Effects of all-trans-retinoic acid on skeletal pattern, 5′HoxD gene expression, and RAR β2/β4 promoter activity in embryonic mouse limbs

Abstract

Mouse embryos were exposed to all-trans-retinoic acid on day 11 or day 12 of development and the resulting skeletal pattern alterations compared with early effects on Hoxd-11 and Hoxd-13 expression domains and RAR-beta 2/beta 4 promoter activity. The effects on skeletal pattern showed a clear correlation between the timing of retinoic acid exposure and the sequence of mesenchymal condensation. Ectopic RAR-beta 2/beta 4 promoter activity was detected within 2 hr of exposure to retinoic acid, and was present throughout the limb bud after 5 hr; it remained high in the apical ectodermal ridge and proximal mesenchyme after 12 hr, by which time the abnormal digital pattern could be seen. HoxD gene expression domains in the distal handplate were narrowed by 5 hr after maternal retinoic acid administration on day 11. Following retinoic acid treatment on both day 11 and day 12, the normal downregulation of Hoxd-11 and Hoxd-13 in the digital mesenchymal condensations was retarded. There was no evidence to suggest that RAR-beta 2/beta 4 promoter activity mediates the effects of RA on HoxD gene expression, but ectopic promoter activity is a useful indicator of at least some of the sites in which RA levels are raised. We suggest (1) that the apical ectodermal ridge is the most functionally significant of these sites, (2) that raised retinoic acid levels in the ridge result in altered gene expression and/or altered cell proliferation within this epithelium, (3) that both altered HoxD gene expression domains and altered skeletal pattern formation are secondary to this effect. There was a good correlation between the effects of retinoic acid on Hoxd-11 and Hoxd-13 expression and delay of skeletal differentiation, suggesting that this may be a direct effect.