FGF signaling modulates mechanotransduction/WNT signaling in progenitors during tooth root development

doi:10.1038/s41413-024-00345-5

. 2024 Jun 24;12(1):37.

doi: 10.1038/s41413-024-00345-5.

FGF signaling modulates mechanotransduction/WNT signaling in progenitors during tooth root development

Fei Pei^#^{1

2}, Tingwei Guo^#¹, Mingyi Zhang¹, Li Ma¹, Junjun Jing¹, Jifan Feng¹, Thach-Vu Ho¹, Quan Wen¹, Yang Chai³

Affiliations

¹ Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA, 90033, USA.
² State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China.
³ Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA, 90033, USA. ychai@usc.edu.

^# Contributed equally.

PMID: 38910207
PMCID: PMC11194271
DOI: 10.1038/s41413-024-00345-5

FGF signaling modulates mechanotransduction/WNT signaling in progenitors during tooth root development

Fei Pei et al. Bone Res. 2024.

. 2024 Jun 24;12(1):37.

doi: 10.1038/s41413-024-00345-5.

Authors

Fei Pei^#^{1

2}, Tingwei Guo^#¹, Mingyi Zhang¹, Li Ma¹, Junjun Jing¹, Jifan Feng¹, Thach-Vu Ho¹, Quan Wen¹, Yang Chai³

Affiliations

¹ Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA, 90033, USA.
² State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China.
³ Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA, 90033, USA. ychai@usc.edu.

^# Contributed equally.

PMID: 38910207
PMCID: PMC11194271
DOI: 10.1038/s41413-024-00345-5

Abstract

Stem/progenitor cells differentiate into different cell lineages during organ development and morphogenesis. Signaling pathway networks and mechanotransduction are important factors to guide the lineage commitment of stem/progenitor cells during craniofacial tissue morphogenesis. Here, we used tooth root development as a model to explore the roles of FGF signaling and mechanotransduction as well as their interaction in regulating the progenitor cell fate decision. We show that Fgfr1 is expressed in the mesenchymal progenitor cells and their progeny during tooth root development. Loss of Fgfr1 in Gli1⁺ progenitors leads to hyperproliferation and differentiation, which causes narrowed periodontal ligament (PDL) space with abnormal cementum/bone formation leading to ankylosis. We further show that aberrant activation of WNT signaling and mechanosensitive channel Piezo2 occurs after loss of FGF signaling in Gli1-Cre^ER;Fgfr1^fl/fl mice. Overexpression of Piezo2 leads to increased osteoblastic differentiation and decreased Piezo2 leads to downregulation of WNT signaling. Mechanistically, an FGF/PIEZO2/WNT signaling cascade plays a crucial role in modulating the fate of progenitors during root morphogenesis. Downregulation of WNT signaling rescues tooth ankylosis in Fgfr1 mutant mice. Collectively, our findings uncover the mechanism by which FGF signaling regulates the fate decisions of stem/progenitor cells, and the interactions among signaling pathways and mechanotransduction during tooth root development, providing insights for future tooth root regeneration.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1**
*Fgfr1* is expressed in *Gli1*⁺ progenitor cells and their progeny during tooth root development. a–h Expression of *Fgfr1* in mandibular first molars from wild-type mice at PN3.5, PN7.5, PN14 and PN28. White arrows point to the expression of *Fgfr1* in apical papilla and odontoblasts; yellow arrows point to the expression of *Fgfr1* in follicle cells. i–l Expression of *Fgfr1* and tdTomato at PN18 in *Gli1-Cre*^ER;tdT mouse model. White arrows point to the expression of *Fgfr1* in periodontal ligament; white arrowheads point to the expression of *Fgfr1* in odontoblasts. PDL, periodontal ligament; OD, odontoblasts; DPC, dental pulp cells. White dashed lines outline Hertwig’s epithelial root sheath (HERS). Scale bars, 100 μm

**Fig. 2**
Loss of *Fgfr1* in *Gli1*⁺ progenitor cells leads to tooth ankylosis. a–f MicroCT analysis of the first mandibular molars in *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice at PN30, PN60, and postnatal 9 months (PN9M). White arrowheads point to the periodontal ligament space; white arrows point to the narrowed periodontal ligament space; white asterisk points to the narrowed root pulp cavity. g–i Relative periodontal ligament space in control and *Fgfr1* mutant mice at PN30, PN60, and postnatal 9 months (PN9M). P < 0.000 1, unpaired Student’s t-test, n = 3 and each point represents one animal. j–l Relative tooth root pulp cavity area in control and *Fgfr1* mutant mice at PN30, PN60, and postnatal 9 months (PN9M). j P = 0.017 5, k, l P < 0.000 1, unpaired Student’s t-test, n = 3 and each point represents one animal. m–n MicroCT overlays: superimposition of control (white) and *Gli1-Cre*^ER;Fgfr1^fl/fl (blue) mouse mandibles. o, q Overlay of the mandibular first molars of control (white) and *Gli1-Cre*^ER;Fgfr1^fl/fl (blue) using the mandibular border as reference. p, r Overlay of the mandibular first molars of control (white) and *Gli1-Cre*^ER;Fgfr1^fl/fl (blue) using the crown as reference. s, t Relative tooth eruption in control and *Fgfr1* mutant mice at PN60 and postnatal 9 months (PN9M). s P = 0.000 9, t P = 0.000 2. n = 3 and each point represents one animal, with an unpaired Student’s t-test performed. Schematic at the bottom indicates the induction protocol. *P < 0.05, ***P < 0.001, ****P < 0.000 1, Scale bars, 1 mm

**Fig. 3**
Narrowed PDL space with ankylosed tooth root in *Gli1-Cre*^ER;Fgfr1^fl/fl mice. a–d Histological analysis of *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice at PN30. Black arrows point to abnormal cementum. Black dashed lines outline the interface between root dentin and cementum. e–h Expression of *Dspp* and periostin in *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice. White arrows point to abnormal periodontal ligaments. i–l Histological analysis of *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice at PN60. The black asterisk points to periodontal ligament space; the black arrowhead points to narrowed periodontal ligament space in furcation; the black arrow points to the absence of periodontal ligament space where the tooth root connects to alveolar bone; Line with arrows indicates root pulp cavity. m–p Periostin expression in *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice at PN60. The white asterisk points to the periodontal ligament; the white arrow points to the absence of periostin expression. The Schematic at the bottom indicates the induction protocol. Scale bars, b, d, f, h, j, l, n, p, 100 μm; others, 500 μm

**Fig. 4**
Loss of *Fgfr1* leads to hyperproliferation and differentiation of *Gli1*⁺ progenitor cells. a–d, f–i Proliferating cells stained with Ki67 in *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice at PN7.5 and PN14. The white arrow points to Ki67⁺ cells in the papilla and follicle. e, j Quantification of Ki67⁺ cells in control and *Fgfr1* mutant mice at PN7.5 and PN14. e P = 0.007 3, j P = 0.001 5, n = 3, and each point represents one animal, with unpaired Student’s t-test performed. k–n Expression of *Pthlh* in control and *Fgfr1* mutant mice at PN14. The white arrow points to cementoblasts expressing *Pthlh* along the tooth root surface. o Quantification of *Pthlh*⁺ cells in control and *Fgfr1* mutant mice at PN14. P = 0.000 8, n = 3, and each point represents one animal, with unpaired Student’s t-test performed. p–s Expression of *Ibsp* and Sp7 in control and *Fgfr1* mutant mice at PN14. The white arrowhead points to cementoblasts expressing *Ibsp* and Sp7 along the tooth root surface; the white arrow points to increased *Ibsp* and Sp7 in cementoblasts and periodontium. t, u Quantification of *Ibsp*⁺ and Sp7⁺ cells in control and *Fgfr1* mutant mice at PN14. t P = 0.000 4, (u) P = 0.000 5, n = 3, and each point represents one animal, with unpaired Student’s t-test performed. The Schematic at the bottom indicates the induction protocol. **P < 0.01, ***P < 0.001, Scale bars, 100 μm

**Fig. 5**
Loss of FGF signaling in tooth root mesenchymal progenitors leads to increased WNT signaling. a Hierarchical clustering showing the gene expression profiles of control and *Gli1-Cre*^ER;Fgfr1^fl/fl mice. Z-scores were used to compare expression levels between samples. b Volcano plot showing 1 043 upregulated genes and 309 downregulated genes in *Fgfr1* mutant relative to control. c GO analysis showing the signaling pathways involved. d–g Expression of *Axin2* in *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice at PN7.5. White and yellow arrows point to the expression of *Axin2* in the dental papilla surrounding HERS and furcation region; white and yellow arrowheads point to increased *Axin2* in the dental papilla, follicle, and furcation region. Orange boxes in d and f indicate furcation region and are shown enlarged in orange dashed insets in e and g. White boxes in (d) and (f) are shown enlarged as (e) and (g), respectively. White dashed lines outline HERS. h Relative fluorescent intensity of *Axin2* at PN7.5 and PN14. (PN7.5) P = 0.001 7, (PN14) P < 0.000 1, n = 3, and each point represents one animal, with unpaired Student’s t-test performed. i–n Expression of *Axin2* and Col1a1 in *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice at PN14. White arrows point to the expression of *Axin2* in apical papilla and periodontium; white arrowheads point to the increased expression of *Axin2*. The white dashed line indicates the interface between the papilla and follicle. The Schematic at the bottom indicates the induction protocol. **P < 0.01, ****P < 0.000 1. Scale bars, 100 μm

**Fig. 6**
FGF signaling modulates mechanotransduction genes *Piezo2* to regulate differentiation of progenitors and WNT signaling. a–f Expression of *Piezo2* in *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice at PN7.5. White arrowheads point to the expression of *Piezo2* in the apical papilla and follicle; white arrows point to increased *Piezo2* in the apical, middle, and coronal papilla and apical follicle. White dashed lines outline HERS. The Schematic at the bottom indicates the induction protocol. g Relative expression of *Piezo2* in pCMV6 and pPiezo2-treated group with qPRC in vitro. P = 0.000 3, unpaired Student’s t-test, n = 3 and each point represents one biological replicate. h–k Expression of *Piezo2* and *Ibsp* in pCMV6- and pPiezo2-treated apical mesenchymal cells from control mice. l–q Mineralized nodules with Alizarin red staining in control, pCMV6- and pPiezo2-treated apical mesenchymal cells from control mice. r Quantification of calcium deposition in the three groups. P < 0.000 1, one-way ANOVA, n = 3 biologically independent samples. s, t Knockdown of *Piezo2* in apical mesenchymal cells from *Gli1-Cre*^ER;Fgfr1^fl/fl mice with *Piezo2* siRNA treatment. u, v Expression of *Ctnnb1* after *Piezo2* siRNA treatment. ***P < 0.001, ****P < 0.000 1. Scale bars, 100 μm

**Fig. 7**
FGF/ETV5 signaling regulates *Piezo2* expression. a Prediction of ETV5 binding with *Piezo2* promoter region. b ChIP-qPCR showed that ETV5 can bind to the genomic locus of *Piezo2*. P = 0.023 1, unpaired Student’s t-test, n = 3, and each point represents one biological replicate. c–f Expression of *Etv5* in *Fgfr1*^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl mice at PN7.5. White arrows point to the expression of *Etv5*. White dashed lines outline HERS. g Relative expression of *Etv5* in Negative control (NC) and *Etv5* siRNA-treated apical mesenchymal cells with qPRC in vitro. P < 0.000 1, unpaired Student’s t-test, n = 3 and each point represents one biological replicate. h, i Expression of *Etv5* in NC and *Etv5* siRNA-treated group. j, k Expression of *Piezo2* in NC and *Etv5* siRNA-treated group. *P < 0.05, ****P < 0.000 1. Scale bars, 100 μm

**Fig. 8**
Downregulation of WNT signaling rescues tooth ankylosis in *Gli1-Cre*^ER;Fgfr1^fl/fl mice. a–c MicroCT analysis of the first mandibular molars in *Fgfr1*^fl/fl, *Gli1-Cre*^ER;Fgfr1^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl;*β-catenin*^fl/+ mice at PN50. White arrows point to the periodontal ligament space; white arrowheads point to the narrowed periodontal ligament space. d–l Histological analysis of *Fgfr1*^fl/fl, *Gli1-Cre*^ER;Fgfr1^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl;*β-catenin*^fl/+ mice. m–r Periostin expression in *Fgfr1*^fl/fl, *Gli1-Cre*^ER;Fgfr1^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl;*β-catenin*^fl/+ mice. Space between white dashed lines (n, p, and r) indicates periodontal ligament space. s–x Proliferation stained with Ki67 in *Fgfr1*^fl/fl, *Gli1-Cre*^ER;Fgfr1^fl/fl and *Gli1-Cre*^ER;Fgfr1^fl/fl;*β-catenin*^fl/+ mice. White arrows point to Ki67⁺ cells. y Quantification of Ki67⁺ cells in three groups. *Fgfr1*^fl/fl versus *Gli1-Cre*^ER;Fgfr1^fl/fl: P = 0.000 4; *Gli1-Cre*^ER;Fgfr1^fl/fl versus *Gli1-Cre*^ER;Fgfr1^fl/fl;*β-catenin*^fl/+: P = 0.001, n = 3 biologically independent samples, with one-way ANOVA performed. The Schematic at the bottom indicates the induction protocol. **P < 0.01, ***P < 0.001. Scale bars, a–c, 1 mm; d, g, j, m, o, q, 500 μm; others, 100 μm

**Fig. 9**
Schematic of FGF signaling in progenitor cells regulating mechanotransduction and WNT signaling to modulate proliferation and differentiation during tooth root development. Loss of Fgfr1 in *Gli1*⁺ progenitor cells leads to increased and ectopic *Piezo2* expression, which activates WNT signaling through Fzd6/*β-catenin* and *Piezo2*/*β-catenin*. Schematic was created with BioRender

See this image and copyright information in PMC

References

1. Scadden DT. The stem-cell niche as an entity of action. Nature. 2006;441:1075–1079. doi: 10.1038/nature04957. - DOI - PubMed
1. Yuan Y, et al. Spatiotemporal cellular movement and fate decisions during first pharyngeal arch morphogenesis. Sci. Adv. 2020;6:eabb0119. doi: 10.1126/sciadv.abb0119. - DOI - PMC - PubMed
1. van der Kooy D, Weiss S. Why stem cells? Science. 2000;287:1439–1441. doi: 10.1126/science.287.5457.1439. - DOI - PubMed
1. Vining KH, Mooney DJ. Mechanical forces direct stem cell behaviour in development and regeneration. Nat. Rev. Mol. Cell Biol. 2017;18:728–742. doi: 10.1038/nrm.2017.108. - DOI - PMC - PubMed
1. Jing JJ, et al. Spatiotemporal single-cell regulatory atlas reveals neural crest lineage diversification and cellular function during tooth morphogenesis. Nat. Commun. 2022;13:4803. doi: 10.1038/s41467-022-32490-y. - DOI - PMC - PubMed

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[1] Scadden DT. The stem-cell niche as an entity of action. Nature. 2006;441:1075–1079. doi: 10.1038/nature04957. - DOI - PubMed

[2] Scadden DT. The stem-cell niche as an entity of action. Nature. 2006;441:1075–1079. doi: 10.1038/nature04957. - DOI - PubMed

[3] Yuan Y, et al. Spatiotemporal cellular movement and fate decisions during first pharyngeal arch morphogenesis. Sci. Adv. 2020;6:eabb0119. doi: 10.1126/sciadv.abb0119. - DOI - PMC - PubMed

[4] Yuan Y, et al. Spatiotemporal cellular movement and fate decisions during first pharyngeal arch morphogenesis. Sci. Adv. 2020;6:eabb0119. doi: 10.1126/sciadv.abb0119. - DOI - PMC - PubMed

[5] van der Kooy D, Weiss S. Why stem cells? Science. 2000;287:1439–1441. doi: 10.1126/science.287.5457.1439. - DOI - PubMed

[6] van der Kooy D, Weiss S. Why stem cells? Science. 2000;287:1439–1441. doi: 10.1126/science.287.5457.1439. - DOI - PubMed

[7] Vining KH, Mooney DJ. Mechanical forces direct stem cell behaviour in development and regeneration. Nat. Rev. Mol. Cell Biol. 2017;18:728–742. doi: 10.1038/nrm.2017.108. - DOI - PMC - PubMed

[8] Vining KH, Mooney DJ. Mechanical forces direct stem cell behaviour in development and regeneration. Nat. Rev. Mol. Cell Biol. 2017;18:728–742. doi: 10.1038/nrm.2017.108. - DOI - PMC - PubMed

[9] Jing JJ, et al. Spatiotemporal single-cell regulatory atlas reveals neural crest lineage diversification and cellular function during tooth morphogenesis. Nat. Commun. 2022;13:4803. doi: 10.1038/s41467-022-32490-y. - DOI - PMC - PubMed

[10] Jing JJ, et al. Spatiotemporal single-cell regulatory atlas reveals neural crest lineage diversification and cellular function during tooth morphogenesis. Nat. Commun. 2022;13:4803. doi: 10.1038/s41467-022-32490-y. - DOI - PMC - PubMed

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FGF signaling modulates mechanotransduction/WNT signaling in progenitors during tooth root development

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FGF signaling modulates mechanotransduction/WNT signaling in progenitors during tooth root development

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