Tryptophan 5-hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of serotonin. cDNA cloning of TPH was carried out, and the occurrence of spatiotemporal transcription of TPH message was examined in larvae of the sea urchin, Hemicentrotus pulcherrimus (HpTPH), with in situ hybridization by using the tyramide signal amplification (TSA) technique and Northern hybridization. Based on deduced amino acids sequence of HpTPH, phylogenetically sea urchin locates at the closest position to vertebrates among invertebrates, and HpTPH had common conserved sequences in a catalytic domain. Initiation of HpTPH transcription occurred at the late gastrula stage exclusively in serotonin cells of apical ganglion (SAG) that was composed of a cluster of HpTPH-positive cells and the negative cells in between. In situ hybridization showed that the mRNA expression pattern was similar to the immunohistochemical localization of serotonin cells reported before (Bisgrove and Burke [1986] Dev. Growth Differ. 28:557-569; Yaguchi et al. [2000] Dev. Growth Differ. 42:479-488). p-Chlorophenylalanine (CPA), an irreversible inhibitor of TPH activity, considerably decreased serotonin content in the serotonin cells, whereas the HpTPH expression pattern and timing, and the extension of neurofibers from SAG cells were apparently unaffected, suggesting CPA exclusively perturbed synthesis of serotonin but not nervous system organization. CPA-treated larvae did not swim, despite the occurrence of ciliary beating in culture chamber, suggesting that proper serotonin synthesis is necessary for normal swimming of the larvae.
Copyright 2003 Wiley-Liss, Inc.