Sonic hedgehog and FGF8: Inadequate signals for the differentiation of a dopamine phenotype in mouse and human neurons in culture

Embryonic mouse striatal neurons and human neurons derived from the NT2/hNT stem cell line can be induced, in culture, to express the dopaminergic (DA ) biosynthetic enzyme tyrosine hydroxylase (TH). The novel expression of TH in these cells is signaled by the synergistic interaction of factors prese nt in the media, such as fibroblast growth factor 1 (FGF1) and one of sever al possible coactivators [DA, phorbol 12-myris-tate 13-acetate (TPA), isobu tylmethylxanthine (IBMX), or forskolin]. Similarly, in vivo it has recently been reported that the expression of TH in the developing midbrain is medi ated by the synergy of FGF8 and the patterning molecule sonic hedgehog (Shh ). In the present study, we examined whether the putative in vivo DA differ entiation factors can similarly signal TH in our in vitro cell systems. We found that FGF8 and Shh induced TH expression in fewer than 2% of NT2/ hNT cells and less than 5% of striatal neurons. The latter could be amplified t o as much as 30% by increasing the concentration of growth factor 10-fold o r by the addition of other competent coactivators (IBMX/ forskolin, TPA, an d DA). Additivity/inhibitor experiments indicated that FGF8 worked through traditional tyrosine kinase-initiated MAP/MEK signaling pathways. However, the Shh signal transduction cascade remained unclear. These data suggest th at cues effective in vivo may be less successful in promoting the different iation of a DA phenotype in mouse and human neurons in culture. Thus, our a bility to generate DA neurons from different cell lines, for use in the tre atment of Parkinson's disease, will depend on the identification of appropr iate differentiation signals for each cell type under investigation. (C) 20 01 Academic Press.