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Epigenetic regulation of Tbx18 gene expression during endochondral bone formation

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Abstract

Endochondral bone formation is tightly regulated by the spatial and sequential expression of a series of transcription factors. To disclose the roles of TBX18, a member of the T-box transcription factor family, during endochondral bone formation, its spatial and temporal expression patterns were characterized in the limb skeletal region of the developing mouse together with those of established osteochondrogenic markers Sox9, Col2a1, and Runx2. TBX18 expression first appeared in condensed mesenchymal cells (chondro-progenitors) in embryonic-day-10.5 (E10.5) limb bud and was co-localized with Sox9 expression, whereas at E11.5 and E12.5, it became undetectable in mesenchymal cells committed to the chondrocyte lineage. From E13.5 to E18.5, TBX18 expression reappeared in chondrocytes, correlating strongly with Col2a1 expression; furthermore, low level TBX18 expression was found in the Runx2-positive perichondral osteoblastic cell lineage. At the postnatal stage, TBX18 expression was observed in epiphyseal chondrocytes and osteocytes within the lacunae of mature trabecular bone. On the assumption that such characteristic Tbx18 gene expression is epigenetically regulated during mouse limb development, we examined the methylation status of the CpG-island in the mouse Tbx18 gene by methylation-specific polymerase chain reaction. Hypermethylation of the Tbx18 gene promoter became evident at an early embryonic stage in TBX18-negative cells and then disappeared at a late embryonic stage in TBX18-positive cells. Therefore, the temporal suppression of Tbx18 gene expression by the hypermethylation of its promoter seems to trigger the differentiation of mesenchymal cells into hypertrophic chondrocytes in the early stages of endochondral ossification.

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Acknowledgments

We express our appreciation to Ms. Yuki Takaoka and Ms. Chie Arimatsu for their valuable assistance.

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Authors and Affiliations

  1. Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan

    Ryuma Haraguchi, Riko Kitazawa & Sohei Kitazawa

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  1. Ryuma Haraguchi
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  2. Riko Kitazawa
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  3. Sohei Kitazawa
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Correspondence to Sohei Kitazawa.

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This work was supported by a Grant-in-Aid for Young Scientists B (23790228 and 25860143) and by the Tenure-Track System at Ehime University.

The experiments were concieved and designed by R.H., R.K., and S.K. and performed by R.H. The reagents/materials/analysis tools were contributed by R.H., R.K., and S.K. The data were analyzed by R.H. and S.K. The paper was written by R.H. and S.K.

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Haraguchi, R., Kitazawa, R. & Kitazawa, S. Epigenetic regulation of Tbx18 gene expression during endochondral bone formation. Cell Tissue Res 359, 503–512 (2015). https://doi.org/10.1007/s00441-014-2028-0

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