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Targeted differentiation of ES cell into serotonergic neurons

Serotonin is a neurotransmitter in the central nervous system (CNS), which has a wide range of functions in human physiology. Serotonergic neurons are located in the raphe nuclei of the brain and project to the spinal cord and almost to all areas of the forebrain. We aimed at directing the differentiation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into an enriched population of serotonin producing cells to identify novel genes that are essential for the development and function of serotonergic system. To this purpose we differentiated ES cells into serotonin producing neurons. Using RNA isolated at different time points during the course of ES cell differentiation we identified genes specifically enriched in the serotonergic lineage by Affymetrix gene array. To evaluate candidate genes we reprogrammed mouse and rat embryonic fibroblast to iPS cells and subsequently differentiated them to serotonergic neurons. We selected Cacna2d1, coding for an α2/δ subunit of voltage dependent calcium channels as a most prominent candidate among these genes. Cacna2d1 is expressed in skeletal muscle, aorta, and in the central nervous system (CNS) in mammals and may be essential for the release of neurotransmitters. Our own immunohistochemical analysis of the Cacna2d1 expression pattern in mouse brain coincided with the Allen brain atlas and revealed a partial overlap with Tph2-expressing cells confirming the relevance of our in vitro data and pointing to a possible importance of this channel for serotonergic function. To analyse this role of the Cacna2d1 protein we used Cacna2d1 knockout mice and morpholino-knockdown in zebrafish but we did not see any direct influence on the serotonergic phenotype upon depletion of Cacna2d1. However, our immunostaining for Cacna2d1 in zebrafish revealed a time-dependent pattern during early development. Cacna2d1 expression starts at day3 at the lateral midline of the trunk; presumably in neuromast cells. Concordantly with their characterization as neuromasts, these Cacna2d1-positive cells are migrating towards the tail. This spatial and time-dependent expression was confirmed by RT-PCR using specific Cacna2d1 primers. Moreover, zebrafish showed disturbed migration behaviours of neuromasts after morpholino-knockdown of Cacna2d1. Thus, this study clarified that Cacna2d1 is essential for zebrafish lateral line development but does not affect the establishment of the serotonergic system.