1. Developmental Biology
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Antagonistic interaction between Ezh2 and Arid1a coordinates root patterning and development via Cdkn2a in mouse molars

  1. Junjun Jing
  2. Jifan Feng
  3. Jingyuan Li
  4. Xia Han
  5. Jinzhi He
  6. Thach-Vu Ho
  7. Jiahui Du
  8. Xuedong Zhou
  9. Mark Urata
  10. Yang Chai  Is a corresponding author
  1. University of Southern California, United States
  2. West China Hospital of Stomatology, Sichuan University, China
Research Article
Cite this article as: eLife 2019;8:e46426 doi: 10.7554/eLife.46426
11 figures, 1 table, 1 data set and 1 additional file

Figures

Loss of Ezh2 in the dental mesenchyme results in decreased H3K27Me3 histone methylation.

H&E staining (A–B), Ezh2 immunofluorescence (C–D), and H3K27Me3 immunofluorescence (E–F) of newborn control and Osr2-Cre;Ezh2fl/fl molars. Arrows indicate positive signal and asterisks indicate absence of signal. n ≥ 3 histological sections were examined from multiple littermate mice per group. Scale bars, 100 μm.

https://doi.org/10.7554/eLife.46426.003
Figure 2 with 2 supplements
Loss of Ezh2 in the dental mesenchyme leads to single-rooted molars.

MicroCT images of control (white) and Osr2-Cre;Ezh2fl/fl (blue) molars at postnatal (PN) 2 and 4 weeks of age. A, F, K, P, lateral view of mandibular molars; B, G, L, Q, apical view of mandibular first molars; C, H, M, R, sagittal sections of mandibular molars; D, I, N, S, coronal sections of mandibular molars in the furcation region; E, J, O, T, coronal sections of mandibular molars in the root forming region. The schematic drawings indicate where the CT section were taken. Arrows indicate furcation and asterisks indicate absence of furcation. Scale bars, 200 μm.

https://doi.org/10.7554/eLife.46426.004
Figure 2—figure supplement 1
Loss of Ezh2 in the dental mesenchyme leads to single-rooted molars in the upper jaw.

(A-F) MicroCT images of control (white) and Osr2-Cre;Ezh2fl/fl (blue) mandibular molars at 1 week of age. A, D, lateral view of mandibular molars; B, E, sagittal sections of mandibular molars; C, F, coronal sections of mandibular molars; (G-N) MicroCT images of control (white) and Osr2-Cre;Ezh2fl/fl (blue) maxillary molars at 2 weeks of age. G, K, lateral view of maxillary molars; H, L, apical view of maxillary first molars; I, M, sagittal sections of maxillary molars; J, N, transverse sections of maxillary molars. Arrows indicate furcation and asterisks indicate absence of furcation. Scale bars, 200 μm.

https://doi.org/10.7554/eLife.46426.005
Figure 2—figure supplement 2
Loss of Ezh2 in the dental mesenchyme has no effect on odontoblast differentiation.

(A-F) Dspp staining of control and Osr2-Cre;Ezh2fl/fl at 2 weeks of age. Boxes in A and D are shown magnified in B, C, E, and F. Arrows indicate odontoblasts. n ≥ 3 histological sections were examined from multiple littermate mice per group. Scale bars, 100 μm.

https://doi.org/10.7554/eLife.46426.006
Figure 3 with 1 supplement
Loss of Ezh2 in the dental mesenchyme affects epithelial diaphragm formation.

A-K, H&E staining (A, D, G, J), Krt14 immunofluorescence (B, E, H, K) and Ki67 staining (C, F) of coronal sections of control and Osr2-Cre;Ezh2fl/fl (mutant) molars at postnatal 7 days and 8 days. I, Quantitation of Ki67+ cells in boxed areas of C and F presented as mean ± SD with n = 4. Arrows indicate positive signal and asterisks indicate absence of signal. ***, p<0.001. Scale bars, 100 μm. Statistical analyses were performed using two-tailed Student’s t-test.

https://doi.org/10.7554/eLife.46426.007
Figure 3—figure supplement 1
Loss of Ezh2 in the dental mesenchyme has no effect on cell survival.

(A-B) TUNEL assay of molars in control and Osr2-Cre;Ezh2fl/fl mice at PN 1 week; (C-D) TUNEL assay of molars in control and Osr2-Cre;Ezh2fl/fl mice at PN 3 weeks. n ≥ 3 histological sections were examined from multiple littermate mice per group. Scale bars, 100 μm.

https://doi.org/10.7554/eLife.46426.008
The alveolar bone and periodontal ligament are affected in Osr2-Cre;Ezh2fl/fl molars.

(A-F) H&E staining (A, B, D, E) and Periostin immunofluorescence (C, F) of control and Osr2-Cre;Ezh2fl/fl molars at 2 weeks of age. B and E are magnified images of the boxed areas in A and D, respectively. (G-J) H&E staining of control and Osr2-Cre;Ezh2fl/fl molars at 4 weeks of age. (H and J) are magnified images of the boxed areas in G and I, respectively. Arrows indicate normal alveolar bone and asterisks indicate defective alveolar bone. n ≥ 3 histological sections were examined from multiple littermate mice per group. Scale bars, 100 μm.

https://doi.org/10.7554/eLife.46426.010
Figure 5 with 3 supplements
Loss of Ezh2 in the epithelium leads to delayed furcation development.

MicroCT images of control (white) and Krt14-Cre;Ezh2fl/fl (blue) molars at 2 and 3 weeks of age. A, F, K, P, lateral view of mandibular molars; B, G, L, Q, apical view of mandibular first molars; C, H, M, R, sagittal sections of mandibular molars; D, I, N, S, coronal sections of mandibular molars in the furcation region; E, J, O, T, coronal sections of mandibular molars in the root forming region. The schematic drawings indicate where the CT section were taken. Arrows indicate furcation and asterisks indicate absence of furcation. Scale bars, 200 μm.

https://doi.org/10.7554/eLife.46426.011
Figure 5—figure supplement 1
H&E staining of molars in control and Krt14-Cre;Ezh2fl/fl mice.

(A-B) H&E staining of molars from control and Krt14-Cre;Ezh2fl/fl mice at PN 2 weeks; (C-F) H&E staining of molars from control and Krt14-Cre;Ezh2fl/fl mice at PN 3 weeks. E and F are magnified images of the boxed areas in C and D, respectively. Arrows indicate alveolar bone and asterisks indicate defect of alveolar bone. n ≥ 3 histological sections were examined from multiple littermate mice per group. Scale bars, 100 μm.

https://doi.org/10.7554/eLife.46426.012
Figure 5—figure supplement 2
Root furcation development is unaffected after loss of Ezh2 in odontoblasts.

(A-H) MicroCT images of control (white) and Dmp1-Cre;Ezh2fl/fl (blue) molars at 3 weeks of age. A, B, lateral view of mandibular molars; C, D, apical view of mandibular first molars; E, F, sagittal sections of mandibular molars; G, H, coronal sections of mandibular molars. (I-J) H&E staining of control and Dmp1-Cre;Ezh2fl/fl molars at 3 weeks of age. n ≥ 3 histological sections were examined from multiple littermate mice per group. Arrows indicate furcation. Scale bars, 200 μm.

https://doi.org/10.7554/eLife.46426.013
Figure 5—figure supplement 3
Arid1a expression is not affected in Osr2-Cre;Ezh2fl/fl molars.

(A-B) expression of Arid1a in coronal sections of molars from control and Osr2-Cre;Ezh2fl/fl mice. n ≥ 3 histological sections were examined from multiple littermate mice per group. Scale bars, 100 μm.

https://doi.org/10.7554/eLife.46426.014
Figure 6 with 1 supplement
Furcation development is rescued in Osr2-Cre;Ezh2fl/fl;Arid1afl/+ mice.

MicroCT images of control, Osr2-Cre;Ezh2fl/fl and Osr2-Cre;Ezh2fl/fl;Arid1afl/+ molars at 3 weeks of age. A, E, I, lateral view of mandibular molars; B, F, J, apical view of mandibular first molars; C, G, K, coronal sections of molars in the furcation region; D, H, L, coronal sections of mandibular molars in the root forming region. The schematic drawings indicate where the CT section were taken. Arrows indicate furcation and asterisks indicate absence of furcation. Scale bars, 200 μm.

https://doi.org/10.7554/eLife.46426.015
Figure 6—figure supplement 1
Monoallelic deletion of Arid1a has no effect on furcation formation.

(A-H) MicroCT images of control (white) and Osr2-Cre;Arid1afl/+ (blue) molars at 4 weeks of age. A, E, lateral view of mandibular molars; B, F, apical view of mandibular first molars; C, G, sagittal sections of mandibular molars; D, H, coronal sections of mandibular molars. Arrows indicate furcation. Scale bars, 200 μm.

https://doi.org/10.7554/eLife.46426.016
Figure 7 with 1 supplement
RNA-sequencing analysis from control and Osr2-Cre;Ezh2fl/fl molars.

(A) Heatmap and list of top twenty upregulated genes (red highlights Cdkn2a) generated from RNA-sequencing. (B-G) Cdkn2a and Ki67 staining of control, Osr2-Cre;Ezh2fl/fl and Osr2-Cre;Ezh2fl/fl;Arid1afl/+ molars at 1 week of age. (H) quantitation of Ki67+ cells in boxed areas of C, E and G presented as mean ± SD with n = 4. Arrows indicate positive signal. ns, not significant. ***, p<0.001. Scale bars, 100 μm. Statistical analyses were performed using one-way ANOVA.

https://doi.org/10.7554/eLife.46426.017
Figure 7—figure supplement 1
ChIP-seq signals of H3K27Me3 ChIP-sequencing from wild type mouse molars.

(A-D) ChIP-seq signals of Hoxc4, Cdkn2a, Eda and Arid1a from H3K27Me3 ChIP-sequencing of mouse molars from three-day-old wild type mice.

https://doi.org/10.7554/eLife.46426.018

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (M. musculus)Arid1aflox/floxJackson LaboratoryStock No. 027717;
RRID:IMSR_JAX:027717
Strain, strain background (M. musculus)Dmp1-CreJackson LaboratoryStock No. 023047;
RRID:IMSR_JAX:023047
Strain, strain background (M. musculus)Ezh2flox/floxJackson LaboratoryStock No. 022616;
RRID:IMSR_JAX:022616
Strain, strain background (M. musculus) Krt14-CreJackson LaboratoryStock No. 018964;
RRID:IMSR_JAX:018964
Strain, strain background (M. musculus) Osr2-CreRulang Jiang, Cincinnati Children’s Hospital
Genetic reagent (M. musculus)anti-Cdkn2a probeAdvanced Cell DiagnosticsCat# 411011
AntibodyRabbit monoclonal anti-Arid1aAbcamCat# ab182561(1:100)
AntibodyRabbit monoclonal anti-Ki67AbcamCat# ab16667;
RRID:AB_302459
(1:200)
AntibodyRabbit monoclonal anti-Ezh2Cell Signaling TechnologyCat# 5246S;
RRID:AB_10694683
(1:200)
AntibodyRabbit monoclonal anti-H3K27Me3Cell Signaling TechnologyCat# 9733S;
RRID:AB_2616029
(1:100)
AntibodyRabbit polyclonal anti-PeriostinAbcamCat# ab14041;
RRID:AB_2299859
(1:100)
Antibodyanti-Rabbit Alexa Fluor 568Life TechnologiesCat# A-11011;
RRID:AB_143157
(1:200)
Antibodyanti-Mouse Alexa Fluor 568Life TechnologiesCat# A-11004;
RRID:AB_2534072
(1:200)
Commercial assay or kitIn Situ Cell Death Detection KitRoche Life ScienceCat# 11684795910
Commercial assay or kitRNeasy Micro KitQIAGENCat# 74004
Commercial assay or kitChromatrap Enzymatic Shearing KitChromatrapCat# 500165
Software, algorithmImageJNIHRRID:SCR_003070
Software,
algorithm
GraphPad PrismGraphPad SoftwareRRID:SCR_002798

Data availability

The GEO accession number for the RNA sequencing and ChIP sequencing data reported in this paper is GSE131684.

The following data sets were generated
  1. 1
    NCBI Gene Expression Omnibus
    1. Jing Junjun
    2. Feng Jifan
    3. Li Jingyuan
    4. He Jinzhi
    5. Ho Thach-Vu
    6. Zhou Xuedong
    7. Urata Mark
    8. Chai Yang
    (2019)
    ID GSE131684. Antagonistic interaction between Ezh2 and Arid1a coordinates root patterning and development via Cdkn2a in mouse molars.

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