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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.
• Our approach is to analyze teeth from Epfn KO and Tbx1 cKO mice.
Epfn and Tbx1 regulate dental epithelial stem cell fate to the ameloblast lineage Brittany Spivey; Yuta Chiba, DDS; and Yoshihiko Yamada, PhDMolecular Biology Section, Laboratory of Cell and Developmental Biology, NIDCR
Epfn expression in developing mouse incisor
Cervical loop(stem cell niche)
P1 incisor
Mesenchyme
EpfnDAPI
Ameloblast
IDEPre-ameloblast Mature-AmeloblastODE
Odontoblast
Nakamura et al., J Biol Chem. (2008) Feb 22;283(8):4825-33.
Epfn KO
WT
Molars
enameldentin
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 222. 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 epithelialstem cell niche
IDEPre-ameloblast
Cervical loop
Secretory Stage
MaturationStage
Proliferationstage
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
IDEPre-ameloblast
ODE
IDEPre-ameloblast
ODE
EpfnTbx1
100nm
Sox2PerlecanDAPI
Control Tbx1 cKO incisorsP1 incisor Cervical loop
Cervical loop
Stem Cell Biology and Tissue Engineering in Dental SciencesEdited by Vishwakarma et al.
Stellatereticulum Stem cell
niche
OEE
Stratumintermedium
Ameloblast
Enamel
Dentin
Odontoblast
IEEInner 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(mesenchymal stem cells)
secre
tion
secre
tion
Mineralization
Mineralization
Ameloblast differentiation processes
differentiationSecretory
Stage
Ameloblast Dental epithelial stem cells
Sox2+
Stem cell makerMaturation
Stage
apoptosis
ProliferationStage
Committed cell toameloblast lineage
self renewal
Inner dental epithelium (IDE)Pre-ameloblast
Introduction
Purpose
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
MorphogenesisTooth
Hair
Limb
Genitals
Glande.g. salivary gland
Basement membrane
. .. . .. Epithelium
Mesenchyme
Epithelial-mesenchymal interaction
Tooth development
Epfn KO and Tbx1 cKO teeth express Sox2 differently
Epfn and Tbx1 are co-expressed in the earlystages of ameloblast development
Tbx1 cKO teeth show Epfn expression
Dental epithelial cell lineages
Sp zinc-finger transcription factor family Activation/suppression domain
(protein interaction)
N- -C
zinc finger motif(DNA binding domain)
Secretory Stage
MaturationStage
ProliferationStage
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 epithelialstem cell niche
ODE
IDEPre-ameloblast odontoblast
Secretory Stage
maturationstage
Proliferationstage
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 (Tbx1).
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 K14Cre; Tbx1flox/flox (Tbx1K14cKO)
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 incisorsControl
Cervical loop
IDE
Mesenchyme Dental epithelium
mesenchyme
ODE
multiple incisors
Tbx1DAPI
Ameloblast
Epfn KO teeth show reduced Tbx1 expression
P1 incisorP1 incisor
P1 incisor P1 incisor
Sox2Epfn
DAPI
100nm
Cervicalloop
Cervicalloop
Epfn KO incisorsControlP1 incisor P1 incisor
• Epfn may regulate Tbx1 expression.
Primary Antibody
Secondary Antibody
Fluorescent Marker
Indirect Immunofluorescence
Protein