title: Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro.
abstract: Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.
authors.affiliation: Department of Anatomy, Faculty of Medicine, University of Ottawa, Ontario, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Sakai
authors.forename: R M
authors.initials: RM
authors.lastname: Langille
mesh_terms: D000818:Animals; D002356:Cartilage / Q000187:drug effects / Q000196:embryology*; D002455:Cell Division; D002642:Chick Embryo; D003094:Collagen / Q000096:biosynthesis*; D005147:Facial Bones / Q000166:cytology / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D008334:Mandible / Q000166:cytology / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism; D009295:Nasal Bone / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D006025:Glycosaminoglycans; D014212:Tretinoin; D003094:Collagen