
==== Front
Stem Cell Res Ther
Stem Cell Res Ther
Stem Cell Research & Therapy
1757-6512
BioMed Central London

39256850
3894
10.1186/s13287-024-03894-y
Research
Fgf9 promotes incisor dental epithelial stem cell survival and enamel formation
Tang Lingyun 1
Chen Mingmei 1
Wu Min 2
Liang Hui 1
Ge Haoyang 1
Ma Yan 3
Shen Yan 1
Lu Shunyuan 1
Shen Chunling 1
Zhang Hongxin 1
Zhang Chenping zhang.chenping@hotmail.com

4
http://orcid.org/0000-0002-2626-4122
Wang Zhugang zhugangw@shsmu.edu.cn

1
1 grid.16821.3c 0000 0004 0368 8293 State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui- Jin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin Road II, Building #17, Shanghai, 200025 P.R. China
2 grid.410656.0 0000 0004 7647 3728 Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital Affiliated to SJTUSM, Shanghai, China
3 https://ror.org/0220qvk04 grid.16821.3c 0000 0004 0368 8293 Ruijin Hospital Lu Wan Branch, Shanghai Jiaotong University School of Medicine, Shanghai, P.R. China
4 https://ror.org/0144s0951 grid.417397.f 0000 0004 1808 0985 Key Laboratory of Head & Neck Cancer Translational Research of Zhejiang Province, Department of Head and Neck Surgery, Zhejiang Cancer Hospital, Hangzhou, 310022 Zhejiang P.R. China
11 9 2024
11 9 2024
2024
15 29312 7 2024
25 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Understanding the role of cytokines in tooth development is critical for advancing dental tissue engineering. Fibroblast growth factor 9 (FGF9) is the only FGF consistently expressed throughout dental epithelial tissue, from the initiation of tooth bud formation to tooth maturation. However, mice lacking Fgf9 (Fgf9−/−) surprisingly show no obvious abnormalities in tooth development, suggesting potential compensation by other FGFs. Here we report findings from an Fgf9S99N mutation mouse model, a loss-of-function mutation with a dominant negative effect. Our study reveals that Fgf9 is crucial for dental epithelial stem cell (DESC) survival and enamel formation.

Methods

To dissect the role of Fgf9 in tooth development, we performed the micro-CT, histomorphological analysis and gene expression assay in mice and embryos with S99N mutation. In addition, we assessed the effect of FGF9 on the DESC survival and dental epithelial differentiation by DESC sphere formation assay and tooth explant culture. Cell/tissue culture methods, gene expression analysis, specific inhibitors, and antibody blockage analysis were employed to explore how Fgf9 regulates enamel differentiation and DESC survival through both direct and indirect mechanisms.

Results

The Fgf9S99N mutation in mice led to reduced ameloblasts, impaired enamel formation, and increased apoptosis in the cervical loop (CL). DESC sphere culture experiments revealed that FGF9 facilitated DESC survival via activating ERK/CREB signaling, without affecting cell proliferation. Furthermore, in vitro tissue culture experiments demonstrated that FGF9 promoted enamel formation in a manner dependent on the presence of mesenchyme. Interestingly, FGF9 stimulation inhibited enamel formation in isolated enamel epithelia and DESC spheres. Further investigation revealed that FGF9 supports DESC survival and promotes amelogenesis by stimulating the secretion of FGF3 and FGF10 in dental mesenchymal cells via the MAPK/ERK signaling pathway.

Conclusions

Our study demonstrates that Fgf9 is essential for DESC survival and enamel formation. Fgf9 performs as a dual-directional regulator of the dental enamel epithelium, not only inhibiting DESC differentiation into ameloblasts to preserve the stemness of DESC, but also promoting ameloblast differentiation through epithelial-mesenchymal interactions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-024-03894-y.

Keywords

Fibroblast growth factors
Dental epithelial stem cells
Cell survival
Enamel
Epithelial-mesenchymal interaction
http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 81900799 82100163 81971462 81901529 Tang Lingyun Wu Min Shen Chunling Zhang Hongxin http://dx.doi.org/10.13039/501100003399 Science and Technology Commission of Shanghai Municipality 19YF1430400 19YF1429700 Tang Lingyun Wu Min http://dx.doi.org/10.13039/100017950 Shanghai Municipal Health Commission 2020YJZX0135 Ma Yan issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
==== Body
pmcBackground

The mammalian dentition is a finely regulated process involving the proliferation, cytodifferentiation, and migration of stem cells [1]. Tooth development is a complex process that involves the formation of different types of cells and tissues, such as enamel, dentin, cementum and pulp. These cells and tissues are derived from two embryonic origins: the oral epithelium and the neural crest-derived mesenchyme. The oral epithelium gives rise to ameloblasts, which produce enamel, while the mesenchyme gives rise to odontoblasts, which produce dentin, as well as other cells that contribute to cementum, pulp, and periodontium [2]. Reciprocal interactions between epithelial and mesenchymal tissues (epithelial-mesenchymal interaction) play a fundamental role in the morphogenesis of teeth and regulate all aspects of tooth development [3]. Understanding the extracellular signals that regulate tooth development is important, as it not only uncovers the fundamental mechanisms behind mammalian dentition but also facilitates the development of new methods for tooth tissue engineering.

Multiple regulatory factors and signaling pathways are reported to regulate the early tooth development, morphogenesis, and homeostasis maintenance, including BMP, Wnt, Shh, and Eda [1]. In particular, FGFs have been well recognized as crucial epithelial-mesenchymal interacted growth factors for tooth development [4]. FGF family contains 22 genes encoding structurally related proteins and includes canonical, hormone-like, and intracellular subfamilies [5]. Most FGFs mediate their biological responses by binding to and activating cell surface tyrosine kinase FGF receptors (FGFRs) [6]. Alternative mRNA splicing of Fgfr1–3 generates IIIb and IIIc isoforms with specific ligand-binding and tissue distributions [7]. IIIb splice isoforms are predominantly expressed in epithelial lineages and transduce signals from FGF ligands in the mesenchyme, while the IIIc splice variant is restricted to mesenchymal lineages and transduces signaling from FGF ligands in the epithelium [8–12]. The spatiotemporal expression of FGFs and FGFRs in tooth and the specific FGF-FGFR interaction are the basis for the participation of epithelial-mesenchymal interactions [3, 13, 14]. In total, 10 FGFs (FGF3, FGF4, FGF8, FGF9, FGF10, FGF15-18, and FGF20) and 2 FGFRs (FGFR1 and FGFR2) are specifically and dynamically expressed in the epithelium and mesenchyme during tooth development [4, 14, 15]. Among the tooth-expressed FGFs, FGF9 is the only FGF family member with continuous epithelial expression throughout tooth development [15, 16]. Fgf9 is expressed in the epithelium during the initiation and bud stage, becomes restricted to the primary enamel knot (pEK) in the cap stage, and persists in the inner enamel epithelium (IEE) from the bell stage to the maturation stage [4, 17–19]. Interestingly, Fgf9 null mice do not exhibit any defects in tooth initiation, bud invagination, tooth number, or shape [20, 21]. This might be due to compensatory effects from other FGF members, such as Fgf20, in the Fgf9 knockout mouse model [21]. Therefore, to further elucidate the role of Fgf9 in tooth development, new animal models that can overcome compensatory effects may be necessary.

Our previous study has identified a missense mutation (p.Ser99Asn, S99N) in exon 2 of the FGF9 gene, which is responsible for human multiple synostoses syndrome-3 [22]. The S99N mutation in Fgf9 is considered a loss-of-function mutation that disrupts important hydrogen bonds between FGF9-FGFRs, resulting in impaired receptor binding capacity and signaling transduction [23–25]. Additionally, this mutation also exhibits a dominant negative effect, interfering with the signaling of wild-type FGF9 [26]. In prior studies, we established a Fgf9S99N knock-in mouse model, which impaired synovial joint development and disturbed bone homeostasis by affecting osteoblast and osteoclast formation [23, 26]. Interestingly, we also observed that the mutant mice present abnormal incisors with misalignment and impaired enamel formation, indicating that Fgf9 participates in enamel formation and tooth development.

In this study, we utilized the Fgf9S99N mutation mouse model and in vitro cell/tissue culture experiments to investigate the role of Fgf9 in amelogenesis and DESC maintenance. The in vivo and in vitro results underscore the crucial role of Fgf9 as a growth factor that promotes DESC survival and inhibits DESC differentiation through an autocrine mechanism and indirectly supports DESC survival and promotes amelogenesis through epithelial-mesenchymal interactions.

Materials and methods

Mice

Mice carrying the S99N mutation in the Fgf9 gene were generated as previously described [23] and maintained on a 129S6 background. The mice were group-housed with littermates in breeding pairs or a breeding harem (2 females to 1 male) and kept in specific pathogen-free (SPF) conditions with free access to food and water. Most homozygous mice were neonatally lethal, with only a few surviving up to 16 days post-birth with special care. The control subjects for the experimental mice and embryos involved in the experiment all come from the same litter. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Rui-jin Hospital affiliated to Shanghai Jiao Tong University School of Medicine (approval title: “Study of the role of Fgf9 gene in skeletal development and homeostasis”; approval number: RJ2023076; Date: June 8, 2023). The work has been reported in line with the ARRIVE guidelines 2.0.

Histomorphology analysis and micro-CT

The teeth from postnatal day 16 (P16) mice were fixed with 4% paraformaldehyde for 24 h at 4 °C. The bone was decalcified with 12.5% EDTA 2Na solution for 2 weeks at 4 °C, and the solution was changed every 3 days. Embryonic and newborn teeth were fixed and sectioned as described above without decalcification. Samples were embedded in paraffin and sectioned at a thickness of 5 μm. DESC spheres embedded in Matrigel were fixed with a fixative solution (2% paraformaldehyde and 1% glutaraldehyde in PBS) for 30 min at room temperature. The DESC spheres were dehydrated using 30% sucrose overnight, embedded in OCT, and sliced into 20 μm slices using a cryostat microtome (Shandon Cryotome SME). Sections were stained with hematoxylin and eosin. All images were captured by a Nikon Eclipse 8Vi. For µ-CT scanning, mouse skull was scanned in PBS on a Skyscan 1275 (Bruker, Belgium) at an 8 μm resolution with 50 kV source voltage, 60 µA source current, 110 ms exposure, and 0.2º rotation step. The 3D image of the mouse skull was generated by Burker 3D.suite software. The incisor and molar images were achieved by three orthogonal projections.

Incisor DESC primary culture and sphere formation assay

Incisor DESC sphere culture was performed as described previously with modifications [27, 28]. Firstly, 3-month-old mice were sacrificed by CO2 and immersed in 75% ethanol for 3 min. The lower incisors were dissected from mandibles and digested with 1% collagenase I (Sigma, C0103) in PBS with 2% FBS at room temperature for 30 min. The epithelium was separated from the tooth with forceps, and the cervical loop was excised by an insulin syringe needle under the stereomicroscope. The collected cervical loops were dissociated with Accumax (Sigma, A7089) at 37 °C for 30 min and dispersed by pipetting using a low-adhesion pipette tip to generate a single-cell suspension. The cell number was counted by hemocytometer, and the cell concentration was adjusted to 1 × 105 cells/ml. Next, the cell suspension was mixed with Matrigel (Corning, 354234) at a ratio of 1:1 and plated in ultralow attachment plates, with 100 µl per drop, followed by incubation at 37 °C for 30 min to allow the Matrigel to solidify. After the solidification of Matrigel, the culture medium [DMEM/F12 (Gibco, 11330032) + 20 ng/ml mEGF (Roche, 2028-EG-200) + 25 ng/ml mFGF2 (Roche, 3139-FB-025) + 1X B27 supplement (Gibico, 17504-044) + PS] was added. For the sphere formation assay, the single-cell suspension was plated at a density of 1000 cells/ml. The culture medium was half-replaced for the first time and replaced every two days. After being cultured for 7 days, the DESC spheres were formed and captured under a stereomicroscope. The number and diameter of the spheres were measured using Image-pro Plus. To induce differentiation, DESC spheres were harvested and attached to the plate for 24 h. They were then cultured in a differentiation medium (DMEM + 10%FBS + 3.0 mM CaCl2 + 100 nM dexamethasone + 10 mM β-glycerolphosphate + 50 µg/ml L-ascorbic acid.) for 14 days with the medium replaced every two days.

DPSC primary culture

3-month-old mice were euthanized by CO2 and immersed in 75% ethanol for 3 min for sterilization. The lower mandible was isolated using forceps and a scalpel, ensuring the removal of surrounding tissue as thoroughly as possible with gauze. The incisor was isolated by breaking the mandible bone using the scalpel. The dental pulp was then separated from the tooth root and digested into single cells using 0.25% Trypsin-EDTA (Gibco, 25200072) for 5 min with pipetting. After neutralization and a wash with culture medium, the cells were cultured in a-MEM medium (HyClone, SH30265.01) supplemented with 10% fetal bovine serum (Gibco, 12664025) and penicillin/streptomycin solution (P/S, 100IU/mL) at 37 °C and 5% CO2. The DPSCs used in this experiment were within 3 passages.

Incisor and dental epithelium explants culture

Incisor and dental epithelium explant culture was performed as previously described with modifications [29]. Three-month-old mice were euthanized by CO2 and immersed in 75% ethanol for 3 min. The lower mandible was isolated using forceps and a scalpel, ensuring the removal of surrounding tissue as thoroughly as possible with gauze. The incisor was separated by carefully breaking the mandible bone using the scalpel. For explant culture, the isolated incisors were placed on the surface of a 70 μm cell strainer with culture medium [DMEM/F12 (Gibco, 11330032) + 10% FBS (Gibco, 12664025) + 150 µg/ml ascorbic acid (Sigma, A8960) + P/S] to ensure the explants were at the liquid/air surface. For dental epithelium explants, the isolated incisor was incubated with 1% collagenase I for 20 min at room temperature. The dental epithelium could be easily isolated and cultured under the same conditions as the incisor explants. For antibody blockage analysis, 2 µg/ml anti-FGF3 and/or anti-FGF10 were added into the culture medium. The medium was changed every other day, carefully avoiding the formation of air bubbles.

Immunofluorescence and TUNEL staining

The paraffin sections were dewaxed, and microwave antigen retrieval was performed in 0.01 M citrate sodium (PH 6.0). The cryosections were washed with PBS without antigen retrieval. For BrdU staining, the slides were incubated with 2 N HCl for 15 min at 37 °C and neutralized with 0.1 M Sodium borate for 10 min before blocking. After washing, slides were blocked in 10% goat serum for 30 min and incubated with primary antibodies (Supplemental Table S2) overnight at 4 °C. Subsequently, the slides were incubated with secondary antibodies for 2 h at room temperature. TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche, 1168475910) according to the manufacturer’s protocol. The slide was mounted, and the nuclei were counterstained with mounting medium (Abcam, ab104139). All images were captured using a fluorescence microscope (NIKON ECLIPSE 8Vi).

BrdU and annexin V analysis by flow cytometry

For in-vivo BrdU labeling, pregnant mice received an intraperitoneal injection of BrdU at a dose of 100 µg/g body weight. After a 2-hour labeling period, the mice were euthanized, and their embryonic lower incisors were collected for histological sectioning. BrdU-labeled cells were detected in the tissues using immunofluorescence staining with anti-BrdU antibodies. For BrdU analysis of DESCs, 10 µM BrdU was added into the culture medium for 1 h. The DESCs spheres were released from Matrigel with 1 mg/ml dispase (StemCell, 07923) for 10 min at 37 °C. The collected DESCs spheres were dissociated with Accumax at 37 °C for 15 min and dispersed by pipetting with a low-adhesion pipette tip to generate a single cell suspension. The viable cell number was counted using a hemocytometer with trypan blue and adjusted to a concentration of 1 × 106 cells/ml. For cell apoptosis analysis, we used the Annexin V Apoptosis Detection Kit (eBioscience, 88-8007-74) following the manufacturer’s protocol. For BrdU analysis, the Phase-Flow™ Alexa Fluor® 647 BrdU Kit (BioLegend, 370706) was used according to the manufacturer’s protocol. After staining, the samples were analyzed on a FACSVerse flow cytometer (BD Bioscience, Franklin Lake, NJ, USA), and the data were processed using FlowJo software.

qRT-PCR

Total RNA was extracted from cells and tissues following the Trizol reagent RNA extraction protocol (Roche, 11418467001). cDNA was synthesized by reverse transcription of 0.5–1 µg of RNA according to the manual protocol (Takara, RR036A). Gene expression levels were determined using real-time PCR with a RealPlex instrument (Eppendorf). The primers used are listed in Supplemental Table S1. β-actin was employed as an internal control.

Western blotting

Proteins were extracted from cells and tissues using NETN lysis buffer (100 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.5 mM EDTA, and 0.5% Nonidet P-40) supplemented with protease (Roche, 11836153001) and phosphatase inhibitor cocktails (Roche, 4906837001). The protein samples were separated by SDS-PAGE and probed with specific primary antibodies and fluorescent secondary antibodies (Supplemental Table S2). Western blot images were captured using an Odyssey near-infrared fluorescence imaging system (Li-COR). Each western blot result presented is representative of a minimum of 3 independent experiments.

Statistical analysis

In statistical analysis, each group of samples has at least three biological replicates. Data in graphs are presented as box-and-whisker plots showing all individual data points, median, and interquartile ranges from the maximum to the minimum values. Statistical significance was determined using an unpaired t-test with a significance threshold of p < 0.05, and all p-values are shown in the graphs. All statistical analyses were performed in GraphPad Prism version 9.1.1 software (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Fgf9S99N mutation impaired ameloblast differentiation and enamel formation

In our previous work, we discovered that the S99N mutation of Fgf9 leads to the failure of joint development in the early stages of development and impaired bone homeostasis referring to the dysregulation of osteoblast and osteoclast formation [23, 26]. Interestingly, we also observed that Fgf9S99N homozygous mice displayed misaligned upper and lower incisors and pale-colored incisors compared to wild-type littermates, suggesting incomplete enamel formation (Fig. 1A). Micro-CT 3D reconstruction images revealed a significant reduction in enamel volume and thickness in the incisor of homozygous mice compared to both wild-type and heterozygous mice (Fig. 1B). Sagittal and coronal views of the lower incisors showed that homozygous mice have smaller incisors with impaired enamel compared to wild-type and heterozygous mice (Fig. 1C, D). Quantification analysis also showed that the enamel thickness of lower incisors in homozygous mice was significantly less compared to wild-type and heterozygous mice (Fig. 1E). Sagittal histological sections and H&E staining further confirmed decreased enamel in the incisors of homozygous mice (Fig. 1F). Similarly, there were no significant differences in the number, morphology and enamel thickness of molars between homozygous mice and wild-type mice at P16 (Fig. S1A, B). These results demonstrate that Fgf9S99N mutation impairs incisor enamel formation.

Fig. 1 Fgf9S99N homozygous mutation leads to impaired enamel development in mouse incisors. (A) Photograph of incisors from the wt, heterozygous (het), and homozygous (mut) mice at the age of postnatal day 16. The white arrows indicate the pale and misaligned incisor of homozygous mice. (B) 3D display of enamel from P16 mouse incisors and molars. Scale bar, 1000 μm. (C) Micro-CT sagittal views of lower incisors from wt, het and mut mice at P16. Scale bar, 1000 μm. (D) Micro-CT coronal section image of the distal end of the tooth socket from wt, het and mut mice at P16. Scale bar, 500 μm. (E) Quantitative results of enamel thickness in lower incisors in P16 mice. The individual lower incisor was considered the experimental unit (n = 6). (F) H&E staining of lower incisor of P16 mice. (G-I) H&E staining of lower incisor of E18.5 embryos. Images in (H) represent the magnified view of the left dot box, and images in (I) represent the magnified view of the right dot box in (G). The black dots line in (H) indicates the dental epithelia of CL. The green, black, and red dots line in (I) indicate the boundary of pre-odontoblast (p-Od) and pre-ameloblast (p-Am) layers. (J) Immunofluorescence staining of ABMN (red) and DAPI (Blue) in the incisor of E18.5 embryos. (K) The mRNA levels of enamel formation-related genes in the incisors of E18.5 embryos with the indicated genotypes (n = 3). The individual mouse was considered the experimental unit within the studies, except (E). Scale bar from F-J, 200 μm. Data are shown as box-and-whisker plots (with median and interquartile ranges) from max to min, with all data points shown. Analyses were performed as unpaired Student’s t test within prism

The development and continuous growth of mouse incisors are orchestrated by highly coordinated processes involving stem cell proliferation, migration, and differentiation [1]. In the proximal region of mouse incisors, different types of stem cells, including dental epithelial stem cells (DESCs), are present in the labial cervical loop (laCL) [30–32]. The differentiation and maturation of dental cells occur sequentially, with ameloblasts and odontoblasts maturing progressively from proximal to distal regions [1]. To further investigate the impact of the Fgf9S99N mutation on enamel formation, we performed histological analysis on E18.5 mouse incisors during the late bell stage, which marks the initiation of enamel and dentin formation and mineralization [1]. H&E staining revealed smaller laCL and thinner dental epithelia in Fgf9S99N/S99N embryos compared to wild-type controls, accompanied by a gradual reduction of ameloblasts from proximal to distal, while the odontoblasts remained normal (Fig. 1G-I). Immunofluorescence staining of AMBN on incisor sections showed significantly reduced expression in homozygotes at E18.5 compared to wild-type controls (Fig. 1J). Furthermore, qRT-PCR analysis demonstrated substantial downregulation of key genes associated with tooth enamel formation, including Ambn, Amelx, Enam, Amtn and Mmp20, in a dose-dependent manner with the presence of mutations (Fig. 1K, Fig. S1D). The morphology of the molar in the E18.5 homozygous embryo was not affected (Fig. S1C). These findings indicate that the Fgf9S99N mutation impairs cervical loop development and ameloblast formation, leading to deficient enamel formation.

Fgf9S99N mutation does not affect cell proliferation but increases dental epithelial stem cell apoptosis

DESCs and ameloblast homeostasis in the cervical loop are vital for enamel formation in the proximal region of mouse incisors [30, 31]. To ascertain whether FGF9 regulates enamel formation by influencing the proliferation and/or apoptosis of DESCs, we conducted BrdU labeling and TUNEL staining analysis on E18.5 mouse embryo incisors. Unexpectedly, the results showed that, compared to wild-type embryos, there was no statistically significant difference in the number of BrdU+ cells in the dental epithelium and mesenchyme of lower incisors and molar 1 (M1) in homozygous embryos (Fig. S2A-D). This observation indicated that the Fgf9S99N mutation may not affect the proliferation of stem cells in the epithelium and mesenchyme of incisors. Therefore, we suspect that the Fgf9S99N mutation may reduce the size of the cervical loop by affecting the survival of the enamel epithelium and mesenchymal stem cells. To investigate this, we performed coronal and sagittal serial sections of E18.5 mouse incisors and identified a longitudinal band of apoptotic cells in the stellate reticulum (SR) region of the cervical loop through TUNEL staining (Fig. S3A). As expected, there was a significant increase in apoptotic cells in the SR region of the cervical loop in homozygous embryos compared to wild-type controls (Fig. 2A, C and Fig. S3B). The SR region is known to house DESC cells, and the level of cell apoptosis in this region determines the size of the mouse incisor CL [27, 31, 33]. To further confirm the role of Fgf9 in laCL, we cultured the wild-type incisor in vitro with/without exogenous rFGF9 protein. TUNEL staining and quantitative analysis revealed that rFGF9 effectively inhibited apoptosis in the laCL (Fig. 2B, D). Furthermore, homozygous mice also exhibited an increased number of apoptotic cells in the distal region of the incisor compared to wild-type mice (Fig. 2E). These findings indicate that Fgf9 might play a vital role in the survival of stem cells in laCL.

Fig. 2 Fgf9S99N mutation results in cell apoptosis in the cervical loop and tooth. (A) TUNEL staining (green) was performed on the coronal section of the cervical loop from E18.5 embryos. The dashed line indicates the boundary of the cervical loop. (B) TUNEL staining of the cervical loop of cultured incisors from 3-month-old wild-type mice with/without 100 ng/ml rFGF9 for 4 days. The dashed line indicates the boundary of the cervical loop. (C) Statistic of total TUNEL-positive cells in the series sagittal section of E18.5 embryos’ cervical loop (n = 4). (D) Statistic of the ratio of TUNEL-positive area to cervical loop area from (B, n = 5). (E) TUNEL staining of the lower incisors of E18.5 embryos. (F, G) The expression level of Fgf3, Fgf10, Etv4, and Etv5 in the incisors of wild-type, heterozygous, and homozygous E18.5 embryos (F) and cultured teeth (G) (n = 3). The individual mouse was considered the experimental unit within the studies. Data are shown as box-and-whisker plots (with median and interquartile ranges) from max to min, with all data points shown. Analyses were performed as unpaired Student’s t test within prism

Mesenchymal-expressed FGF3 and FGF10 are crucial for DESC survival and proliferation [34–36]. To determine the hypothesized that epithelium-derived FGF9 (Fig. S4A) promotes DESC survival by upregulating mesenchymal-derived FGF3 and/or FGF10 expression, we analyzed the expression of genes associated with FGF signaling in the lower incisors of E18.5 embryos. The results showed significant downregulation of FGF signaling target genes Etv4 and Etv5, as well as mesenchymal-expressed Fgf3 and Fgf10, in the incisors of homozygotes compared to wild-type controls (Fig. 2F). Furthermore, in cultured incisor explants, rFGF9 stimulation significantly upregulated Etv4, Etv5, Fgf3, and Fgf10 (Fig. 2G). These findings suggest that Fgf9 is essential for maintaining FGF signaling and the expression of Fgf3 and Fgf10 in the tooth, which in turn promotes the survival of stem cells in the cervical loop.

FGF9 directly promotes DESC sphere formation by inhibiting cell apoptosis without affecting cell proliferation

The aforementioned observations suggest that FGF9 potentially indirectly promote DESCs survival by upregulating FGF3 and FGF10. Interestingly, spatiotemporal expression analysis in mice revealed the presence of Fgfr1c, a high-affinity receptor for FGF9, in the dental epithelium [13, 37]. Additionally, qRT-PCR analysis of Fgfr1-4 expression and their isotypes in DESCs and dental pulp stem cells (DPSCs) showed the expression of Fgfr1c in DESCs (Fig. S4B-D). Therefore, it is reasonable to hypothesize that FGF9 might directly regulate DESCs via an autocrine pathway, in addition to its indirect epithelial-mesenchymal interactions. To investigate this, we conducted DESC sphere formation assays in vitro, revealing that exogenous rFGF9 dose-dependently increased DESC sphere numbers and sizes without the presence of mesenchyme (Fig. 3A-C). Further investigations indicated that FGF9 promotes DESC sphere formation mainly by inhibiting apoptosis rather than promoting cell proliferation. Ki67 immunofluorescence staining and statistical analysis revealed that FGF9 did not affect the proliferation of DESCs (Fig. 3D, E). BrdU incorporation and flow cytometry assay also showed that FGF9 did not affect the proportion of cells in the S phase and G2/M phase of DESC spheres (Fig. S5A, B). To further investigate whether FGF9 promotes DESC sphere formation by protecting DESC cells from apoptosis, we performed Cleaved-Caspase3 immunofluorescence staining on DESC sphere sections. The staining and statistical results showed that rFGF9 treatment significantly reduced the number of apoptotic cells in the center of the DESC spheres in a dose-dependent manner (Fig. 3F, G). Annexin V flow cytometry assay also revealed that rFGF9 significantly promoted the cell survival of DESCs spheres (Fig. S5 C, D). Furthermore, immunoblotting and quantitative analysis of DESC spheres demonstrated elevated phosphorylation of CREB and BCL2 (anti-apoptotic proteins) and decreased BAX levels (pro-apoptotic protein) upon rFGF9 treatment (Fig. 3H, Fig. S5E). Additionally, qRT-PCR results revealed downregulated expression of pro-apoptotic (Bax, Bid, Fas) and upregulated expression of anti-apoptotic (Bcl2, Bcl-xl) genes in DESC spheres with rFGF9 treatment (Fig. 3I). To elucidate the signaling pathway responsible for the anti-apoptotic effect of FGF9 in DESC cells, we stimulated DESCs with different concentrations of rFGF9. Studies have shown that ERK/CREB/BCL2 pathway activation protects stem cells from apoptosis [38]. Immunoblotting and quantitative results revealed that rFGF9 stimulation elevated the phosphorylation of ERK, AKT, and CREB in DESC cells to play an anti-apoptotic effect (Fig. 3J, K). These findings provide evidence that epithelium-derived FGF9 can directly promote the survival of DESCs in an autocrine manner without affecting their proliferation.

Fig. 3 FGF9 promotes DESC sphere formation by inhibiting cell apoptosis via the ERK/ CREB signaling pathway. (A-C) Recombinant mouse FGF9 promotes DESC sphere formation and growth in dose-dependent manners. Single cells of DESCs were cultured in Matrigel with a gradient rFGF9 (0, 10, 20, 50 ng/ml) for 7 days. The number (B, n = 4) and diameter (C, n > 120) of DESC spheres were measured by Image-Pro plus 6. (D, E) Immunofluorescence staining (D) and quantitative analysis (E) of Ki67 were performed on the frozen section of the 7-day cultured DESC sphere with the indicated concentration of rFGF9 (n = 10). (F, G) Immunofluorescence staining (F) and quantitative analysis of cleaved-Caspase 3 (G) were performed on the frozen section of the 7-day cultured DESC sphere with the indicated concentration of rFGF9. Quantitative analysis of the ratio of C-Caspase3 positive cells to the total area of the DESC sphere (G, n > 29). (H) Immunoblot analysis shows the expression level of BCL2, BAX, CREB, and p-CREB in DESC spheres after 20 ng/ml FGF9 treatment for 24 h. (I) qRT-PCR analysis shows the relative mRNA level of pro-apoptotic (Bax, Bid, and Fas) and anti-apoptotic (Bcl2, and Bcl-xl) genes in DESCs treated with 20 ng/ml FGF9 for 24 h (n = 4). (J, K) Immunoblot analysis (J) and quantitative analysis (K) revealed the expression level of MEK, p-MEK, ERK, p-ERK, AKT, p-AKT, CREB, and p-CREB in DESCs after FGF9 stimulation for 5 min (n = 3). GAPDH was used as the internal control. Full-length blots are presented in Supplementary figures of full-length blots. Data are shown as box-and-whisker plots (with median and interquartile ranges) from max to min, with all data points shown. Analyses were performed as unpaired Student’s t test within prism

FGF9 promotes enamel formation in the presence of mesenchyme but inhibits enamel differentiation without mesenchyme

Previous findings have indicated that an S99N mutation in Fgf9 impaired enamel formation in mice, implying a critical role of FGF9 in ameloblast differentiation and maturation. To explore this further, lower incisors from 3-month-old wild-type mice were dissected, and the left and right sides of each mouse were randomly divided into two groups, which were cultured in a medium with or without rFGF9 proteins for five days. Immunofluorescence staining showed that FGF9 enhances AMBN secretion in the dental epithelium (Fig. 4A). qRT-PCR showed a significant upregulation of genes related to the secretion stage (Ambn, Amelx, and Enam) and maturation stage (Amtn, Klk4, CK14, Mmp20, and Odam) of ameloblast in the presence of rFGF9 (Fig. 4B, C). Immunoblotting and quantitative analysis further confirmed that FGF9 stimulation up-regulated AMBN and AMELX protein levels (Fig. 4 D, E). These findings suggest that FGF9 promotes ameloblast differentiation and enamel formation in cultured incisors.

Fig. 4 FGF9 promotes enamel formation in cultured teeth but inhibits enamel formation in cultured enamel epithelium. (A) Immunofluorescence staining of AMBN (green) was performed in the cervical loop of cultured incisors from 3-month-old wild-type mice with/without 100ng/ml rFGF9 for 5 days. The dashed line indicates the boundary of the cervical loop. (B, C) FGF9 promotes the expression level of enamel genes at the secretory stage (B, Ambn, Amelx, and Enam) and maturation stage (C, Amtn, Klk4, Ck14, Mmp20, and Odam) in cultured teeth (n = 3). (D, E) Immunoblot analysis (D) and quantitative analysis (E) revealed that FGF9 promotes the protein level of AMBN and AMELX in cultured teeth (n = 3). (F, G) FGF9 inhibits the mRNA level of enamel-related genes in the cultured enamel epithelium (n = 3). (H) Immunofluorescence staining of AMBN (green) on the attached DESCs after differentiation for 14 days with gradient FGF9 stimulation. DAPI, blue. Full-length blots are presented in Supplementary figures of full-length blots. Data are shown as box-and-whisker plots (with median and interquartile ranges) from max to min, with all data points shown. Analyses were performed as unpaired Student’s t test within prism

To explore whether FGF9 directly or indirectly promotes enamel formation, we isolated and cultured enamel epithelium from wild-type mouse lower incisors. Surprisingly, rFGF9 stimulation significantly reduced the expression of genes involved in enamel formation, particularly those expressed during the maturation stage (Amtn, Klk4, CK14, Mmp20) (Fig. 4F, G). This phenomenon was also observed in the differentiated DESC cells. FGF9 inhibited AMBN secretion in DESCs under differentiation conditions in a gradient-dependent manner (Fig. 4H, Fig. S6A) and suppressed mRNA levels of Ambn, Amtn, and Ck14 (Fig. S6B). These results implied that FGF9 might act as a bidirectional regulatory factor in ameloblast differentiation. It directly inhibits DESC differentiation into ameloblasts, maintaining DESC stemness while promoting ameloblast differentiation through epithelial-mesenchymal interactions.

FGF9 promotes enamel formation by upregulating the expression of mesenchymal FGF3 and FGF10 via MAPK/ERK signaling

Previous results demonstrated that FGF9 promotes ameloblast differentiation in the presence of mesenchymal tissue. Additionally, we found that FGF9 upregulates the expression levels of FGF3 and FGF10, which are specifically expressed in dental mesenchyme, suggesting that FGF9 promotes ameloblast differentiation through the FGF9-FGF3/FGF10 loop in epithelial-mesenchymal interactions. To further confirm this hypothesis, DPSCs were isolated and stimulated with rFGF9. qRT-PCR and ELISA analysis revealed that rFGF9 stimulation significantly upregulated the mRNA and secreted protein levels of FGF3 and FGF10 in a dose-dependent manner (Fig. 5A, B). Additionally, we co-cultured the supernatant of DPSCs with enamel epithelium and detected the expression of enamel differentiation marker genes. The results showed that the supernatant of DPSCs, stimulated by rFGF9, significantly promoted the expression of Ambn, Amelx, Enam, Amtn, Klk4, CK14, Mmp20, and Odam (Fig. 5C, Fig. S7A) compared to the control supernatant. To further confirm that FGF9-stimulated DPSC supernatant promotes ameloblast differentiation through FGF3 and FGF10, neutralizing antibodies for FGF3 and FGF10 were added. Gene expression results indicated that FGF3 and FGF10 antibodies can inhibit, to some degree, the up-regulatory effect of the FGF9-stimulated DPSC supernatant on enamel marker genes in cultured enamel epithelium (Fig. 5D, E, Fig. S7B, C). Interestingly, the FGF3 neutralizing antibody exhibits a broader inhibitory effect (Ambn, Amelx, Enam, Amtn, and Klk4) compared to the FGF10 neutralizing antibody (Ambn, Amtn, and klk4), indicating a slight difference in the regulation of dental epithelium differentiation by these two mesenchymal-secreted cytokines. Furthermore, the combined use of FGF3 and FGF10 antibodies exhibited a stronger inhibitory effect than using either FGF3 or FGF10 antibodies alone (Fig. 5F, Fig. S7D). These results demonstrate that FGF9 promotes the differentiation of ameloblast by promoting the expression of FGF3 and FGF10 in mesenchymal cells.

Fig. 5 FGF9 promotes enamel formation via upregulation of FGF3/FGF10 expressions in DPSCs. (A) The mRNA expression levels of Fgf3 and Fgf10 in DPSCs increase with the indicated concentration of rFGF9 treatment for 48 h (n = 3). (B) The protein levels of FGF3 and FGF10 in the DPSC culture medium were detected by ELISA. The relative ratio to control is shown (n > 5). (C) The primary cultured enamel epithelium was incubated with the DPSC culture medium from (B) for 48 h. The expression levels of the enamel-related genes in the enamel epithelium, were detected by qRT-PCR (n = 3). (D-F) FGF3 (D), FGF10 (E), FGF3 and FGF10 (F) neutralized antibodies were added into the culture medium to block the rFGF9-treated DPSC culture medium-induced enamel formation in cultured enamel epithelium. The expression levels of the enamel-related genes were detected by qRT-PCR (n = 3). (G) DPSCs were stimulated with 5 and 20 ng/ml rFGF9 for 5 min. The expression levels of MEK, p-MEK, ERK, p-ERK, AKT, p-AKT, P38, and p-P38 were detected by Western blot. (H, I) The relative mRNA levels of Fgf3 and Fgf10 in DPSCs with 20ng/ml rFGF9 and 5 µM BGJ398 (pan-FGFR inhibitor), 10 µM U0126 (MEK inhibitor) and 2 µM MK2206 (AKT1/2/3 inhibitor) for 24 h (n = 3). (J) A schematic diagram of FGF9 regulating DESC survival and enamel formation. IEE, inner enamel epithelium; OEE, outer enamel epithelium; SR: stellate reticulum. Full-length blots are presented in Supplementary figures of full-length blots. Data are shown as box-and-whisker plots (with median and interquartile ranges) from max to min, with all data points shown. Analyses were performed as unpaired Student’s t test within prism

FGF9 binds FGFRs, activating MAPK, PI3K/AKT, and PLCγ cascades that regulate cell proliferation and differentiation [39]. A signal transduction assay and quantitative results revealed that FGF9 treatment activated ERK and AKT phosphorylation in a dose-dependent manner (Fig. 5G, Fig. S7E). Specific inhibitors revealed the MAPK/ERK pathway is crucial in the FGF9-mediated upregulation of FGF3 and FGF10 in DPSC cells. The pan-FGFR inhibitor (BGJ398) completely blocked the FGF9-induced elevation of Fgf3 and Fgf10 in DPSC cells, and MEK inhibitor (U0126) significantly inhibited it, and there was no effect from the AKT inhibitor (MK2206) (Fig. 5H, I). These results demonstrate that FGF9 promotes enamel formation by upregulating mesenchymal FGF3 and FGF10 through the MAPK/ERK signaling pathway.

Discussion

In this study, we have presented genetic and molecular evidence establishing the critical role of Fgf9 in mouse incisor dental epithelium development. The Fgf9S99N mutation impairs ameloblast differentiation and increases apoptosis of dental epithelial stem cells, resulting in impaired enamel formation of the incisors in mice. Additionally, we demonstrate that FGF9 directly promotes DESC survival and inhibits DESC differentiation through an autocrine mechanism and indirectly supports DESC survival and promotes amelogenesis by upregulating FGF3 and FGF10 expression in dental mesenchymal cells through the MAPK/ERK signaling pathway (Fig. 5J).

Both paracrine and autocrine aspects of FGF9 are involved in the differentiation and homeostasis of mouse incisor epithelial cells. Fgf9 is a canonical FGF that plays a crucial role in organ development dependent on epithelial-mesenchymal interactions, such as lung, kidney, and gonad development [40–47]. In tooth development, the interaction between epithelial-derived FGF9 and mesenchymal-derived FGF3/FGF10 has been observed [36]. It has been reported that the continuous expression of Fgf10 in the mesenchyme is required for the maintenance of the epithelial stem cell [48], and Fgf9 promotes the expression of Fgf10 expression in constantly growing incisors [36]. However, our research unveils that epithelial-derived FGF9 not only functions through the FGF9-FGF3/FGF10 interaction loop but also directly promotes survival and inhibits the differentiation of DESCs. The spatiotemporal expression of FGFRs and their isotypes in dental epithelium and dental mesenchyme, along with selective FGF-FGFR interactions, underlies FGF signaling in epithelial-mesenchymal interactions [1, 3]. Kettunen and colleagues have shown that Fgfr1c is highly expressed in enamel epithelium and mesenchyme during the cap and bell stages in mice [13]. Additionally, our analysis of FGFR receptor subtypes expressed in DESC cells revealed that DESC cells not only express the classic epithelial-specific Fgfr1b, Fgfr2b, and Fgfr3b, but also the mesenchymal-specific Fgfr1c (Supplementary Fig. S4B-D). These expression patterns indicate that epithelial-derived FGF9 can potentially activate the Fgfr1c receptor on the surface of DESCs in an autocrine manner. Furthermore, DPSCs express not only the typical mesenchymal-specific FGFR receptors (Fgfr1c, Fgfr2c, and Fgfr3c) but also an epithelial-specific Fgfr1b, the high-affinity receptor for FGF3 and FGF10 secreted by DPSCs (Supplementary Fig. S4B-D). This implies that both epithelial and mesenchymal cells may have an autocrine pathway contributing to tooth development and homeostasis. More precise mouse models, such as inducible Fgfr1b dental mesenchyme-specific knockout and Fgfr1c dental epithelium-specific knockout mouse models, warrant investigation to confirm this hypothesis.

The dual regulatory role of FGF9 in ameloblast differentiation maintains enamel epithelium homeostasis. Enamel epithelium development involves DESC cell proliferation, migration towards the T-A region of the IEE, and sequential differentiation into ameloblast cells from proximal to distal [1]. A precise balance between DESC stemness and cytodifferentiation is crucial for the proper development of incisors. The antagonistic effect of signals is a vital mechanism within tissues for constraining and finely regulating signaling. For instance, FGF signals can upregulate Spry genes, which prevent lingual ameloblast formation by inhibiting an FGF-mediated epithelial-mesenchymal signaling loop in mice [35]. Bmp4 prompts the expression of Sostdc1, a Wnt and BMP antagonist, which in turn acts on Bmp4 through a negative feedback loop, leading to restricted enamel knots [49]. Some studies have indicated that FGF9 also plays a crucial role in amelogenesis and dental epithelium homeostasis. Fgf9 deletion results in a smaller labial cervical loop and posterior extension of Shh expression, while ectopic FGF9 reduces Shh mRNA expression, indicating a potential role for Fgf9 in protecting dental progenitor cells from Shh signaling and maintaining their undifferentiated state within the cervical loop [20, 50]. Our findings suggest that FGF9, as a single factor, has an antagonistic dual effect on dental epithelium differentiation, which could hold significant implications for maintaining the quantity and sustainability of the DESC pool. Certainly, the mechanistic underpinnings behind this phenomenon warrant further elucidation. Specifically, an important question is whether the negative effect of FGF9 on the ameloblast differentiation is direct, entailing direct regulatory influence on the differentiation process, or indirect, potentially mediated through its impact on other genes expressed within the dental epithelium, such as Shh and Spry2/4 [20, 35]. Consequently, future research endeavors may involve the utilization of meticulously designed mouse models featuring conditional Fgf9 overexpression/knockout and Fgfr-specific knockout strategies, thereby enabling a comprehensive investigation into the intricacies of FGF9’s regulatory role in ameloblast differentiation. Furthermore, investigating the effects of FGF9 on human dental epithelial stem cells and their organoids will enhance our understanding of the role of FGF9 in human tooth development. This will be of great importance for translating fundamental research on FGF9 in mouse teeth to clinical and dental tissue engineering applications.

Conclusion

In conclusion, our study demonstrates that Fgf9 is essential for dental epithelial stem cell survival and enamel formation. We also uncover that Fgf9 is a bidirectional regulatory growth factor in ameloblast differentiation (Fig. 5J). It directly inhibits DESC differentiation into ameloblasts, maintaining DESC stemness, while promoting ameloblast differentiation through epithelial-mesenchymal interactions. Our research provides new insights into the role of Fgf9 in tooth development and facilitates the development of new methods for tooth tissue engineering.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Additional file 1. Supplementary materials.

Additional file 2. Supplementary figures of full-length blots/gels.

Acknowledgements

The authors declare that they have not used Artificial Intelligence in this study.

Author contributions

Z.W.: Conceptualization, Investigation, Validation, Writing-review and editing. C.Z.: Conceptualization, Investigation, Writing-review and editing. L.T.: Conceptualization, Investigation, Validation, Formal analysis, Project administration, Visualization, Writing-original draft, Writing-review and editing, Funding acquisition. M.C.: Investigation, Data Curation, Formal analysis, Visualization. M.W.: Resources, Formal analysis, Validation, Writing-review and editing, Funding acquisition. H.L.: Investigation, Visualization. H.G.: Investigation, Visualization. Y.M.: Resources, Funding acquisition. Y.S.: Investigation, Visualization, Methodology. S.L.: Investigation, Software, Visualization. C.S: Resources, Funding acquisition. H.Z: Resources, Validation, Funding acquisition.

Funding

This work was supported by the National Natural Science Foundation of China (81900799, 2019; 82100163, 2021; 81971462, 2019 and 81901529, 2019), the Science and Technology Commission of Shanghai Municipality (19YF1430400, 2019 and 19YF1429700, 2019) and Shanghai Municipal Health Commission (2020YJZX0135, 2019). The authors deny any conflicts of interest related to this study.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Rui-jin Hospital affiliated to Shanghai Jiao Tong University School of Medicine (approval title: “Study of the role of Fgf9 gene in skeletal development and homeostasis”; approval number: RJ2023076; Date: June 8, 2023).

Consent for publication

All authors confirm their consent for publication.

Competing interests

The authors declare that they have no competing interests.

Abbreviations

DESC Dental epithelial stem cell

laCL Labial cervical loop

FGF Fibroblast growth factor

FGFR Fibroblast growth factor receptor

pEK Primary enamel knot

IEE Inner enamel epithelium

OEE Outer enamel epithelium

WT Wild-type

HET Heterozygote

SPF Specific pathogen-free

µ-CT Micro computed tomography

DPSC Dental pulp stem cell

H&E Hematoxylin and eosin

AMBN Ameloblastin

M1 Molar 1

TUNEL Terminal-deoxynucleoitidyl transferase mediated nick end labeling

SR Stellate reticulum

P Postnatal day

E Embryonic day

ELISA Enzyme-linked immuno sorbent assay

MAPK Mitogen-activated protein kinase

ERK Extracellular signal regulated kinase

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lingyun Tang and Mingmei Chen contributed equally to this work.
==== Refs
References

1. Yu T, Klein OD. Molecular and cellular mechanisms of tooth development, homeostasis and repair. Development. 2020;147(2). 10.1242/dev.184754.
2. Yu T Volponi AA Babb R An Z Sharpe PT Stem cells in tooth development, growth, repair, and regeneration Curr Top Dev Biol 2015 115 187 212 10.1016/bs.ctdb.2015.07.010 26589926
Yu T, Volponi AA, Babb R, An Z, Sharpe PT. Stem cells in tooth development, growth, repair, and regeneration. Curr Top Dev Biol. 2015;115:187–212. 10.1016/bs.ctdb.2015.07.010.26589926 10.1016/bs.ctdb.2015.07.010
3. Balic A Thesleff I Tissue interactions regulating tooth development and Renewal Curr Top Dev Biol 2015 115 157 86 10.1016/bs.ctdb.2015.07.006 26589925
Balic A, Thesleff I. Tissue interactions regulating tooth development and Renewal. Curr Top Dev Biol. 2015;115:157–86. 10.1016/bs.ctdb.2015.07.006.26589925 10.1016/bs.ctdb.2015.07.006
4. Du W Du W Yu H The role of fibroblast growth factors in tooth development and Incisor Renewal Stem Cells Int 2018 2018 7549160 10.1155/2018/7549160 29713351
Du W, Du W, Yu H. The role of fibroblast growth factors in tooth development and Incisor Renewal. Stem Cells Int. 2018;2018:7549160. 10.1155/2018/7549160.29713351 10.1155/2018/7549160
5. Ornitz DM Itoh N Fibroblast growth factors Genome Biol 2001 2 3 REVIEWS3005 10.1186/gb-2001-2-3-reviews3005 11276432
Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biol. 2001;2(3):REVIEWS3005. 10.1186/gb-2001-2-3-reviews3005.11276432 10.1186/gb-2001-2-3-reviews3005
6. Ornitz DM Itoh N The fibroblast growth factor signaling pathway Wiley Interdiscip Rev Dev Biol 2015 4 3 215 66 10.1002/wdev.176 25772309
Ornitz DM, Itoh N. The fibroblast growth factor signaling pathway. Wiley Interdiscip Rev Dev Biol. 2015;4(3):215–66. 10.1002/wdev.176.25772309 10.1002/wdev.176
7. Itoh N Ornitz DM Evolution of the Fgf and Fgfr gene families Trends Genet 2004 20 11 563 9 10.1016/j.tig.2004.08.007 15475116
Itoh N, Ornitz DM. Evolution of the Fgf and Fgfr gene families. Trends Genet. 2004;20(11):563–9. 10.1016/j.tig.2004.08.007.15475116 10.1016/j.tig.2004.08.007
8. Avivi A Yayon A Givol D A novel form of FGF receptor-3 using an alternative exon in the immunoglobulin domain III FEBS Lett 1993 330 3 249 52 10.1016/0014-5793(93)80882-u 8375495
Avivi A, Yayon A, Givol D. A novel form of FGF receptor-3 using an alternative exon in the immunoglobulin domain III. FEBS Lett. 1993;330(3):249–52. 10.1016/0014-5793(93)80882-u.8375495 10.1016/0014-5793(93)80882-u
9. Orr-Urtreger A Bedford MT Burakova T Arman E Zimmer Y Yayon A Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2) Dev Biol 1993 158 2 475 86 10.1006/dbio.1993.1205 8393815
Orr-Urtreger A, Bedford MT, Burakova T, Arman E, Zimmer Y, Yayon A, et al. Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev Biol. 1993;158(2):475–86. 10.1006/dbio.1993.1205.8393815 10.1006/dbio.1993.1205
10. Alarid ET, Rubin JS, Young P, Chedid M, Ron D, Aaronson SA et al. Keratinocyte growth factor functions in epithelial induction during seminal vesicle development. Proc. Natl. Acad. Sci. U. S. A. 1994;91(3):1074–1078. 10.1073/pnas.91.3.1074
11. Yan G Fukabori Y McBride G Nikolaropolous S McKeehan WL Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy Mol Cell Biol 1993 13 8 4513 22 10.1128/mcb.13.8.4513-4522.1993 7687739
Yan G, Fukabori Y, McBride G, Nikolaropolous S, McKeehan WL. Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy. Mol Cell Biol. 1993;13(8):4513–22. 10.1128/mcb.13.8.4513-4522.1993.7687739 10.1128/mcb.13.8.4513-4522.1993
12. Gilbert E Del Gatto F Champion-Arnaud P Gesnel MC Breathnach R Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA Mol Cell Biol 1993 13 9 5461 8 10.1128/mcb.13.9.5461-5468.1993 8355693
Gilbert E, Del Gatto F, Champion-Arnaud P, Gesnel MC, Breathnach R. Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA. Mol Cell Biol. 1993;13(9):5461–8. 10.1128/mcb.13.9.5461-5468.1993.8355693 10.1128/mcb.13.9.5461-5468.1993
13. Kettunen P Karavanova I Thesleff I Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and – 9 Dev Genet 1998 22 4 374 85 10.1002/(SICI)1520-6408(1998)22:4<374::AID-DVG7>3.0.CO;2-3 9664689
Kettunen P, Karavanova I, Thesleff I. Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and – 9. Dev Genet. 1998;22(4):374–85. 10.1002/(SICI)1520–6408(1998)22:4 < 374::AID-DVG7 > 3.0.CO;2–3.9664689 10.1002/(SICI)1520-6408(1998)22:4<374::AID-DVG7>3.0.CO;2-3
14. Porntaveetus T Otsuka-Tanaka Y Basson MA Moon AM Sharpe PT Ohazama A Expression of fibroblast growth factors (Fgfs) in murine tooth development J Anat 2011 218 5 534 43 10.1111/j.1469-7580.2011.01352.x 21332717
Porntaveetus T, Otsuka-Tanaka Y, Basson MA, Moon AM, Sharpe PT, Ohazama A. Expression of fibroblast growth factors (Fgfs) in murine tooth development. J Anat. 2011;218(5):534–43. 10.1111/j.1469-7580.2011.01352.x.21332717 10.1111/j.1469-7580.2011.01352.x
15. Li CY Prochazka J Goodwin AF Klein OD Fibroblast growth factor signaling in mammalian tooth development Odontology 2014 102 1 1 13 10.1007/s10266-013-0142-1 24343791
Li CY, Prochazka J, Goodwin AF, Klein OD. Fibroblast growth factor signaling in mammalian tooth development. Odontology. 2014;102(1):1–13. 10.1007/s10266-013-0142-1.24343791 10.1007/s10266-013-0142-1
16. Nakamura T Jimenez-Rojo L Koyama E Pacifici M de Vega S Iwamoto M Epiprofin regulates enamel formation and tooth morphogenesis by Controlling epithelial-mesenchymal interactions during tooth development J Bone Min Res 2017 32 3 601 10 10.1002/jbmr.3024
Nakamura T, Jimenez-Rojo L, Koyama E, Pacifici M, de Vega S, Iwamoto M, et al. Epiprofin regulates enamel formation and tooth morphogenesis by Controlling epithelial-mesenchymal interactions during tooth development. J Bone Min Res. 2017;32(3):601–10. 10.1002/jbmr.3024.10.1002/jbmr.3024
17. Kettunen P Thesleff I Expression and function of FGFs-4, -8, and – 9 suggest functional redundancy and repetitive use as epithelial signals during tooth morphogenesis Dev Dyn 1998 211 3 256 68 10.1002/(SICI)1097-0177(199803)211:3<256::AID-AJA7>3.0.CO;2-G 9520113
Kettunen P, Thesleff I. Expression and function of FGFs-4, -8, and – 9 suggest functional redundancy and repetitive use as epithelial signals during tooth morphogenesis. Dev Dyn. 1998;211(3):256–68. 10.1002/(SICI)1097 – 0177(199803)211:3 < 256::AID-AJA7 > 3.0.CO;2-G.9520113 10.1002/(SICI)1097-0177(199803)211:3<256::AID-AJA7>3.0.CO;2-G
18. Huang F Hu X Fang C Liu H Lin C Zhang Y Expression profile of critical genes involved in FGF signaling pathway in the developing human primary dentition Histochem Cell Biol 2015 144 5 457 69 10.1007/s00418-015-1358-7 26266341
Huang F, Hu X, Fang C, Liu H, Lin C, Zhang Y, et al. Expression profile of critical genes involved in FGF signaling pathway in the developing human primary dentition. Histochem Cell Biol. 2015;144(5):457–69. 10.1007/s00418-015-1358-7.26266341 10.1007/s00418-015-1358-7
19. Colvin JS Feldman B Nadeau JH Goldfarb M Ornitz DM Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene Dev Dyn 1999 216 1 72 88 10.1002/(SICI)1097-0177(199909)216:1<72::AID-DVDY9>3.0.CO;2-9 10474167
Colvin JS, Feldman B, Nadeau JH, Goldfarb M, Ornitz DM. Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene. Dev Dyn. 1999;216(1):72–88. 10.1002/(SICI)1097 – 0177(199909)216:1 < 72::AID-DVDY9 > 3.0.CO;2–9.10474167 10.1002/(SICI)1097-0177(199909)216:1<72::AID-DVDY9>3.0.CO;2-9
20. Kurosaka H Islam MN Kuremoto K Hayano S Nakamura M Kawanabe N Core binding factor beta functions in the maintenance of stem cells and orchestrates continuous proliferation and differentiation in mouse incisors Stem Cells 2011 29 11 1792 803 10.1002/stem.722 21898689
Kurosaka H, Islam MN, Kuremoto K, Hayano S, Nakamura M, Kawanabe N, et al. Core binding factor beta functions in the maintenance of stem cells and orchestrates continuous proliferation and differentiation in mouse incisors. Stem Cells. 2011;29(11):1792–803. 10.1002/stem.722.21898689 10.1002/stem.722
21. Haara O Harjunmaa E Lindfors PH Huh SH Fliniaux I Aberg T Ectodysplasin regulates activator-inhibitor balance in murine tooth development through Fgf20 signaling Development 2012 139 17 3189 99 10.1242/dev.079558 22833125
Haara O, Harjunmaa E, Lindfors PH, Huh SH, Fliniaux I, Aberg T, et al. Ectodysplasin regulates activator-inhibitor balance in murine tooth development through Fgf20 signaling. Development. 2012;139(17):3189–99. 10.1242/dev.079558.22833125 10.1242/dev.079558
22. Wu XL Gu MM Huang L Liu XS Zhang HX Ding XY Multiple synostoses syndrome is due to a missense mutation in exon 2 of FGF9 gene Am J Hum Genet 2009 85 1 53 63 10.1016/j.ajhg.2009.06.007 19589401
Wu XL, Gu MM, Huang L, Liu XS, Zhang HX, Ding XY, et al. Multiple synostoses syndrome is due to a missense mutation in exon 2 of FGF9 gene. Am J Hum Genet. 2009;85(1):53–63. 10.1016/j.ajhg.2009.06.007.19589401 10.1016/j.ajhg.2009.06.007
23. Tang L Wu X Zhang H Lu S Wu M Shen C A point mutation in Fgf9 impedes joint interzone formation leading to multiple synostoses syndrome Hum Mol Genet 2017 26 7 1280 93 10.1093/hmg/ddx029 28169396
Tang L, Wu X, Zhang H, Lu S, Wu M, Shen C, et al. A point mutation in Fgf9 impedes joint interzone formation leading to multiple synostoses syndrome. Hum Mol Genet. 2017;26(7):1280–93. 10.1093/hmg/ddx029.28169396 10.1093/hmg/ddx029
24. Liu Y Ma J Beenken A Srinivasan L Eliseenkova AV Mohammadi M Regulation of receptor binding specificity of FGF9 by an Autoinhibitory Homodimerization Structure 2017 25 9 1325 e13361323 10.1016/j.str.2017.06.016 28757146
Liu Y, Ma J, Beenken A, Srinivasan L, Eliseenkova AV, Mohammadi M. Regulation of receptor binding specificity of FGF9 by an Autoinhibitory Homodimerization. Structure. 2017;25(9):1325–e13361323. 10.1016/j.str.2017.06.016.28757146 10.1016/j.str.2017.06.016
25. Bird AD Croft BM Harada M Tang L Zhao L Ming Z Ovotesticular disorders of sex development (DSD) in FGF9 mouse models of human synostosis syndromes Hum Mol Genet 2020 10.1093/hmg/ddaa100 32761094
Bird AD, Croft BM, Harada M, Tang L, Zhao L, Ming Z, et al. Ovotesticular disorders of sex development (DSD) in FGF9 mouse models of human synostosis syndromes. Hum Mol Genet. 2020. 10.1093/hmg/ddaa100.32761094 10.1093/hmg/ddaa100
26. Tang L Wu M Lu S Zhang H Shen Y Shen C Fgf9 negatively regulates Bone Mass by inhibiting Osteogenesis and promoting Osteoclastogenesis Via MAPK and PI3K/AKT signaling J Bone Min Res 2021 36 4 779 91 10.1002/jbmr.4230
Tang L, Wu M, Lu S, Zhang H, Shen Y, Shen C, et al. Fgf9 negatively regulates Bone Mass by inhibiting Osteogenesis and promoting Osteoclastogenesis Via MAPK and PI3K/AKT signaling. J Bone Min Res. 2021;36(4):779–91. 10.1002/jbmr.4230.10.1002/jbmr.4230
27. Chang JY Wang C Jin C Yang C Huang Y Liu J Self-renewal and multilineage differentiation of mouse dental epithelial stem cells Stem cell Res 2013 11 3 990 1002 10.1016/j.scr.2013.06.008 23906788
Chang JY, Wang C, Jin C, Yang C, Huang Y, Liu J, et al. Self-renewal and multilineage differentiation of mouse dental epithelial stem cells. Stem cell Res. 2013;11(3):990–1002. 10.1016/j.scr.2013.06.008.23906788 10.1016/j.scr.2013.06.008
28. Chavez MG Yu W Biehs B Harada H Snead ML Lee JS Characterization of dental epithelial stem cells from the mouse incisor with two-dimensional and three-dimensional platforms Tissue Eng Part C Methods 2013 19 1 15 24 10.1089/ten.TEC.2012.0232 22742471
Chavez MG, Yu W, Biehs B, Harada H, Snead ML, Lee JS, et al. Characterization of dental epithelial stem cells from the mouse incisor with two-dimensional and three-dimensional platforms. Tissue Eng Part C Methods. 2013;19(1):15–24. 10.1089/ten.TEC.2012.0232.22742471 10.1089/ten.TEC.2012.0232
29. Juuri E, Balic A. Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem cells. J Visualized Experiments: JoVE. 2021;17310.3791/62462.
30. Fresia R Marangoni P Burstyn-Cohen T Sharir A From bite to byte: Dental structures resolved at a single-cell resolution J Dent Res 2021 100 9 897 905 10.1177/00220345211001848 33764175
Fresia R, Marangoni P, Burstyn-Cohen T, Sharir A. From bite to byte: Dental structures resolved at a single-cell resolution. J Dent Res. 2021;100(9):897–905. 10.1177/00220345211001848.33764175 10.1177/00220345211001848
31. Yang Z Balic A Michon F Juuri E Thesleff I Mesenchymal Wnt/beta-Catenin signaling controls epithelial stem cell homeostasis in Teeth by inhibiting the Antiapoptotic Effect of Fgf10 Stem Cells 2015 33 5 1670 81 10.1002/stem.1972 25693510
Yang Z, Balic A, Michon F, Juuri E, Thesleff I. Mesenchymal Wnt/beta-Catenin signaling controls epithelial stem cell homeostasis in Teeth by inhibiting the Antiapoptotic Effect of Fgf10. Stem Cells. 2015;33(5):1670–81. 10.1002/stem.1972.25693510 10.1002/stem.1972
32. Sanz-Navarro M, Seidel K, Sun Z, Bertonnier-Brouty L, Amendt BA, Klein OD, et al. Plasticity within the niche ensures the maintenance of a Sox2(+) stem cell population in the mouse incisor. Development. 2018;145(1). 10.1242/dev.155929.
33. Harada H Kettunen P Jung HS Mustonen T Wang YA Thesleff I Localization of putative stem cells in dental epithelium and their association with notch and FGF signaling J Cell Biol 1999 147 1 105 20 10.1083/jcb.147.1.105 10508859
Harada H, Kettunen P, Jung HS, Mustonen T, Wang YA, Thesleff I. Localization of putative stem cells in dental epithelium and their association with notch and FGF signaling. J Cell Biol. 1999;147(1):105–20. 10.1083/jcb.147.1.105.10508859 10.1083/jcb.147.1.105
34. Harada H Toyono T Toyoshima K Yamasaki M Itoh N Kato S FGF10 maintains stem cell compartment in developing mouse incisors Development 2002 129 6 1533 41 10.1242/dev.129.6.1533 11880361
Harada H, Toyono T, Toyoshima K, Yamasaki M, Itoh N, Kato S, et al. FGF10 maintains stem cell compartment in developing mouse incisors. Development. 2002;129(6):1533–41.11880361 10.1242/dev.129.6.1533
35. Klein OD Lyons DB Balooch G Marshall GW Basson MA Peterka M An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors Development 2008 135 2 377 85 10.1242/dev.015081 18077585
Klein OD, Lyons DB, Balooch G, Marshall GW, Basson MA, Peterka M, et al. An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors. Development. 2008;135(2):377–85. 10.1242/dev.015081.18077585 10.1242/dev.015081
36. Yokohama-Tamaki T Fujiwara N Shibata S Wakisaka S Harada H The epithelial-mesenchymal Interaction plays a role in the maintenance of the stem cell niche of mouse incisors via Fgf10 and Fgf9 signaling Open Biotechnol J 2008 2 111 5 10.2174/1874070700802010111
Yokohama-Tamaki T, Fujiwara N, Shibata S, Wakisaka S, Harada H. The epithelial-mesenchymal Interaction plays a role in the maintenance of the stem cell niche of mouse incisors via Fgf10 and Fgf9 signaling. Open Biotechnol J. 2008;2:111–5.10.2174/1874070700802010111
37. Zhang X Ibrahimi OA Olsen SK Umemori H Mohammadi M Ornitz DM Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family J Biol Chem 2006 281 23 15694 700 10.1074/jbc.M601252200 16597617
Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM. Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem. 2006;281(23):15694–700. 10.1074/jbc.M601252200.16597617 10.1074/jbc.M601252200
38. Fu X Feng Y Shao B Zhang Y Activation of the ERK/Creb/Bcl2 pathway protects periodontal ligament stem cells against hydrogen peroxideinduced oxidative stress Mol Med Rep 2019 19 5 3649 57 10.3892/mmr.2019.10027 30896883
Fu X, Feng Y, Shao B, Zhang Y. Activation of the ERK/Creb/Bcl2 pathway protects periodontal ligament stem cells against hydrogen peroxideinduced oxidative stress. Mol Med Rep. 2019;19(5):3649–57. 10.3892/mmr.2019.10027.30896883 10.3892/mmr.2019.10027
39. Xie Y Su N Yang J Tan Q Huang S Jin M FGF/FGFR signaling in health and disease Signal Transduct Target Ther 2020 5 1 181 10.1038/s41392-020-00222-7 32879300
Xie Y, Su N, Yang J, Tan Q, Huang S, Jin M, et al. FGF/FGFR signaling in health and disease. Signal Transduct Target Ther. 2020;5(1):181. 10.1038/s41392-020-00222-7.32879300 10.1038/s41392-020-00222-7
40. Colvin JS White AC Pratt SJ Ornitz DM Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme Development 2001 128 11 2095 106 10.1242/dev.128.11.2095 11493531
Colvin JS, White AC, Pratt SJ, Ornitz DM. Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme. Development. 2001;128(11):2095–106.11493531 10.1242/dev.128.11.2095
41. Colvin JS Green RP Schmahl J Capel B Ornitz DM Male-to-female sex reversal in mice lacking fibroblast growth factor 9 Cell 2001 104 6 875 89 10.1016/s0092-8674(01)00284-7 11290325
Colvin JS, Green RP, Schmahl J, Capel B, Ornitz DM. Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Cell. 2001;104(6):875–89. 10.1016/s0092-8674(01)00284-7.11290325 10.1016/s0092-8674(01)00284-7
42. Murakami H Okawa A Yoshida H Nishikawa S Moriya H Koseki H Elbow knee synostosis (eks): a new mutation on mouse chromosome 14 Mamm Genome 2002 13 7 341 4 10.1007/s00335-001-2143-6 12140681
Murakami H, Okawa A, Yoshida H, Nishikawa S, Moriya H, Koseki H. Elbow knee synostosis (eks): a new mutation on mouse chromosome 14. Mamm Genome. 2002;13(7):341–4. 10.1007/s00335-001-2143-6.12140681 10.1007/s00335-001-2143-6
43. Pirvola U Zhang X Mantela J Ornitz DM Ylikoski J Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions Dev Biol 2004 273 2 350 60 10.1016/j.ydbio.2004.06.010 15328018
Pirvola U, Zhang X, Mantela J, Ornitz DM, Ylikoski J. Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions. Dev Biol. 2004;273(2):350–60. 10.1016/j.ydbio.2004.06.010.15328018 10.1016/j.ydbio.2004.06.010
44. Hung IH Yu K Lavine KJ Ornitz DM FGF9 regulates early hypertrophic chondrocyte differentiation and skeletal vascularization in the developing stylopod Dev Biol 2007 307 2 300 13 10.1016/j.ydbio.2007.04.048 17544391
Hung IH, Yu K, Lavine KJ, Ornitz DM. FGF9 regulates early hypertrophic chondrocyte differentiation and skeletal vascularization in the developing stylopod. Dev Biol. 2007;307(2):300–13. 10.1016/j.ydbio.2007.04.048.17544391 10.1016/j.ydbio.2007.04.048
45. Geske MJ Zhang X Patel KK Ornitz DM Stappenbeck TS Fgf9 signaling regulates small intestinal elongation and mesenchymal development Development 2008 135 17 2959 68 10.1242/dev.020453 18653563
Geske MJ, Zhang X, Patel KK, Ornitz DM, Stappenbeck TS. Fgf9 signaling regulates small intestinal elongation and mesenchymal development. Development. 2008;135(17):2959–68. 10.1242/dev.020453.18653563 10.1242/dev.020453
46. Barak H Huh SH Chen S Jeanpierre C Martinovic J Parisot M FGF9 and FGF20 maintain the stemness of nephron progenitors in mice and man Dev Cell 2012 22 6 1191 207 10.1016/j.devcel.2012.04.018 22698282
Barak H, Huh SH, Chen S, Jeanpierre C, Martinovic J, Parisot M, et al. FGF9 and FGF20 maintain the stemness of nephron progenitors in mice and man. Dev Cell. 2012;22(6):1191–207. 10.1016/j.devcel.2012.04.018.22698282 10.1016/j.devcel.2012.04.018
47. Yin H Staples SCR Pickering JG The fundamentals of fibroblast growth factor 9 Differentiation 2023 10.1016/j.diff.2023.09.004 37783652
Yin H, Staples SCR, Pickering JG. The fundamentals of fibroblast growth factor 9. Differentiation. 2023. 10.1016/j.diff.2023.09.004.37783652 10.1016/j.diff.2023.09.004
48. Harada H Toyono T Toyoshima K Ohuchi H FGF10 maintains stem cell population during mouse incisor development Connect Tissue Res 2002 43 2–3 201 4 10.1080/03008200290000989 12489159
Harada H, Toyono T, Toyoshima K, Ohuchi H. FGF10 maintains stem cell population during mouse incisor development. Connect Tissue Res. 2002;43(2–3):201–4. 10.1080/03008200290000989.12489159 10.1080/03008200290000989
49. Laurikkala J Kassai Y Pakkasjarvi L Thesleff I Itoh N Identification of a secreted BMP antagonist, ectodin, integrating BMP, FGF, and SHH signals from the tooth enamel knot Dev Biol 2003 264 1 91 105 10.1016/j.ydbio.2003.08.011 14623234
Laurikkala J, Kassai Y, Pakkasjarvi L, Thesleff I, Itoh N. Identification of a secreted BMP antagonist, ectodin, integrating BMP, FGF, and SHH signals from the tooth enamel knot. Dev Biol. 2003;264(1):91–105. 10.1016/j.ydbio.2003.08.011.14623234 10.1016/j.ydbio.2003.08.011
50. Seidel K Ahn CP Lyons D Nee A Ting K Brownell I Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor Development 2010 137 22 3753 61 10.1242/dev.056358 20978073
Seidel K, Ahn CP, Lyons D, Nee A, Ting K, Brownell I, et al. Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor. Development. 2010;137(22):3753–61. 10.1242/dev.056358.20978073 10.1242/dev.056358
