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Root formaon

Cementum Dentin Cementoblast Odontoblast

Role of PTH/PTHrP Receptor Signaling on Root Epithelial rests Formaon of Malassez (ERM) Dental Wanida Ono papilla cells

AGE Orthodoncs, Department of Developmental Biology, Harvard School of Dental Medicine

Endocrine Unit, Massachuses General Hospital and Harvard Medical School Dental follicle cells

Hertwig’s epithelial root sheath (HERS)

Osterix PTHrP-PPR system • Transcripon factor essenal to osteoblast differenaon PTHrP (Nakashima K et al. 2002) PTH/PTHrP receptor (PPR) • Expressed in odontoblasts and alveolar osteoblasts during Gαs Gq tooth development (Chen S et al. 2009)

• Controls cellular cementum formaon (Cao Z et al. 2012) • Mediates epithelial-mesenchymal interacons • Osterix-expressing precursors in the perichondrium move • PTHrP is expressed in enamel epithelia/HERS? to bone marrow and become osteoblasts during fetal • PPR is expressed in dental mesenchymes development (Maes C, Kronenberg HM et al. 2010) (Beck et al 1995; Lee Deeds and Segre 1995; Liu et al 1998) • PTHrP is required for tooth erupon in mice (Philbrick WM, Karaplis AC et al. PNAS 1998) Osterix+ Root-forming • PPR haploinsufficiency is associated with primary cells progenitors failure of tooth erupon (PFE) in humans ? (Decker E, Weber BH et al. Am J Hum Gen 2008)

PTHrP expression paern during root morphogenesis Aims of this study PTHrPLacZ/+ x40 P7 P14 P49 • Idenfy how osterix-expressing progenitors contribute to murine root morphogenesis

• Understand how PTH/PTHrP receptor signal regulates root-forming progenitors PTHrP-LacZ x200 P7 x400 P14 ? PTHrP Epithelial root sheath PPR

PTHrP-LacZ Osx+ progenitors Odontoblast PTHrP parcipates in …….. Dental follicle/papilla cells Cementoblast - dental (root) development - homeostasis of root-bone interface ( composed by PDL, cementum)

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Experimental approach (1) Working hypothesis • Idenfy how osterix-expressing progenitors contribute to murine root formaon PTHrP HERS (Epithelial root sheath) Tamoxifen Osx-CreERT2

PPR ? Molar root formaon P3 P4 P7 P11 P25 Root-forming progenitors Cementoblast CAG Tomato Dental follicle cells X loxP Stop loxP Cre

CAG Tomato Rosa26-Tomato

In vivo fate mapping of Osx+ cells Osx+ cells are root-forming progenitors Tam

P3 P4 P7 P14 P25 Osx+ cells Dental pulp cells Osx-CreER; Tomato

P4 (+1d) Dental papilla Odontoblasts

Osx+ cells Periodontal ligament cells

Dental follicle Postnatal day 4 Cementoblasts Mandibular M1, mid-sagial secon Alveolar osteoblasts

Experimental approach (2) Osx-Cre; PPRflox/+ PPRflox/flox • Understand how PTH/PTHrP receptor signal (PPR) X regulates root-forming progenitors P7 Control Condional Het Condional KO Osx-Cre; PPRflox/+

PTH/PTHrP receptor (PPR) locus

E1 E2 PPR floxed allele X Cre (Kobayashi T, Kronenberg HM et al. 2002)

E2 PPR null allele PPRflox/flox Osx-Cre; PPRflox/+ Osx-Cre; PPRflox/flox PPRflox/flox

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PPR signal in Osx+ progenitors are Conclusion

essenal for root formaon • Osterix-expressing cells are dental root-forming progenitors during morphogenesis: Osx+ cells PPR signal Osx+ cells become dental pulp cells and odontoblasts, Periodontal ligament cells periodontal ligament cells, cementoblasts and alveolar osteoblasts

Dental follicle • Deleon of PTH/PTHrP receptor in Osx+ cells leads to

Cementoblasts dental root malformaon and failure of erupon 1) Truncaon of the dental root PPR signal 2) Hypogenesis of periodontal ligament 3) Ankylosis of cementum to bone 4) Underdeveloped mandible 5) Failure of molar erupon “malformed/ truncated root”

Acknowledgement Endocrine Unit / Massachuses General Hospital Hank Kronenberg Noriaki Ono Shigeki Nishimori Tatsuya Kobayashi Marc Wein Forest Lai Everyone in the Endocrine Unit

HSDM AGE Orthodoncs Program Acknowledgement for Important Mice… Arthur Broadus PTHrP-LacZ Andrew McMahon Osx-Cre::GFP

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