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Quiescent adult stem cells in murine teeth are regulated by Shh signaling

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Abstract

The mechanisms regulating the maintenance of quiescent adult stem cells in teeth remain to be fully elucidated. Our aim is to clarify the relationship between BrdU label-retaining cells (LRCs) and sonic hedgehog (Shh) signaling in murine teeth. After prenatal BrdU labeling, mouse pups were analyzed during postnatal day 1 (P1) to week 5 (P5W). Paraffin sections were processed for immunohistochemistry for BrdU, Sox2, Gli1, Shh, Patched1 (Ptch1) and Ki67 and for in situ hybridization for Shh and Ptch1. Dense LRCs, Gli1-(+) cells and Ptch1-(+) cells were co-localized in the outer enamel epithelium of the apical bud and apical dental papilla of incisors. In developing molars, dense LRCs were numerous at P1 but then decreased in number over the course of odontogenesis and were maintained in the center of pulp tissue. Gli1-(+) cells were maintained in the pulp horn during the examined stages, while they increased in number and were maintained in the center of pulp tissue during P2-5W. Ptch1-(+) cells were localized in the pulp horn at P1 and increased in number in the center of the pulp after P3W. Shh mRNA was first expressed in the enamel epithelium and then shifted to odontoblasts and other pulp cells. Shh protein was distributed in the epithelial and mesenchymal tissues of incisors and molars. These findings suggest that quiescent dental stem cells are regulated by Shh signaling, and that Shh signaling plays a crucial role in the differentiation and integrity of odontoblasts during epithelial-mesenchymal interactions and dentinogenesis.

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References

  • Ahn S, Joyner AL (2005) In vivo analysis of quiescent adult neural stem cells responding to sonic hedgehog. Nature 437:894–897

    Article  CAS  PubMed  Google Scholar 

  • Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P (2015) Molecular biology of the cell. Garland Science, New York

    Google Scholar 

  • Bhardwaj G, Murdoch B, Wu D, Baker DP, Williams KP, Chadwick K, Ling LE, Karanu FN, Bhatia M (2001) Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation. Nat Immunol 2:172–180

    Article  CAS  PubMed  Google Scholar 

  • Bitgood MJ, McMahon AP (1995) Hedgehog and bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev Biol 172:126–138

    Article  CAS  PubMed  Google Scholar 

  • Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E (2004) Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118:635–648

    Article  CAS  PubMed  Google Scholar 

  • Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317

    Article  CAS  PubMed  Google Scholar 

  • Gilbert SF (2014) Developmental biology, 10th edn. Sinauer, Sunderland

    Google Scholar 

  • Gritli-Linde A, Bei M, Maas R, Zhang XM, Linde A, McMahon AP (2002) Shh signaling within the dental epithelium is necessary for cell proliferation, growth and polarization. Development 129:5323–5337

    Article  CAS  PubMed  Google Scholar 

  • Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 97:13625–13630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harada H, Ohshima H (2004) New perspectives on tooth development and the dental stem cell niche. Arch Histol Cytol 67:1–11

    Article  PubMed  Google Scholar 

  • Ida-Yonemochi H, Nakatomi M, Ohshima H (2014) Establishment of in vitro culture system for evaluating dentin-pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells. Histochem Cell Biol 142:323–333

    Article  CAS  PubMed  Google Scholar 

  • Ishikawa Y, Ida-Yonemochi H, Suzuki H, Nakakura-Ohshima K, Jung HS, Honda MJ, Ishii Y, Watanabe N, Ohshima H (2010) Mapping of BrdU label-retaining dental pulp cells in growing teeth and their regenerative capacity after injuries. Histochem Cell Biol 134:227–241

    Article  CAS  PubMed  Google Scholar 

  • Ishikawa Y, Ida-Yonemochi H, Nakakura-Ohshima K, Ohshima H (2012) The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling. Cell Tissue Res 348:95–107

    Article  CAS  PubMed  Google Scholar 

  • Jaks V, Barker N, Kasper M, Es JH van, Snippert HJ, Clevers H, Toftgard R (2008) Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 40:1291–1299

  • Juuri E, Saito K, Ahtiainen L, Seidel K, Tummers M, Hochedlinger K, Klein OD, Thesleff I, Michon F (2012) Sox2+ stem cells contribute to all epithelial lineages of the tooth via Sfrp5+ progenitors. Dev Cell 23:317–328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Katsuragi Y, Anraku J, Nakatomi M, Ida-Yonemochi H, Obata M, Mishima Y, Sakuraba Y, Gondo Y, Kodama Y, Nishikawa A, Takagi R, Ohshima H, Kominami R (2013) Bcl11b transcription factor plays a role in the maintenance of the ameloblast-progenitors in mouse adult maxillary incisors. Mech Dev 130:482–492

    Article  CAS  PubMed  Google Scholar 

  • Khan M, Seppala M, Zoupa M, Cobourne MT (2007) Hedgehog pathway gene expression during early development of the molar tooth root in the mouse. Gene Expr Patterns 7:239–243

    Article  CAS  PubMed  Google Scholar 

  • Li J, Feng J, Liu Y, Ho TV, Grimes W, Ho HA, Park S, Wang S, Chai Y (2015) BMP-SHH signaling network controls epithelial stem cell fate via regulation of its niche in the developing tooth. Dev Cell 33:125–135

    Article  PubMed  PubMed Central  Google Scholar 

  • Li L, Clevers H (2010) Coexistence of quiescent and active adult stem cells in mammals. Science 327:542–545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li L, Kwon HJ, Harada H, Ohshima H, Cho SW, Jung HS (2011) Expression patterns of ABCG2, Bmi-1, Oct-3/4, and yap in the developing mouse incisor. Gene Expr Patterns 11:163–170

    Article  CAS  PubMed  Google Scholar 

  • Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, Suri P, Wicha MS (2006) Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res 66:6063–6071

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Milenkovic L, Goodrich LV, Higgins KM, Scott MP (1999) Mouse patched1 controls body size determination and limb patterning. Development 126:4431–4440

    CAS  PubMed  Google Scholar 

  • Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S (2003) SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 100:5807–5812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Morris RJ, Liu Y, Marles L, Yang Z, Trempus C, Li S, Lin JS, Sawicki JA, Cotsarelis G (2004) Capturing and profiling adult hair follicle stem cells. Nat Biotechnol 22:411–417

    Article  CAS  PubMed  Google Scholar 

  • Nakagawa E, Zhang L, Kim EJ, Shin JO, Cho SW, Ohshima H, Jung HS (2012a) The novel function of Oct3/4 in mouse tooth development. Histochem Cell Biol 137:367–376

    Article  CAS  PubMed  Google Scholar 

  • Nakagawa E, Zhang L, Shin JO, Kim EJ, Cho SW, Ohshima H, Chen Z, Jung HS (2012b) The novel expression of Oct3/4 and Bmi1 in the root development of mouse molars. Cell Tissue Res 347:479–484

    Article  CAS  PubMed  Google Scholar 

  • Nakatomi C, Nakatomi M, Saito K, Harada H, Ohshima H (2015) The enamel knot-like structure is eternally maintained in the apical bud of postnatal mouse incisors. Arch Oral Biol 60:1122–1130

    Article  CAS  PubMed  Google Scholar 

  • Nakatomi M, Morita I, Eto K, Ota MS (2006) Sonic hedgehog signaling is important in tooth root development. J Dent Res 85:427–431

    Article  CAS  PubMed  Google Scholar 

  • Ohshima H, Nakasone N, Hashimoto E, Sakai H, Nakakura-Ohshima K, Harada H (2005) The eternal tooth germ is formed at the apical end of continuously growing teeth. Arch Oral Biol 50:153–157

    Article  PubMed  Google Scholar 

  • Paladini RD, Saleh J, Qian C, Xu GX, Rubin LL (2005) Modulation of hair growth with small molecule agonists of the hedgehog signaling pathway. J Invest Dermatol 125:638–646

    Article  CAS  PubMed  Google Scholar 

  • Pandolfi S, Stecca B (2015) Cooperative integration between HEDGEHOG-GLI signalling and other oncogenic pathways: implications for cancer therapy. Expert Rev Mol Med 17:e5

    Article  PubMed  PubMed Central  Google Scholar 

  • Saito K, Nakatomi M, Ohshima H (2013) Dynamics of bromodeoxyuridine label-retaining dental pulp cells during pulpal healing after cavity preparation in mice. J Endod 39:1250–1255

    Article  PubMed  Google Scholar 

  • Saito K, Ohshima H (2017) Differentiation capacity and maintenance of dental pulp stem/progenitor cells in the process of pulpal healing following tooth injuries. J Oral Biosci 59:63–70

    Article  Google Scholar 

  • Seidel K, Ahn CP, Lyons D, Nee A, Ting K, Brownell I, Cao T, Carano RA, Curran T, Schober M, Fuchs E, Joyner A, Martin GR, Sauvage FJ de, Klein OD (2010) Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor. Development 137:3753–3761

  • Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, Liu H, Gronthos S, Wang CY, Shi S, Wang S (2006) Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One 1:e79

    Article  PubMed  PubMed Central  Google Scholar 

  • Sousa BR, Parreira RC, Fonseca EA, Amaya MJ, Tonelli FM, Lacerda SM, Lalwani P, Santos AK, Gomes KN, Ulrich H, Kihara AH, Resende RR (2014) Human adult stem cells from diverse origins: an overview from multiparametric immunophenotyping to clinical applications. Cytometry A J Int Soc Anal Cytol 85:43–77

    Article  Google Scholar 

  • Trempus CS, Morris RJ, Bortner CD, Cotsarelis G, Faircloth RS, Reece JM, Tennant RW (2003) Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 120:501–511

    CAS  PubMed  Google Scholar 

  • Vaahtokari A, Aberg T, Jernvall J, Keranen S, Thesleff I (1996) The enamel knot as a signaling center in the developing mouse tooth. Mech Dev 54:39–43

    Article  CAS  PubMed  Google Scholar 

  • Wang XP, O’Connell DJ, Lund JJ, Saadi I, Kuraguchi M, Turbe-Doan A, Cavallesco R, Kim H, Park PJ, Harada H, Kucherlapati R, Maas RL (2009) Apc inhibition of Wnt signaling regulates supernumerary tooth formation during embryogenesis and throughout adulthood. Development 136:1939–1949

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yue Z, Jiang TX, Widelitz RB, Chuong CM (2005) Mapping stem cell activities in the feather follicle. Nature 438:1026–1029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang L, Yuan G, Liu H, Lin H, Wan C, Chen Z (2012) Expression pattern of Sox2 during mouse tooth development. Gene Expr Patterns 12:273–281

    Article  CAS  PubMed  Google Scholar 

  • Zhao H, Feng J, Seidel K, Shi S, Klein O, Sharpe P, Chai Y (2014) Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor. Cell Stem Cell 14:160–173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We cordially thank Dr. A. P. McMahon and Dr. M. P. Scott for providing riboprobes and Mr. S. Kenmotsu for his technical assistance. This work was supported by JSPS KAKENHI (grant nos. 25462955 and 17K11730 to YI and nos. 25293371 and 17H04366 to HO).

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Authors and Affiliations

  1. Department of Oral Health Sciences, Faculty of Nursing and Welfare, Kyushu University of Nursing and Social Welfare, 888 Tominoo, Tamana, Kumamoto, 865-0062, Japan

    Yuko Ishikawa

  2. Division of Oral Science for Health Promotion, Department of Oral Health and Welfare, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan

    Yuko Ishikawa

  3. Division of Anatomy, Department of Oral Health Improvement, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan

    Mitsushiro Nakatomi

  4. Division of Anatomy and Cell Biology of the Hard Tissue, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan

    Hiroko Ida-Yonemochi & Hayato Ohshima

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  1. Yuko Ishikawa
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Correspondence to Hayato Ohshima.

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Ethics

All the animal experiments were conducted in compliance with a protocol that was reviewed by the Institutional Animal Care and Use Committee and approved by the President of Niigata University (permit number: 27 Niigata Univ. Res.282–5).

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The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Electronic supplementary material

Suppl. Fig. 1

Immunohistochemical negative controls performed by replacing the primary antibodies (a Gli1, b Shh, c Ptch1, d Sox2) with phosphate-buffered saline, showing no specific immunoreaction (D dentin, DF dental follicle, DP dental papilla or pulp, asterisk apical bud). Bar 100 μm. (JPEG 739 kb)

Suppl. Fig. 2

Sections stained by hematoxylin and eosin (H&E; a, d) and in situ hybridization with an Shh antisense probe (b), Shh sense probe (c), Ptch1 antisense probe (e) and Ptch1 sense probe (f) in the frontally sectioned upper first molar at embryonic day 15 (DF dental follicle, DP dental papilla, EK enamel knot, EO enamel organ, OEE outer enamel epithelium). The lingual side is right. In accordance with past reports, Shh expression is restricted in the EK, whereas Ptch1 transcripts are mainly identified in the DP, DF and OEE, indicating the usefulness of the antisense probes used in this study. Sense probes as negative controls do not show any significant staining. Bar 50 μm. (JPEG 1015 kb)

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Ishikawa, Y., Nakatomi, M., Ida-Yonemochi, H. et al. Quiescent adult stem cells in murine teeth are regulated by Shh signaling. Cell Tissue Res 369, 497–512 (2017). https://doi.org/10.1007/s00441-017-2632-x

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  • DOI: https://doi.org/10.1007/s00441-017-2632-x

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