Detection of retinoic acid catabolism with reporter systems and by in situ hybridization for CYP26 enzymes

Abstract

Retinoic acid (RA), an active form of vitamin A, is essential for life in vertebrates, owing to its capacity of influencing expression of a sizable fraction of all genes and proteins. It functions via two modes: (1) as controlling ligand for specific transcription factors in the nucleus it stimulates or inhibits gene expression from RA response elements in gene promoters; (2) in non-genomic pathways it activates kinase-signaling cascades that converge with additional influences to regulate gene expression and mRNA translation. RA performs a critical role in morphogenesis of the developing embryo, which is reflected in spatio-temporally changing expression patterns of RA-synthesizing and RA-degrading enzymes and in its biophysical characteristics as a small diffusible lipid. Because its histological localization cannot be directly visualized for technical reasons, its sites of action in vivo are inferred from the locations of the metabolic enzymes and through use of two kinds of RA reporter systems. Here we explain techniques for use of RA reporter cells and RA reporter mice, and we describe in situ hybridization methods for the three major RA-degrading enzymes: CYP26A1, CYP26B1, and CYP26C1. Comparisons of the different indicators for sites of RA signaling demonstrate that local RA peaks and troughs are important for inferring some but not all locations of RA actions. When integrated within cells of living mice, expression of the RA reporter construct is rarely a simple measure of local RA levels, especially in the developing brain, but it appears to provide cues to an RA involvement in site-specific regulatory networks in combination with other spatial determinants.