DEVELOPMENTAL BIOLOGY 117, 435-441 (1986) Retinoic Acid-Binding Protein in the Axolotl: Distribution in Mature Tissues and Time of Appearance during Limb Regeneration S. KEEBLE AND M. MADEN Naticmal Institute for Medical Research, The Ridgeway, Mill Hill, Lcmdrm, NW7 IAA, United Kingdom Received February 25, 1986; accepted in revised form April 21, 1986 Analysis of cytoplasmic protein preparations from axolotl tissues revealed the presence of a cytoplasmic retinoic acid-binding protein (CRABP), of approximate molecular weight 17K. This protein was found to be present at various concentrations in skin, muscle, and limb tissue preparations, but not in liver and serum preparations. The distribution and molecular weight of this protein agrees with that reported in mammalian studies. The level of CRABP in cone stage blastemas was found to be significantly higher than that found in nonregenerating whole limb preparations. The level falls gradually, to approach normal, towards the completion of regeneration. Such an increase, at the start of regeneration, was not altered by 4 days pretreatment with 36 mg/liter all-trans-retinoic acid, a sufficient dose to produce pattern effects. Competition experiments confirmed that the all-tram and 13-cis isomers of retinoic acid bind to CRABP with similar high efficiencies, and that the arotinoid, Ro 13-6298, exhibits only a fraction of this binding activity. Retinol, retinol palmitate, and retinol acetate were unable to compete with [3H]retinoic acid for binding to CRABP. The results presented here are discussed in terms of their possible value to understanding pattern specification in the regenerating urodele limb. e 1986 Academic PWS, IX. INTRODUCTION Retinoids alter the tissue patterns produced during development and regeneration. In the developing chick limb local application of retinoids to the anterior border of the limb bud cause duplications in the anteroposterior (AP) axis of the limb (Tickle et al., 1982; Summerbell, 1983). In regenerating urodele (axolotls and newts) limbs both systemic (Maden, 1983a) and local (Maden et al., 1985) applications of retinoids produce pattern dupli- cations in the proximodistal (PD) axis of the limb, the AP axis being apparently unaffected. However, manip- ulations of the regenerating limb (Stocum and Thorns, 1984; Kim and Stocum, 1986), to produce either anterior half and double anterior half, or posterior and double posterior half regenerates, have demonstrated that ret- inoids are also able to affect the AP axis of the urodele limb. Effects on both the PD and AP axes are seen fol- lowing administration to the regenerating limbs of an- uran (frogs and toads) tadpoles. PD effects are seen at low doses, but if the dosage is increased a second limb of reversed handedness is regenerated (Maden, 198313; Niazi and Alam, 1984; Niazi and Rathasamy, 1984). Ret- inoids, therefore, provide a useful tool with which to study the mechanisms of normal pattern production in such systems. The mechanism of retinoid action is of central im- portance to understanding these effects. Several mech- anisms have been proposed (Roberts and Sporn, 1984), but the most popular is one in which retinoids interact with specific binding proteins and, ultimately, the cell genome, rather like the suggested mechanism of steroid hormone action. It is envisaged that retinoic acid (RA) passesinto the cell, binds to its specific receptor CRABP, is transported to the nucleus, and changes the pattern of gene transcription. A considerable body of experi- mental data has been accumulated to support this idea. Cytoplasmic binding proteins specific for retinol and re- tinoic acid have been identified in a range of tissue types, their characteristics and distribution in mammalian tissues being well documented. Foetal and neonatal tis- sues provide excellent sources of both proteins (Ong and Chytil, 1976) but levels drop in adult tissues, indicating a change in gene expression during development (re- viewed in Chytil and Ong, 1984). Nuclear receptors for retinol and RA have been reported (Takase et al., 1979; Cope et al., 1984) but it appears that the cytoplasmic binding proteins deliver the retinoids to the nucleus without the proteins themselves binding (Liau et ah, 1981) and in this respect the mechanism differs from the steroid hormone model. Retinoids are, however, widely reported to alter gene expression both in vitro and in vivo (Chytil, 1984). A steroid hormone type mechanism provides a basis with which to initiate a molecular analysis of retinoid effects in the regenerating urodele limb. We report the identification of an amphibian cytoplasmic binding pro- tein specific for retinoic acid, similar in character and distribution to that found in mammalian studies, and that the level of this protein rises during early stages 435 0012-1606/86 $3.00 Copyright Q 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.