Differences in pathways contributing to thyroid hormone effects on postnatal cartilage calcification versus secondary ossification center development
- Musculoskeletal Disease Centre, Jerry L. Pettis VA Medical Center, United States
- Fulgent Genetics, United States
- Departments of Medicine, Loma Linda University, United States
- Departments of Biochemistry, Loma Linda University, United States
- Departments of Physiology, Loma Linda University, United States
Figures
Figure 1
Tshr-/- proximal femur phenotype and onset of mineralization.
(Panel A) Ventral X-ray view of postnatal (P) 21-day-old mice. Arrows point to joint between hip (h) and femur (f). (Panel B) Lateral view of three-dimensional (3D) μCT scans from P21 proximal …
Figure 2 with 1 supplement
Histological analysis of femur head (FH) in euthyroid Tshr+/- and hypothyroid Tshr-/- mice.
Safranin O stained sections showing cartilage in red (Panel A), toluidine blue stained sections showing cartilage in violet (Panel B), Alizarin red stained sections showing mineral in red (Panel C), …
Figure 2—figure supplement 1
Delayed mineralization in Tshr-/- femur head (FH).
Postnatal week 8 proximal femur sections stained in red for Safranin O (Panel A), violet for toluidine blue (Panel B), dark blue-mineral, light blue-collagen, red-nuclei for trichrome (Panel C).
Figure 3
Thyroid hormone (TH)-dependent transcriptional differences at proximal and distal femur (DF).
(Panels A–G) Gene expression changes from femur head (FH) or DF between Tshr-/- injected with vehicle, or T3/T4, plotted as fold-change relative to anatomical and stage matched region in euthyroid Ts…
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Figure 3—source data 1
Source data for Figure 3A–D.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data for day 10 Tshr femur head (FH) and distal femur (DF). Average fold changes plotted and t-test values are highlighted in yellow.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig3-data1-v2.xlsx
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Figure 3—source data 2
Source data for Figure 3E–H.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data for day 21 Tshr femur head (FH) and distal femur (DF). Average fold changes plotted and t-test values are highlighted in yellow.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig3-data2-v2.xlsx
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Figure 3—source data 3
Source data for Figure 3I.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data for day 10 and day 21 comparing femur head (FH) versus distal femur (DF) from euthyroid mice. Average fold changes plotted and t-test values are highlighted in yellow.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig3-data3-v2.xlsx
Figure 4
Immunohistochemical characterization of femur head (FH) development.
Longitudinal sections of Tshr+/- were probed for protein expression in FH on days 10, 17, 21, proximal tibia epiphysis on day 10, and FH of Tshr-/- on day 17 by immunofluorescence for collagens: …
Figure 5
Immunohistochemical characterization of femur head (FH) development.
Longitudinal sections of Tshr+/- were probed for protein expression in FH on days 10, 17, 21, proximal tibia epiphysis on day 10, and FH of Tshr-/- on day 17 by immunofluorescence for enzymes: …
Figure 6 with 1 supplement
Immunohistochemical characterization of femur head (FH) development.
Longitudinal sections of Tshr+/- were probed for protein expression in FH on days 10, 17, 21, proximal tibia epiphysis on day 10, and FH of Tshr-/- on day 17 by immunofluorescence for …
Figure 6—figure supplement 1
Alkaline phosphatase (ALPL) activity in femur head (FH) on days (D) 10, 17, 21, proximal tibia epiphysis on day 10 of Tshr+/-, and FH of Tshr-/- on day 17 FH (red stain).
Figure 7
Immunohistochemical characterization of femur head (FH) development.
Longitudinal sections of Tshr+/- were probed for protein expression in FH on days 10, 17, 21, proximal tibia epiphysis on day 10, and FH of Tshr-/- on day 17 by immunofluorescence for transcription …
Figure 8
Immunohistochemical characterization of femur head (FH) development.
Longitudinal sections of Tshr+/- were probed for protein expression in FH on days 10, 17, 21, proximal tibia epiphysis on day 10, and FH of Tshr-/- on day 17 by immunofluorescence for thyroid …
Figure 9 with 3 supplements
Thyroid hormone (TH) and transcription factor regulation of chondroprogenitor genes.
(Panels A–G) Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) on day 3 for ATDC5 cell line represented as fold-change for genes labeled on X-axis. (Panel A) ATDC5 with control …
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Figure 9—source data 1
Source data for Figure 9A.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data for control shRNA. Average fold changes plotted and t-test values are highlighted in yellow.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-data1-v2.xlsx
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Figure 9—source data 2
Source data for Figure 9C.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data for RUNX2 shRNA. Average fold changes plotted and t-test values are highlighted in yellow.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-data2-v2.xlsx
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Figure 9—source data 3
Source data for Figure 9D.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data for SP7 shRNA. Average fold changes plotted and t-test values are highlighted in yellow.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-data3-v2.xlsx
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Figure 9—source data 4
Soure data for Figure 9E.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data for DLX3 shRNA. Average fold changes plotted and t-test values are highlighted in yellow.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-data4-v2.xlsx
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Figure 9—source data 5
Source data for Figure 9F.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data for DLX5 shRNA. Average fold changes plotted and t-test values are highlighted in yellow.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-data5-v2.xlsx
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Figure 9—source data 6
Source data for Figure 9G.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data for SP7 shRNA without or with either DLX3 shRNA and/or DLX5 shRNA. Average fold changes plotted and t-test values are highlighted in yellow.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-data6-v2.xlsx
Figure 9—figure supplement 1
Effect of individual transcription factor knockdown in vehicle treatment.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) on day 3 for ATDC5 transduced with shRNA for individual transcription factors RUNX2, SP7, DLX3, or DLX5 comparing fold changes …
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Figure 9—figure supplement 1—source data 1
RUNX2 shRNA.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-figsupp1-data1-v2.xlsx
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Figure 9—figure supplement 1—source data 2
SP7 shRNA.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-figsupp1-data2-v2.xlsx
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Figure 9—figure supplement 1—source data 3
DLX3 shRNA.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-figsupp1-data3-v2.xlsx
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Figure 9—figure supplement 1—source data 4
DLX5 shRNA.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-figsupp1-data4-v2.xlsx
Figure 9—figure supplement 2
Regulation of mineralization enzymes by SP7.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) on day 3 for ATDC5 with SP7 overexpression (OE) or knockdown with SP7 shRNA. Fold changes are relative to empty vector GFP …
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Figure 9—figure supplement 2—source data 1
SP7 OE and shRNA.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-figsupp2-data1-v2.xlsx
Figure 9—figure supplement 3
Thyroid hormone (TH) regulation of Sp7 and Dlx3 expression in chondrocytes ex vivo.
Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) for Sp7 or Dlx3 from cultured chondrocytes (CCs) isolated from femur head (FH) or distal femur (DF) treated with TH for 22 …
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Figure 9—figure supplement 3—source data 1
FH vs DF chondrocyte response to TH.
- https://cdn.elifesciences.org/articles/76730/elife-76730-fig9-figsupp3-data1-v2.xlsx
Figure 10
Spatiotemporal model of proximal-distal femoral chondrocyte fate acquisition.
Please see text in the discussion for explanation. Abbreviations: PF, proximal femur; AC, articular cartilage; CC, calcified cartilage; DF, distal femur; IC, immature chondrocyte; MC, mature …
Tables
Table 1
Comparison of genes regulated by different transcription factors with a p-value < 0.05 from Figure 9 panel B after treatment with vehicle, TH, AA + TH, or AA.
‘Negative’ indicates knockdown of a given transcription factor results in a significant increase of gene in question, while ‘positive’ indicates the opposite. 2 = RUNX2; 3 = DLX3; 5 = DLX5; 7 = …
| Vehicle treatment | TH treatment | AA + TH treatment | AA treatment | |||||
|---|---|---|---|---|---|---|---|---|
| Negative | Positive | Negative | Positive | Negative | Positive | Negative | Positive | |
| Sox9 | 2,3,5 | 2,3,5,7 | 7 | 5,7 | ||||
| Runx2 | 7 | |||||||
| Sp7 | 3 | 2 | 3 | 2 | 2 | |||
| Dlx3 | 7 | 7 | 2,5 | 2,5,7 | ||||
| Dlx5 | 3 | 2 | ||||||
| Dlx6 | 2 | 3 | 2 | |||||
| Col2a1 | 2,3 | 7 | 3 | 7 | 2,3,5 | 2,3,5 | ||
| Col10a1 | 2,3,7 | 2 | 5,7 | 2,3,5,7 | 2,3,5,7 | |||
| Col1a1 | 2,3,5 | 2,3,5 | 2,3,5,7 | 2,3,5,7 | ||||
| Mmp13 | 5 | 2,3,7 | 2,3,7 | 5 | 2,3 | 5 | 2,3 | |
| Mmp9 | 2,3 | 2,3 | 5 | 2,3 | 2,3 | 5 | ||
| Ibsp | 2,3 | 2,5,7 | 3 | 2,3,7 | 2,3 | |||
| Spp1 | 2,3,7 | 5 | 2,3,7 | 5 | 2,7 | 5 | 2,7 | 3,5 |
| Bglap2 | 3 | 5 | 2,7 | 5 | 2,3,7 | 5 | 2,7 | 3 |
| Alpl | 3 | 7 | 3 | 2 | 5 | 2,5 | ||
| Dmp1 | 2,7 | 2,7 | 5 | 2,7 | 3,5 | 2,7 | 3 | |
| Mgp | 2,7 | 2,5,7 | 2,5,7 | 3 | 2,5,7 | 3 | ||
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/76730/elife-76730-transrepform1-v2.pdf
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Supplementary file 1
Antibodies.used.
Antigen retrieval with: HD = 2 mg/mL hyaluronidase (Sigma-Aldrich) in PBS at 37°C for 45 min. S = 10 mM sodium citrate with 2 mM EDTA and 0.05% Tween-20 (pH 6.0) at 95°C for 6 min. L = 10 mM sodium citrate with 2 mM EDTA and 0.05% Tween-20 (pH 6.0) at 60°C for 72 hr.
- https://cdn.elifesciences.org/articles/76730/elife-76730-supp1-v2.xlsx
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Supplementary file 2
Real Time quantitative PCR primers used.
- https://cdn.elifesciences.org/articles/76730/elife-76730-supp2-v2.xlsx
