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• Since both Epfn KO and Tbx1 cKO teeth show similar early stage defects in ameloblast development, we hypothesized that genetic and functional interactions of Epfn and Tbx1 regulate dental epithelial stem cell commitment to the ameloblast lineage. Epfn and Tbx1 regulate dental epithelial stem cell fate to the ameloblast lineage Brittany Spivey; Yuta Chiba, DDS; and Yoshihiko Yamada, PhD Molecular Biology Section, Laboratory of Cell and Developmental Biology, NIDCR Epfn expression in developing mouse incisor Cervical loop (stem cell niche) P1 incisor Mesenchyme Epfn DAPI Ameloblast IDE Pre-ameloblast Mature-Ameloblast ODE Odontoblast Nakamura et al., J Biol Chem. (2008) Feb 22;283(8):4825-33. Epfn KO WT Molars enamel dentin dentin enamel no enamel dentin dentin BrdU Incisors Enamel IDE proliferation Epfn KO mice lack enamel Epfn is essential for tooth morphogenesis • Cardiac defects • Thymic hypoplasia • Hypoparathyrodism T-box transcription factor family DiGeorge syndrome 1. A large deletion of human Chr 22 2. Autosomal dominant mutation Oberoi and Vargervik, Am J Med Genet A. (2005) Jan 15;132A(2):194-7. • Abnormal face • Cleft palate • Enamel hypoplasia Tbx1 deletion affects amelogenesis • Epfn KO mice have no enamel, defects in cusp formation, abnormal dentin structure, and supernumerary teeth. • IDE cell proliferation and differentiation are inhibited. • Epfn is a member of the Sp transcription factor family and is a homologue of Sp6. Epfn is expressed in the ameloblast lineage from the early stage to the maturation stage. • Mature odontoblasts also express Epfn. Tbx1 DAPI P1 incisor odontoblast Ameloblast mesenchyme Dental epithelial stem cell niche IDE Pre-ameloblast Cervical loop Secretory Stage Maturatio n Stage Proliferat ion stage Stem Cells Committed Cells ODE •A large deletion of human Chr 22 including the Tbx1 gene results in DiGeorge syndrome. •Tbx1 is expressed in the dental epithelial stem cells and IDE cells. Its expression disappears in differentiated ameloblasts. K14Cre-mediated Tbx1 cKO mice have enamel hypoplasia Immunostaining: • Antibodies Epfn, Tbx1 (abcam), and Perlecan were used for indirect immunofluorescence. Primary antibodies were detected by FITC- conjugated (Invitrogen) and Cy-3-conjugated secondary antibodies (Jackson ImmunoResearch). Antigen Sox2-expressing cells is reduced in Tbx1 cKO mice Hypothesis and Approach Sox2-expressing stem cells remain in Epfn KO mice Control Tbx1 cKO incisors 100um Cervical loop Cervical loop IDE Pre-ameloblast ODE IDE Pre-ameloblast ODE Epfn Tbx1 100nm Sox2 Perlecan DAPI Control Tbx1 cKO incisors P1 incisor Cervical loop Cervical loop Stem Cell Biology and Tissue Engineering in Dental Sciences Edited by Vishwakarma et al. Stellate reticulu m Stem cell niche OEE Stratum intermediu m Ameloblast Enamel Dentin Odontoblast IEE Inner dental epithelium (IDE) Incisor Mandibular Outer dental epithelium (ODE) Tooth Morphogenesis ＊ ＊ Adult Molar Enamel Dentin Odontoblast Ameloblast Enamel Matrix Dentin matrix Capillary and Nerve Dental Pulp (mesenchym al stem cells) secretion secretion Mineralization Mineralization Ameloblast differentiation processes differentiation Secretory Stage Ameloblast Dental epithelial stem cells Sox2 + Stem cell maker Maturation Stage apoptosis Proliferation Stage Committed cell to ameloblast lineage self renewal Inner dental epithelium (IDE) Pre-ameloblast Introduction Purpos e Summary Future Plan Acknowledgements Sox2 + stem cells Outer dental epithelium Inner dental epithelium proliferation stage Differentiation stage Epfn (low levels) Tbx1 (low levels) Epfn (high levels) Stem cell to pre-ameloblast Pre-ameloblast to ameloblast Tbx1 (high levels) Mouse Incisor Cervical Loop Morphogenesis Tooth Hair Limb Genitals Gland e.g. salivary gland Basement membrane . . . . . . Epithelium Mesenchyme Epithelial-mesenchymal interaction Tooth development Epfn KO and Tbx1 cKO teeth express Sox2 different Epfn and Tbx1 are co-expressed in the early stages of ameloblast development Tbx1 cKO teeth show Epfn expression ental epithelial cell lineages Sp zinc-finger transcription factor family Activation/suppression domain (protein interaction) N- -C zinc finger motif (DNA binding domain) Secretory Stage Maturation Stage Proliferat ion Stage Stem Cells Committed Cells Transactivation domain N- -C DNA binding domain Gao S et al. Hum Mol Genet. (2015) Apr 15;24(8):2330-48. Tbx1 cKO incisor Enamel Enamel WT P14 incisor Epfn Tbx1 DAPI Cervical loop P1 incisor Ameloblast Dental epithelial stem cell niche ODE IDE Pre-ameloblast odontoblast Secretory Stage maturatio n stage Proliferat ion stage Stem Cells Committed Cells Materials and Methods Tooth morphogenesis is initiated by epithelial- mesenchymal interaction. The process is similar to that of other ectodermal organs. The tooth is a good model to understand the development of other organs. The reciprocal interaction of the epithelial and mesenchymal tissues results in the successive initiation, bud, cap, and bell stages before the tooth fully develops. Ameloblast cells produce the enamel matrices and then undergo apoptosis with the rest of the dental epithelium. The enamel matrix then becomes mineralized with hydroxylapatite. Our objective is to understand the regulatory mechanism of dental epithelial stem cell commitment to the ameloblast lineage. Two candidate regulatory factors are involved in this process: Epiprofin (Epfn) and T-box 1 in vitro analysis to identify the molecular mechanism: • Identify the role of Epfn and Tbx1 in IDE cell proliferation • Gene expression analysis induced by Epfn and/or Tbx1 in a dental epithelial stem cell line • Epfn-Tbx1 interactions (how Epfn regulates Tbx1 expression) • K14Cre; Tbx1 -/- mice show reduced IDE cell proliferation and enamel hypoplasia. • Epfn and Tbx1 may functionally interact for the early stages of ameloblast development. • Epfn is expressed in Tbx1 cKO teeth. • Tbx1 may be required for stem cell maintenance by inducing Sox2 expression. • Epfn is continuously expressed from the early stage to the mature stage of ameloblast development. • Epfn may downregulate Sox2 and Tbx1 expression. • Tbx1 is expressed from dental epithelial stem cells to the inner dental epithelium cells and its expression disappears in the secretory stage of ameloblasts. • Tbx1 may regulate the stem cell maintenance by inducing Sox2 expression and other stem cell related genes. • The dental epithelium includes these cell types: outer dental epithelium, stellate reticulum, stratum intermedium, and inner dental epithelium (IDE). • The mouse dental epithelial stem cells are located in the cervical loop where they continuously self-renew. If they commit to become IDE cells, they will differentiate into enamel matrix-secreting ameloblasts which form enamel. Genotyping: • Mice from WT, Epfn -/- , and K14 Cre ; Tbx1 flox/flox (Tbx1 K14cKO ) were genotyped with DNA extraction from tail biopsies. PCR was performed using KAPA Mouse Genotyping Kit. Making tissue sections: • Post-natal day 1 (P1) mouse heads were skinned and cut in two along the middle sagittal plane. The samples were fixed with 4% paraformamide (PFA) for 24 hours and were then embedded in an OCT compound. The embedded samples were cut into 8 μm sections using Leica Microdissection. • NIDCR/NIH • Dr. Deborah Philp—NIDCR Office of Education • MBS and LCDB members • Dr. Kiyoshi Sakai, Mr. Darius Marboubi Results Ectodermal origin organ morphogenesis • Epfn may regulate expression of Sox2, a dental epithelial stem cell marker. P1 incisor Epfn KO incisors Control Cervical loop IDE Mesenchyme Dental epithelium mesenchyme ODE multiple incisors Tbx1 DAPI Ameloblast Epfn KO teeth show reduced Tbx1 expression P1 incisor P1 incisor P1 incisor P1 incisor Sox2 Epfn DAPI 100nm Cervical loop Cervical loop Epfn KO incisors Control P1 incisor P1 incisor • Epfn may regulate Tbx1 expression. Primary Antibody Secondary Antibody Fluorescent Marker Indirect Immunofluorescence Protein
