Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain.

We have explored the plastic ability of neuronal precursors to acquire different identities by manipulating their surrounding environment. Specifically, we sought to identify potential signals involved in the specification of forebrain dopaminergic neurons. Here we describe culture conditions under which tyrosine hydroxylase (TH) expression is induced in neuronal precursors, which were derived directly from the embryonic striatum and adult subependyma (SE) of the lateral ventricle or generated from multipotent forebrain stem cells. TH was successfully induced in all of these cell types by 24 hr exposure to basic fibroblast growth factor (FGF2) and glial cell conditioned media (CM). The greatest magnitude of the inductive action was on embryonic striatal precursors. Although FGF2 alone induced limited TH expression in striatal cells (1.1 +/- 0.2% of neurons), these actions were potentiated 17.5-fold (19.6 +/- 1.5% of neurons) when FGF2 was coadministered with B49 glial cell line CM. Of these TH-immunoreactive cells, approximately 15% incorporated bromodeoxyuridine (BrdU), indicating that they were newly generated, and 95% coexpressed the neurotransmitter GABA. To investigate whether precursors of the adult forebrain subependyma were competent to respond to the instructive actions of FGF2+CM, they were first labeled in vivo with a pulse of BrdU. Although none of the cells expressed TH in control, 0.2% of total cells showed TH immunoreactivity in FGF2+CM-treated cultures. Under these same conditions only, in vitro-generated precursors from epidermal growth factor-responsive stem cells exhibited TH expression in 10% of their total neuronal progeny. Regulation of neurotransmitter phenotype in forebrain neuronal precursors, by the synergistic action of FGF2 and glial-derived diffusible factors, may represent a first step in understanding how these cells are generated in the embryonic and adult brain and opens the prospect for their manipulation in vitro and in vivo for therapeutic use.