Transforming growth factor-βs are essential for the development of midbrain dopaminergic neurons in vitro and in vivo

Development of midbrain dopaminergic neurons is known to depend on inductive signals derived from the ventral midline, including Sonic hedgehog (Shh) as one of the identified molecules. Here we show that in addition to Shh, transforming growth factor (TGF)-beta is required for both induction and survival of ventrally located midbrain dopaminergic neurons. Like Shh, TGF-beta is expressed in early. embryonic structures such as notochord and floor plate, as well as in the area where mibrain dopaminergic neurons are developing. Treatment of cells dissociated from the rat embryonic day (E) 12 mibrain floor with TGF-beta significantly increases the number of tyrosine hydroxylase (TH)-positive dopaminergic neurons within 24 hr. Neutralization of TGF-beta in vitro completely abolishes the induction of dopaminergic neurons. In the absence of TGF-beta, Shh cannot induce TH-positive neurons, and vice versa, neutralizing endogenous Shh abolishes the capacity of TGF-beta to induce dopaminergic neurons in vitro. Furthermore, neutralization of TGF-beta in vivo during chick E2-7 but not E4-7 resulted in a significant reduction in TH-positive neurons in the ventral midbrain floor but not in the locus coeruleus or diencephalon, which suggests that the TGF-beta is required for the induction of mesencephalic dopaminergic neurons with a critical time period at E2/E3. Furthermore, neutralization of TGF-beta between E6 and 10, a time period during maturation of mesencephalic dopaminergic neurons when no further inductive cues are required, also resulted in a significant loss of dopaminergic neurons, suggesting that TGF-beta is required for the promotion of survival of ventral midbrain dopaminergic neurons as well. Together, our results identify TGF-beta as an essential mediator for the induction and maintenance of midbrain dopaminergic neurons.