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Abstract
Serotonin (5-HT) is a neurotransmitter that mediates a wide variety of effects, both in the central and peripheral adult nervous system. In the last years, an increasing amount of experimental evidence demonstrates that serotonin can acts as growth-regulatory signal for neuronal and non-neuronal cells by controlling proliferation and sinaptogenesis, modulating the wiring and plasticity of brain circuits during neural development.
We decided to address the developmental roles played by serotonin by using Xenopus laevis as a model system.
First I characterized the spatial and temporal nervous system distribution of serotonin during the Xenopus development, using immunohistochemistry with an antibody recognizing 5-HT. I have detected this neurotransmitter in rombencephalic raphe nuclei starting from stage 35 to the adult brain. I have also found serotonin in retinal amacrine cells and in peripheric tissues.
Both in adult and during embryonic life, all the biological actions of 5-HT are mediated by cognate G-coupled receptors that are widely distributed in central nervous system. Previous studies made in our lab demonstrated that the Xenopus 5-HT2B mRNA is expressed in proliferating regions of the larval nervous system and of the retina.
I have focused my study on the role of 5-HT2B receptor during Xenopus development, performing loss and gain of function experiments. To make functional studies, antisense morpholino oligodeoxynucleotides and capped syntetic X5-HT2B mRNA were injected into one cell of two-cell stage Xenopus embryos. Furthermore, in overexpression experiments, I treated the injected embryos with serotonin to activate ectopic 5-HT2B receptors. To determine whether 5-HT2B receptor plays a role in retinal proliferation, apoptosis and morphogenesis, sections of stage 42 injected embryos were analyzed by immunohistochemistry with antibodies recognizing N-tubulin and phospho-histone-H3; in situ hybridization with cyclin D1, tunel assay and Hoechst staining were also performed. On the whole the results obtained suggest that 5-HT2B receptor plays an important role in retinal morphogenesis by supporting cell proliferation and survival.
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Furthermore I determined the spatial and temporal expression patterns of two genes involved in serotoninergic system homeostasis regulation: XTph that codifies for the serotonin biosynthesis rate-limiting enzyme and X5-HTT, codifying for the only protein able to re-uptake serotonin.
To characterize the X5-HTT and XTph expression patterns I generated two digoxigenin labeled antisense RNA probes and I used them to perform in situ hybridization on whole mount and sectioned embryos starting from early stage to adult.
Expression of X5-HTT is first detected in raphe nuclei at stage 30 and it is still expressed on adult brain. At stage 33 it is also transiently detectable in the pineal gland and at stage 45 it is present also in retinal inner nuclear layer cells.
XTph mRNA is expressed in pineal gland starting from stage 28; at stage 45 I found signal also in retinal photoreceptors, in ventral hypothalamic nuclei, in pharynx and gut. This expression pattern is comparable to that of mammals Tph1 isoform.
In conclusion my results show, the important 5-HT2B-mediated serotonin role in eye morphogenesis. Moreover I describe for the first time, the 5-HTT expression pattern during Xenopus development. The presented XTph expression pattern results suggest that in Xenopus, as in other vertebrates, a second Tph isoform may exists to synthesize serotonin in raphe nuclei.
