Identifying vitamin A signaling by visualizing gene and protein activity, and by quantification of vitamin A metabolites

Abstract Vitamin A (retinol) is an essential nutrient for embryonic development and adult homeostasis. Signaling by vitamin A is carried out by its active metabolite, retinoic acid (RA), following a two-step conversion. RA is a small, lipophilic molecule that can diffuse from its site of synthesis to neighboring RA-responsive cells where it binds retinoic acid receptors within RA response elements of target genes. It is critical that both vitamin A and RA are maintained within a tight physiological range to protect against developmental disorders and disease. Therefore, a series of compensatory mechanisms exist to ensure appropriate levels of each. This strict regulation is provided by a number synthesizing and metabolizing enzymes that facilitate the precise spatiotemporal control of vitamin A metabolism, and RA synthesis and signaling. In this chapter we describe protocols that (1) biochemically isolate and quantify vitamin A and its metabolites and (2) visualize the spatiotemporal activity of genes and proteins involved in the signaling pathway.