Development 110, 343-352 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 343 Localization of specific retinoid-binding sites and expression of cellular retinoic-acid-binding protein (CRABP) in the early mouse embryo L. DENCKER 1 *, E. ANNERWALL 1 , C. BUSCH 2 and U. ERIKSSON 3 1 Department of Toxicology, The Biomedical Centre, University of Uppsala, Box 594 S-751 24, Uppsala Sweden 2 Department of Pathology, University Hospital, University of Uppsala, S-751 85, Uppsala Sweden 3 Ludwtg Institute for Cancer Research, Stockholm Branch, Box 60202, S-104 01 Stockholm Sweden • To whom correspondence should be addressed Summary Retinoids (vitamin A derivatives) are important for normal embryogenesis and retinoic acid, an acidic derivative of vitamin A, was recently proposed to be an endogenous morphogen. Several retinoids are also potent teratogens. Using an autoradiographic technique, we have identified tissues and cells in early mouse embryos that are able to specifically accumulate a radiolabelled synthetic derivative of retinoic acid. Strong accumulation of radioactivity was seen in several neural crest derivatives and in specific areas of the CNS. Gel filtration analyses of cytosols from embryos that received the radiolabelled retinoid in utero suggested that cellular retinoic acid-binding protein (CRABP) was involved in the accumulation mechanism. Immunohisto- chemical localization confirmed that cells accumulating retinoids also expressed CRABP. Strong CRABP immu- noreactivity was found in neural crest-derived mesen- chyme of the craniofacial area, in visceral arches, in dorsal root ganglia and in cells along the gut and the major vessels of the trunk region. In CNS, CRABP expression and retinoid binding was largely restricted to the hindbrain, to a single layer of cells in the roof of the midbrain and to cells in the mantle layer of the neural tube. Our data suggest that cells in the embryo expressing CRABP are target cells for exogenous retinoids as well as endogenous retinoic acid. Retinoic acid may thus play an essential role in normal development of the CNS and of tissues derived from the neural crest. We propose that the teratogenic effects of exogenous retinoids are due to an interference with mechanisms by which endogenous retinoic acid regulates differentiation and pattern formation in these tissues. Key words: retinoic acid, binding, embryo, mouse, teratogenesis. Introduction Retinoic acid (RA), a biologically active form of vitamin A, is known to support embryonic development (Takahashi et al. 1975: Thompson et al. 1964) and to affect differentiation and growth properties of many cell types. (Lotan, 1980; Strickland and Mahdavi, 1978). The presence of RA in limb and its ability to specify the anteroposterior axis during limb develop- ment suggests that RA is an endogenous morphogen (Thaller and Eichele, 1987). In addition, RA is a potent teratogen, drastically affecting formation of the cranio- facial area, brain and vascular system in exposed humans (Rosa, 1983: Lammer et al. 1985) and experimental animals (Durston et al. 1989; Goulding and Pratt, 1986; Kochhar, 1967; Shenefelt, 1972; Wiley et al. 1983; Webster et al. 1986). There is some evidence suggesting that neural crest-derived cells and neuroepi- thelial cells are among the primary targets for RA during early embryonic development, since adminis- tration of RA early in gestation or to early embryos in vitro, predominantly induces malformations in tissues derived from these cells (Durston et al. 1989; Goulding and Pratt, 1986; Lammer et al. 1985: Webster et al. 1986) Furthermore, a previous study indicated that exogenous RA accumulate in neural-crest-derived cells and in neuroepithelium (Dencker et al. 1987). The various biological effects caused by RA treat- ment are likely to be mediated by specific binding proteins. One RA-binding protein, the cellular retinoic- acid-binding protein (CRABP) was identified almost 15 years ago. However, its role in retinoid metabolism remains unclear (for a review see Chytil and Ong, 1984). The close structural similarities to a number of intracellular fatty-acid-binding proteins suggest its possible role as a transport and storage protein for RA (Sundelin et al. 1985a,/?). It is conceivable that CRABP may be involved in regulating the supply of RA to the recently identified family of nuclear receptors for RA. The three RA receptors identified in mammals (RAR,