Characterization of tyramine beta-hydroxylase in planarian Dugesia japonica: cloning and expression.

The planarian Dugesia japonica has a relatively well-organized central nervous system (CNS) consisting of a brain and ventral nerve cords (VNCs), and can completely regenerate it CNS utilizing pluripotent stem cells present in the mesenchymal space. This remarkable capacity has begun to be exploited for research on neural regeneration. Recently, several kinds of molecular markers for labeling of neural subtypes have been reported in planarians. These molecular markers are useful for visualizing the distinct neural populations in planarians. In this study, we isolated a cDNA encoding tyramine beta-hydroxylase (TBH), an octopamine (OA) biosynthetic enzyme, by degenerate PCR in the planarian D. japonica, and named it DjTBH (D. japonica tyramine beta-hydroxylase). In order to examine whether DjTBH contributes to OA biosynthesis, we measured the OA content in DjTBH-knockdown planarians created by RNA interference. In addition, to examine the specificity of DjTBH for OA biosynthesis, we measured not only OA content but also noradrenaline (NA) content, because NA is synthesized by a pathway similar to that for OA. According to high-performance liquid chromatography analysis, the amount of OA, but not NA, was significantly decreased in DjTBH-knockdown planarians. In addition, we produced anti-DjTBH antibody to visualize the octopaminergic neural network. As shown by immunofluorescence analysis using anti-DjTBH antibody, DjTBH-immunopositive neurons were mainly distributed in the head region, and elongated their dendrites and/or axons along the VNCs. In order to visualize octopaminergic and dopaminergic nervous systems (phenolamine/catecholamine nervous system) in the planarian CNS, double-immunofluorescence analysis was carried out using both anti-DjTBH antibody and anti-DjTH (a planarian tyrosine hydroxylase) antibody. DjTBH-immunopositive neurons and DjTH-immunopositive neurons mainly formed distinct neural networks in the head region. Here, we demonstrated that DjTBH clearly contributes to OA biosynthesis, and DjTBH antibody is a useful tool for detecting octopaminergic neurons in planarians.