, and Migration Studies

Pulp tissue was isolated and dental MSCs cultured as detailed in the Appendix. Cell migration assay and quantitative real-time polymerase chain reaction are detailed in the Appendix.

Statistical Analysis

Data are expressed as the mean ± standard error of the mean. Statistical analysis was conducted as outlined in the Appendix.

Results

Sfrp4 Deletion Leads to Shorter Incisors and Reduced Root Length in Developing Molars

We first assessed whether Sfrp4 global deletion affects the size and shape of the skull by measuring several craniofacial parameters as described in Appendix/Figure S1. Unlike the dramatic appendicular skeletal phenotype previously reported in Sfrp4^−/− mice (Simsek Kiper et al 2016; Chen et al 2019a; Chen et al 2023), neither the size nor the shape of the skull were affected by loss of Sfrp4 at all ages examined (Appendix/Table S1 and Table S2).

Dentition in the Sfrp4^−/− mice appeared normal (Fig 1A, Appendix/Fig S2). However, the upper and lower incisor length and arc height were significantly decreased in adult and 20-d-old Sfrp4^−/− mice compared with wt mice in both sexes (Fig 1B–E and Appendix/Fig S2). Although no difference in molar size and root length was observed between wt and Sfrp4^−/− adult mice (Appendix/Table S3), in 20-d-old Sfrp4^−/− mice, the length of the first mandibular molar root was significantly decreased compared with wt mice in both sexes (Fig 1F–G, Appendix/Table S4). In addition, histology and micro–computed tomography (mCT) of the first mandibular molar at postnatal day 12 (p12) also show a reduction in the root length and bifurcation height (Fig 1H–I). These findings suggest a key role for Sfrp4 in both the fast-growing teeth (incisors) and developing molars, where there is a need to balance maintenance and differentiation of stem cells.

Figure 1.

Effect of Sfrp4 deletion on adult incisors and developing molars. (A) Micro–computed tomography (μCT) representative images (A) of the maxilla and mandible of adult wt and Sfrp4^−/− females (n = 6 to 11). Orange and blue lines = upper and lower incisor length, respectively. Yellow and white lines = upper and lower arc height, respectively. (B–E) Measurements of the upper and lower incisor length (B–D) and arc height (C–E) in males and females. All data are expressed as mean ± standard error of the mean (SEM) (n = 6 to 11). Blue dots = wt, and green dots = Sfrp4^−/− mice. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 by Student’s t test. (F, G) Representative μCT images of the first mandibular molar (F) and measurements of the root length (G) in 20-d-old wt and Sfrp4^−/− males and females. All data are expressed as mean ± SEM. Each dot represents a mouse (n = 4 to 8). *P < 0.05, ***P < 0.005 by Student’s t test comparing wt (blue dots) to Sfrp4^−/− (green dots) in each sex. (H, I) Representative hematoxylin and eosin (H) and mCT (I) images of the p12 first mandibular molar of wt and Sfrp4^−/− males (n = 3).

Sfrp4 Is Expressed in the Adult Incisors and Developing Molars

In the adult incisor, Sfrp4 is expressed in the MSCs, mTACs, dental pulp, and odontoblasts (Appendix/Fig S3A). Sfrp4 is also expressed in the slow-cycling EpSCs located in the outer enamel epithelium (OEE), the underlying stellate reticulum (SR), the actively cycling progenitors in the inner enamel epithelium (IEE), as well as in preameloblasts and ameloblasts (Appendix/Fig S3A). Importantly, at p12, when molars have not yet erupted and stem cells are still present, Sfrp4 is expressed in the developing apex of the molar roots, pulp, odontoblasts, and periodontal ligament (Appendix/Fig S3B).

Sfrp4 Deletion Decreases the Gli1⁺ MSCs, Ki67⁺ mTACs, and Preameloblasts and Impairs the Incisor Growth Rate

As shown in Figure 2, Sfrp4^−/− incisors have a significant reduction in Gli1⁺ MSCs and a significant decrease in the percentage of Ki67⁺ mTACs and preameloblasts (Fig 2A, B and Appendix/Fig S4A). A trend to a decrease that did not reach significance was also observed in the IEE (Fig 2B and Appendix/Fig S4B). Furthermore, BrdU pulse-chase experiments revealed that lack of Sfrp4 leads to a marked decrease in mTAC proliferating rate and their migration into the pulp, as indicated by a significant reduction in BrdU⁺ cells along the proximal–distal axis of the pulp 1.5 and 24 h of the chase. These changes were not significant at 48 h of the chase (Fig 2C).

Figure 2.

Sfrp4 deletion reduces epithelial and mesenchymal stem cells and transient amplifying cells. (A) Representative images and quantification of Gli1 levels in 6-wk-old wt and Sfrp4^−/− male mice incisors (n = 5). (B) Representative images and quantification of percentage of Ki67⁺ mTACs (yellow), IEE (green), and pre-aml (blue) of 6-wk-old wt and Sfrp4^−/− male mice incisors (n = 3). (C) Representative images and quantification of BrdU pulse chase of mTACs (n = 5). Dotted areas indicate the regions of interest analyzed. mTAC, mesenchymal transient amplifying cells; IEE, inner enamel epithelium; pre-aml, preameloblasts. Data are the mean ± standard error of the mean. Each dot represents a mouse. *P < 0.05 and **P < 0.01 by Student’s t test.

In addition, we used the clipping approach to stimulate and measure the effect of Sfrp4 deletion on the incisor growth rate and demonstrated that the growth rate of the Sfrp4^−/− incisors was significantly decreased compared with that of wt mice (Appendix/Fig S5). These findings suggest that Sfrp4 contributes to the adult incisor growth by regulating both MSCs and EpSCs and their ability to give rise to their derivatives.

Sfrp4 Deletion Impairs Odontoblast and Ameloblast Function

To assess whether Sfrp4 regulates odontoblast function, we performed histomorphometry analyses of undecalcified incisor sections. We found that Sfrp4 deletion leads to a significant increase in unmineralized dentin and a decrease in dentin mineral apposition (Fig 3). In addition, Sfrp4 deletion leads to a significant decrease in type I collagen levels (Fig 3 and Appendix/Fig S4C).

Figure 3.

Sfrp4 deletion leads to decreased dentin mineral apposition. (A) Representative Von Kossa staining images of undecalcified mandibles of 6-wk-old wt and Sfrp4^−/− females. (B) Representative images of double labeling (calcein in green and alizarine red in red) in wt and Sfrp4^−/− females (n = 3). (C) Representative images of type I collagen (Col1) levels in 6-wk-old wt and Sfrp4^−/− females (n = 5). (D–F) quantification of unmineralized dentin (D), dentin mineral apposition rate (E), and type collagen (F). Data are the mean ± standard error of the mean, with each dot representing a mouse. *P < 0.05 by Student’s t test.

μCT shows that in Sfrp4^−/− mice, the transition from secretory to maturational stage enamel is shifted from the proximal portion of the incisor toward the incisor tip, suggesting a delay in ameloblast function and maturation, additionally supported by a significant decrease in enamel volume compared with wt mice (Fig 4A–B). Furthermore, in Sfrp4^−/− mice, the distal ameloblasts appeared longer, although they remain polarized (Fig 4C). Confirming μCT and histology, scanning electron microscopy analysis shows a longer secretory stage in Sfrp4^−/− incisors and confirms that maturation-stage mineral density is reached later than in wt (Fig 4D–a–b). During enamel mineralization, matrix proteins embedding the mineral are removed to allow crystal expansion. In wt mice, this extensive protein removal transforms enamel from loosely packed crystallites in early maturation (Fig 4D–c–e) to densely packed rods and interrod mature enamel (Fig 4D–g). In Sfrp4^−/− mice, protein removal is delayed, resulting in loosely packed enamel near the incisal edge, resembling earlier wt enamel stages (Fig 4D–e) with incomplete protein removal (Fig 4D–d–f–g). However, Sfrp4 deletion does not affect the structural organization into decussating rods and interrod enamel (Fig 4D).

Figure 4.

Sfrp4 deletion leads to a decrease in enamel volume and enamel protein secretions. (A) Representative μCT images of the maxilla and mandible of 6-wk-old wt and Sfrp4^−/− male mice. The yellow dotted line highlights the proximal end of the incisors. The arrows point to incisor enamel shifted toward the incisor tip. (B) μCT quantification of enamel volume in 6-wk-old wt and Sfrp4^−/− male mice (n = 5). Data are the mean ± standard error of the mean. Each dot represents a mouse. **P < 0.01 by Student’s t test. (C) Representative hematoxylin and eosin images of the ameloblasts in wt (a, c) and Sfrp4^−/− (b, d) male mice (n = 4 to 6). (D) Scanning electron microscopy analyses with secondary electron detection. At low magnification (a, b), higher mineral density is seen as brighter and is reached earlier in wt (a), compared with Sfrp4^−/− (b), as highlighted by the magenta-colored line indicating the transition from the secretory to maturation stage. Inserts indicate regions of interest in the secretory stage (yellow), early maturation stage (pink), and erupted mature enamel (light blue) in the erupted incisor, near the incisal edge. The typical enamel microstructure with decussating rods is visible in the secretory stage wt (c, e) and Sfrp4^−/− enamel (d, f), with enamel rod outlined in the cross section (arrowheads) and seen in the oblique plane (open arrows). The etching step during sample preparation removes some of the mineral, thereby creating a relief that accentuates structural organization and reveals the protein-rich matrix in which most mineral has been removed in the secretory stage (c, d) and early maturation stage in the Sfrp4^−/− (f). Crystals in the rod and interrod pattern are visible in wt early maturation (e) and mature enamel of wt and Sfrp4^−/− (g, h). Note the similarity between the erupted mature Sfrp4^−/− enamel near the incisal edge (h) and the wt early maturation stage enamel (dark blue frames) (e) (n = 5). (E) Representative confocal images and quantification of amelogenin levels in wt and Sfrp4^−/− male mice. a and b show higher magnification (n = 3). (F) Representative confocal images and quantification of Mmp20 levels in wt and Sfrp4^−/− male mice. a and b show higher magnification (n = 3 to 4).

To assess ameloblast function, we next examined amelogenin levels, the most abundant structural enamel matrix protein secreted by secretory ameloblasts, and the levels of matrix metalloproteinase 20 (Mmp20), a key enzyme required for amelogenin cleavage and proper enamel formation. As shown in Figure 4E and Appendix/Figure S4D, not only were amelogenin levels significantly reduced in Sfrp4^−/− mice, but it also appeared that amelogenin was not properly removed and remained in the matrix. Accordingly, Mmp20 levels also were significantly reduced in the secretory stage ameloblasts (Fig 4F and Appendix/Fig S4E). Overall, these findings demonstrate a role for Sfrp4 in both the odontoblast and ameloblast activity.

Sfrp4 Deletion Leads to Decreased Dental Mesenchymal Cell Differentiation and Migration In Vitro

To further investigate the effect of Sfrp4 deletion on dental MSC differentiation and function, we performed a series of in vitro studies. MSC isolated from the Sfrp4^−/− incisors displayed decreased ability to differentiate, as shown by alkaline phosphatase (ALP), Alizarin Red (AR) staining, and Dmp1 expression levels (Fig 5A–C). The increase in Axin2, Lef-1, Tcf1, and c-Jun expression during differentiation supports the activation of canonical and noncanonical Wnt cascades, respectively, in these cultures as we expected in the absence of Sfrp4 (Fig 5D). In addition, in vitro migration studies showed a significantly impaired migration rate as indicated by the increase in the time required for Sfrp4^−/− cells to close the wound compared with wt cells (Fig 5E). Overall, the findings of a decrease in cell differentiation and wound healing support our in vivo study of a decrease in dentin formation and a defect in cell migration along the proximal–distal axis of the pulp.

Figure 5.

Sfrp4 deletion impairs dental mesenchymal stem cell differentiation and migration in vitro. (A) Representative images of alkaline phosphatase (ALP) staining and quantification at 7 d. (B) Representative images of Alizarin Red staining and quantification at 28 d. (C) Dmp1 mRNA levels at different time points. (D) mRNA levels of selected canonical and noncanonical Wnt target genes at different time points. (E) Quantification of the percentage of wound closure and representative images (blue dots = wt and green dots = Sfrp4^−/− cells). Data are the mean ± standard error of the mean. Each dot represents values obtained from cell isolated from 1 mouse (n = 4 to 6). Each experiment was run in triplicate. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 by Student’s t test.

Discussion

Studies of adult mouse incisors are primarily driven by their relevance to human tooth development and regeneration (Volponi and Sharpe 2013; Sharpe 2016; Zhang and Yelick 2021; Jing et al 2022). In these continuously growing teeth, stem cells remain active throughout adulthood, giving rise to cells that replace lost dentin and enamel at the distal tip, compensating for wear at the incisal edge (Krivanek et al 2017; Jing et al 2022). This makes the mouse incisor a valuable model for investigating adult dental stem cells and the local factors that regulate their niche and support tooth regeneration. Despite the enormous clinical importance of Wnt signaling and the rapid expansion of our understanding of the mechanisms by which this signaling governs tooth formation and adult tooth homeostasis, conflicting findings have been reported. For example, human mutations in APC and Axin2, the 2 components of the same β-catenin degradation complex, lead to opposite anomalies, hyperdontia and hypodontia, respectively (Lu et al 2017), and Wnt activation by deletion of Notum, a Wnt inactivating enzyme (Kakugawa et al 2015), results in dentin dysplasia (Vogel et al 2016). These discrepancies likely result from feedback interactions, both positive and negative, between canonical and noncanonical cascade involvement, which vary depending on the cell type (epithelial versus mesenchymal) as well as on the differential expression of receptors, co-receptors agonists, and antagonists (Gori and Baron 2021).

Although the role of Sfrp4 in skeletal homeostasis has been established, its role in tooth biology has not been investigated. We show that in the adult mouse incisors, Sfrp4 is expressed in both the epithelial and mesenchymal compartments and that its deletion leads to shorter incisors with decreased growth rate, type I collagen levels, dentin mineralization, amelogenin, and Mmp20 levels and enamel volume. Consistent with the diminished expression of enamel matrix proteins and protease, in the absence of Sfrp4, the pace of enamel mineralization is slower and the transition from the secretion to maturation stage is delayed. The extensive removal of matrix proteins is critical for the growth of crystallites to achieve a fully mineralized, hard enamel, and therefore, defects in the removal of these proteins hinder the expansion of enamel crystals and prevent their tight packing into rods, ultimately compromising enamel quality. Highlighting the relevance of our studies to dental anomalies seen in Pyle disease, the proper deposition of enamel and mineral composition are important for resistance to caries in childhood and adult life, and defects in these processes might be responsible for the increased susceptibility to dental caries seen in patients with Pyle disease (Narayananan et al 2006; Simsek Kiper et al 2016; Wonkam et al 2016; Chetty et al 2022).

In teeth with limited growth such as in humans and in mouse molars, dental stem cells are typically dormant and are partially activated in response to significant damage (Krivanek et al 2020). We found that, in molars, Sfrp4 is expressed in the developing apex of the roots (where stem cells reside), pulp, odontoblasts, and periodontal ligament and that in Sfrp4^−/− mice, developing molars have significantly shorter molar roots and reduced bifurcation height. It is worth noting that taurodontism, a condition that results from disrupted developmental homeostasis and is characterized by several anomalies including defects in tooth root furcation and short roots of deciduous molars, has been described in individuals with Pyle disease (Chetty et al 2021; Chetty et al 2022). Because dental stem cells become quiescent in adult molars, our findings suggest that Sfrp4 is a needed to balance maintenance and differentiation of the stem cell niche. These findings resonate with our previous studies showing that Sfrp4 in periosteal stem cells is required for their maintenance and differentiation (Chen et al 2023). Further studies investigating whether Sfrp4 regulates stem cell activation in response to damage and induction of reparative dentin formation at the site of pulp exposure will confirm a role for Sfrp4 in the regulation of odontogenesis in the mouse molars. In this context, it has been reported, using a direct pulp-capping model in rats, that Sfrp1, which, like Sfrp4 regulates odontoblast differentiation, induces reparative dentin formation (Ipposhi et al 2021).

The transition from slow-cycling stem cells to transit proliferating cells governs the growth rate of the incisors (Sharpe 2016; Krivanek et al 2020). We report here that Sfrp4 deletion leads to a significant reduction in Gli1⁺ MSCs and in the Ki67 proliferating eTACs and mTACs as well their migration and differentiation into functional ameloblasts and odontoblasts. These effects on the MSCs and EpSCs in turn lead to reduced growth rate and shorter incisors with reduced mineralized dentin and enamel maturation stage and volume. Thus, our findings suggest that Sfrp4 plays a role in the maintenance of the pool of stem cells and/or their ability to give rise to their derivatives. It remains to be resolved whether the cells responsive to Sfrp4 are the cells that also secrete Sfrp4 and whether responsiveness of the stem cells to Sfrp4 secreted by their progeny provided feedback mechanisms for the stem cells. Of relevance to our previous work demonstrating an Sfrp4/Wnt5a/Ror2 signaling axis in the regulation of endosteal osteoclasts within cortical bone (Chen et al 2019a), Sfrp4 may contribute to the Wnt5a/Ror2 feedback loop between mTACs and MSCs in the incisors (Jing et al 2021). It is plausible that Sfrp4, produced by the stem cell pool, supports stem cell maintenance, expansion, and function and/or influences mTAC activity and differentiation. Whether this is a direct (from the EpSCs) or indirect (from the MSCs) effect of Sfrp4 deletion remains to be addressed. To answer these questions, further studies are needed, for example, Sfrp4 deletion in specific dental cells using well-established mouse models (Gli1^CreERT2, Axin2^CreERT2, K14^CreERT2) to target MSCs and EpSCs and our novel mouse model (Sfrp4^fl/fl). Further supporting the role of Sfrp4 in these processes are the findings that Sfrp1 and Sfrp2 knockdown suppresses odontoblastic differentiation of human dental papilla cells and the osteogenic differentiation ability of stem cells isolated from the apical papilla, respectively, and that Sfrp2⁺ fibroblast progenitors were found in mouse incisors and the developing tooth root of human molars (Yu et al 2016; Ipposhi et al 2021; Zhao et al 2025). Deletion of Wnt5a in mTACs or Ror2 in Gli1⁺ MSCs produces a phenotype similar to that of Sfrp4^−/− mice. While this may seem paradoxical, it is important to note a key distinction: Sfrp4 deletion activates both canonical and noncanonical Wnt signaling, whereas deletion of Wnt5a or Ror2 primarily disrupts the noncanonical pathway. In addition, given that Sfrp4 encodes a secreted protein capable of binding to multiple Wnt ligands and Frizzled receptors, its deletion may influence the activity of several Wnt ligands and downstream cascades and not only that of Wnt5a, thereby affecting the overall outcome in complex ways. Canonical and noncanonical pathways are tightly interconnected, and selectively modulating one versus both can lead to distinct outcomes. This aligns with our previous findings in long bones, in which noncanonical Wnt activation in Sfrp4-null mice overrides the anabolic effects of canonical Wnt signaling on cortical bone, ultimately leading to bone loss, while increased trabecular bone mass is due to the activation of canonical Wnt signaling in this bone compartment (Simsek Kiper et al 2016; Chen et al 2019a). Moreover, due to the extensive network of positive- and negative-feedback loops within the Wnt signaling system, both activation and inhibition of different pathway components can sometimes converge on similar phenotypic results. For example, constitutively active β-catenin and full deletion of β-catenin in the dental mesenchyme impairs tooth germ morphogenesis and tooth development (Fujimori et al 2010; Chen et al 2019b). Hence, this highlights the complexity of Wnt signaling cascades in tooth biology and the need for precise regulation of Wnt signaling for proper odontogenesis and tooth development.

Our in vivo and in vitro findings support a role for Sfrp4 in regulating mesenchymal cell migration and differentiation. Consistent with a role for the noncanonical Wnt cascade in these processes, the overexpression of Wnt5a has been shown to inhibit the migration of human dental papilla cells and ALP activity and the mineralization and expression of osteogenic-specific genes in stem cells from the apical papilla (Peng et al 2010; Fu et al 2022). Hence, in other systems/tissues, it has been shown that Wnt5a missense mutations, which result in loss of function, lead to decreased cell migration and mandibula hypoplasia (Hosseini-Farahabadi et al 2017). Similar to our previous findings, it is plausible that in the incisors, the activation of the noncanonical Wnt cascade in these cells overrides the positive effect of canonical Wnt activation on migration and/or differentiation.

In conclusion, our findings underscore the importance of Sfrp4 in maintaining the proper growth and function of dental tissues. Adding to this are the findings that individuals with Pyle disease present with a series of dental conditions, including tooth decay that might be caused by defects in odontoblast and ameloblast differentiation and function (Beighton 1987; Narayananan et al 2006; Adeniji 2009; Simsek Kiper et al 2016; Bhattacharjee et al 2018; Chetty et al 2022). By investigating the molecular mechanisms driving dental tissue development, we can gain deeper insights into the underlying causes of oral diseases and may open novel avenues for regenerative approaches aimed at restoring tooth structure and function in humans.

Supplementary Material

A supplemental appendix to this article is available online.

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