The expression of the cocaine-sensitive serotonin transporter (SerT) is regulated in a complex manner in the developing central nervous system (CNS). Mutations that alter expression of SerT are associated with a number of human mental illnesses. Here we describe a set of experiments in the relatively simple fly CNS that test the role of certain genes in regulating SerT. In addition, we also propose to examine the effects of altering SerT expression on serotonergic cellular anatomy. Our previous experiments showed that the axon guidance molecule robo2 regulates SerT expression via the transcription factor eagle (eg). Here we test whether the cytoplasmic tyrosine kinase abl mediates robo2 signaling to eg. Our preliminary data indicate that members of the FGF signaling pathway function to inhibit SerT activity. In FGF, FGF-receptor, pointed (pnt) andros mutants, precocious SerT activity appears during embryonic development. Here we propose to test where and when these molecules function to attenuate SerT activity. Our working model is that the activation of embryonic SerT expression is a balance between robo2/eg induction and FGF/pnt inhibition. This balance is further revealed by the fact that the phenotype of each single mutant is suppressed in a pnt/eg double mutant. We therefore propose to examine how the balance of robo2 and FGF signaling regulates SerT. While the robo2/eg and FGF/pnt pathways seem to function during initial SerT onset early in development we will also test whether these same molecules continue to function as regulators in the CNS of the behaving larva. For this we will use timed expression of components of these pathways to test for later function. Extracellular serotonin levels, as regulated by SerT, have been hypothesized to regulate serotonergic synapse formation. We have developed a method to quantify serotonergic synaptic varicosities in the CNS. We will use this approach to first test the direct effects of altering SerT and serotonin levels on varicosities. We will then test the effects of altering genes that regulate SerT and serotonin. The long-term goal of this research is to further understand the regulation and function of this clinically relevant molecule.
