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Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis
E Koyama1, E B Golden, T Kirsch
1Department of Anatomy and Histology, School of Dental Medicine, Philadelphia, Pennsylvania, 19104, USA.
Abstract:
Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues.
Insights
Retinoid signaling is crucial for skeletal development, with retinoic acid receptors (RARs) guiding chondrocyte maturation and endochondral ossification in developing limbs. Blocking this pathway severely impairs bone formation.
Area of Science:
- Developmental Biology
- Skeletal Biology
- Molecular Endocrinology
Background:
- Retinoids are known to influence skeletogenesis, but their precise roles and those of their nuclear receptors (retinoic acid receptors, RARs) in skeletal development are not fully understood.
- Key questions include the presence of endogenous retinoids in developing bones, changes in RAR gene expression during chondrocyte maturation, and the impact of retinoid signaling interference on skeletogenesis.
Purpose of the Study:
- To investigate the presence and localization of endogenous retinoids and RARs during chick embryo limb skeletogenesis.
- To determine the effect of retinoid signaling interference on chondrocyte maturation and endochondral ossification.
Main Methods:
- Analysis of retinoic acid receptor (RAR) gene expression (alpha, beta, gamma) in developing chick embryo humerus chondrocytes at different developmental stages.
- Bioassays to detect endogenous retinoids in cartilaginous elements and surrounding perichondrial tissues.
- Implantation of retinoid antagonists (Ro 41-5253, AGN 193109) near developing limb anlagen and subsequent analysis of skeletal morphology and chondrocyte differentiation.
- In vitro studies using chondrocyte cultures to assess the impact of antagonists on retinoic acid-induced terminal cell maturation.
Main Results:
- Retinoic acid receptor gamma (RARgamma) expression was significantly upregulated in hypertrophic chondrocytes during limb development, while RARalpha and RARgamma were low in immature chondrocytes.
- Endogenous retinoids were detected in developing limb cartilages, with particularly high concentrations found in the perichondrial tissues.
- Interference with retinoid signaling via antagonist implantation severely inhibited humerus development, leading to shorter, bent bones and impaired chondrocyte maturation and endochondral ossification, without affecting intramembranous ossification.
Conclusions:
- Retinoid-dependent and RAR-mediated mechanisms are essential for the complete maturation of chondrocytes and successful endochondral ossification during limb development.
- The retinoid-rich perichondrial tissues may play a cooperative role in positively influencing these retinoid signaling pathways.
- These findings clarify the critical role of retinoid signaling in skeletal development and highlight potential therapeutic targets for skeletal disorders.