Endogenous Status of Retinoids and Their Cytosolic Binding Proteins in Limb Buds of Chick vs Mouse Embryos

Abstract

The morphogenetic role of all-trans-retinoic acid (RA) during limb development, especially its activity in directing anterior/posterior pattern formation remains controversial. We have measured retinoids and cytosolic binding proteins in anterior and posterior pieces of limb buds from comparably aged chick and mouse embryos expecting that localization patterns of morphological significance will be evident across species. Many species differences were found including: (1) nondetectable 3,4-didehydroretinoic acid (ddRA) in mouse limb buds, the predominant morphogenetically active retinoid in chick limbs; the precursor of ddRA, 3,4-didehydroretinol (ddROH), was also not present in mouse limb buds but found in high concentration in chick limb buds; (2) a higher concentration of morphogenetically active retinoid (RA + ddRA) in chick compared to mouse limb buds; (3) a high level of retinyl esters (1.5 μM) in chick limb bud, but undetectable concentration in mouse limb buds; and (4) a higher level of cytosolic retinoic acid binding proteins (CRABPs) in chick limb buds, especially CRABP II, which is 10× greater than in mouse limb buds. An interesting finding seen in mouse and chick limb buds was a disparity between the concentration of ligand and cytosolic binding protein. Retinol (ROH) and ddROH were present in much higher concentrations than cytosolic retinol binding protein (CRBP). Conversely, RA and ddRA were in far lower concentration than CRABPs. Any morphological significance of these disparities is unknown. A putative gradient of RA, high posteriorly, was found in chick limb buds as in earlier studies (Thaller and Eichele, 1987), but the magnitude of this gradient was less than previously reported. We also found ddRA in equal concentration in the anterior and posterior pieces of chick limb bud studied here, further weakening the idea of an RA gradient as the direct force in anterior/posterior pattern formation. The "free" concentration of retinoic acid, calculated from ligand and binding protein concentration and affinity, was slightly higher in posterior chick limb tissue (2.0X) and in mouse limb buds at a concentration similar to the Kd of murine nuclear retinoic acid receptors