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| Floriana Volpicelli |
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via Pietro castellino 111, 80131 Naples Italy
e-mail: volpicel@iigb.na.cnr.it |
Poster Presentation: |
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| INDUCTION OF RAT MIDBRAIN DOPAMINERGIC NEURONS IN VITRO BY SONIC HEDGEHOG AND MODULATION OF NURR1 GENE EXPRESSION. |
| Volpicelli F.1, Perrone-Capano C.1,2, di Porzio U.1 | |
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1Institute of genetics and biophysic, CNR, Naples; 2 University of Catanzaro \"Magna Grecia\" |
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| In mammals, midbrain DA neurons are located ventrally to form the retrorubral nucleus, the substantia nigra and the ventral tegmental area (VTA). They form two important neuronal circuits the nigrostriatal and the mesocorticolimbic pathways, involved in motor control, emotion and rerward. The precise anatomical localization and functional differentiation of midbrain DA neurons in the mammalian brain is achieved through the action and gradient disposition of various diffusible molecules and morphogens. Early DA neuronal determination is achieved through the action of two extracellular inducers, Sonic Hedgehog (SHH) and FGF8.
After this first commitment, the function of selectively activated transcription factors, like the orphan steroid nuclear receptor Nurr1, is required for DA final determination. Subsequently, DA function is selectively modulated by specific interaction with the developing striatal target tissue. Committed and determined DA neurons express the key genes involved in DA neurotransmission at different time in development.
We are studying early events involved in the acquisition and maturation of the dopaminergic phenotype using primary cultures of the rat ventral mesencephalon (MES) from early embryonic stages. Addition of bFGF to MES cells stimulates proliferation of DA precursors and delays their differentiation. The addition of recombinant SHH greatly enhances DA differentiation, as monitored by the number of tyrosine hydroxylase-positive cells and by high-affinity DA uptake. These expanded MES cultures are rich in nestine-positive neurons, glial cells are rare, whilst all DA markers are expressed at high levels. We also show that in these cultures, and in normal MES primary cultures, Nurr1 gene expression is plastic and can be modulated in vitro by neuronal activity. To study the function of Nurr1 gene in dopaminergic differentiation, we have transfected A1 cells, a pluripotent dopaminergic precursor generated from the embryonic mouse ventral mesencephalon , with Nurr1 constructs.
Our findings indicate that the combined action of bFGF and SHH can be exploited to expand in vitro the number of committed DA neurons and to study the cellular and molecular mechanisms underlying these events. |
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