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 present in the media, such as fibroblast growth factor 1 (FGF1) and one of several possible coactivators [DA, phorbol 12-myristate 13-acetate (TPA), isobutylmethylxanthine (IBMX), or forskolin]. Similarly, in vivo, it has recently been reported that the expression of TH in the developing midbrain is mediated by the synergy of FGF8 and the patterning molecule sonic hedgehog (Shh). In the present study, we examined whether the putative in vivo DA differentiation 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 to as much as 30% by increasing the concentration of growth factor 10-fold or by the addition of other competent coactivators (IBMX/forskolin, TPA, and 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 that cues effective in vivo may be less successful in promoting the differentiation of a DA phenotype in mouse and human neurons in culture. Thus, our ability to generate DA neurons from different cell lines, for use in the treatment of Parkinson's disease, will depend on the identification of appropriate differentiation signals for each cell type under investigation.
Stull
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Department of Neurology, Thomas Jefferson University Medical College, 1025 Walnut Street, Philadelphia, Pennsylvania, 19107, USA.
Iacovitti
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003342:Corpus Striatum / Q000166:cytology / Q000196:embryology; D004298:Dopamine / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D053823:Hedgehog Proteins; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010641:Phenotype; D011506:Proteins / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D010959:Tissue Plasminogen Activator / Q000494:pharmacology; D015534:Trans-Activators; D014446:Tyrosine 3-Monooxygenase / Q000096:biosynthesis
D004791:Enzyme Inhibitors; C496456:FGF8 protein, human; C496457:Fgf8 protein, mouse; D053823:Hedgehog Proteins; D011506:Proteins; C509239:SHH protein, human; D015534:Trans-Activators; D051524:Fibroblast Growth Factor 8; D005576:Colforsin; D005346:Fibroblast Growth Factors; D014446:Tyrosine 3-Monooxygenase; D010959:Tissue Plasminogen Activator; D015056:1-Methyl-3-isobutylxanthine; D004298:Dopamine