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.

Several lines of evidence suggest that tumor necrosis factor-alpha (TNFalpha) may contribute to the pathogenesis of autoimmune thyroid disease. It is not known, however, whether increased thyroidal TNFalpha levels are associated with changes in thyroid function. The purpose of the present study was to utilize in situ hybridization histochemistry and immunohistochemistry to determine if the expression of TNF-alpha in the thyroid is associated with a decrease in thyroglobulin (Tg) and thyroid peroxidase (TPO) mRNA levels. Lymphocytic thyroiditis was induced in BB/Wor rats by iodide administration, and thyroidal Tg and TPO mRNA levels were assessed by Northern blot analysis and in situ hybridization, and TNFalpha expression by Northern blot analysis and immunohistochemistry. Thyroids were obtained before and 1 and 2 months after iodide administration. Hematoxylin and eosin staining revealed that there was a progressive increase in mononuclear cells in the thyroids of BB/Wor rats ingesting iodide for 1 and 2 months. Northern blot analysis revealed that during the same time course there was a progressive increase in TNFalpha mRNA levels and a progressive decrease in Tg and TPO mRNA levels in the thyroids. In situ hybridization histochemistry was performed to determine if the decrease in Tg and TPO mRNA levels was associated with thyroid follicular cells in contact with infiltrating mononuclear cells. In rats treated with iodide for 1 month, there was a modest decrease in Tg and TPO mRNA levels in follicular cells in contact with infiltrating mononuclear cells. After 2 months of iodide treatment there was clearly a localized decrease in Tg and TPO mRNA levels in follicular cells in contact with infiltrating mononuclear cells. Immunohistochemical analysis did not detect TNFalpha in the thyroids from control rats or from rats treated with iodide for 1 month. In contrast, after 2 months of treatment, TNFalpha was easily detected in infiltrating mononuclear cells and in some thyroid follicular cells. Together, these results suggest that the suppression of Tg and TPO mRNA levels was associated with the expression of TNFalpha and thus are in agreement with in vitro studies demonstrating that TNFalpha inhibits thyroid cell function.