In vitro regulated expression of tyrosine hydroxylase in ventral midbrain neurons from Nurr1-null mouse pups

The transcription factor Nurr1, an orphan member of the steroid-thyroid hormone nuclear receptor superfamily, is essential for the proper terminal differentiation of ventral midbrain dopaminergic neurons. Disruption of the Nurr1 gene in mice by homologous recombination abolishes synthesis of dopamine (DA) and expression of DA biosynthetic enzymes, including tyrosine hydroxylase (TH), in the ventral midbrain without affecting the synthesis of DA in other areas of the brain. At birth, however, dopaminergic neuron precursors in Nurr1 null (-/-) pups remain as shown by continued expression of residual, untranslated Nurr1 mRNA not altered by homologous recombination. Since Nurr1 disruption is lethal shortly after birth, to further investigate the developmental properties of these neurons, dissociated ventral midbrain neurons from newborn pups were grown for 5 days on an astrocyte feeder layer, subjected to various treatments and then evaluated for expression of TH by fluorescent immunocytochemistry. Initially, a small percentage of neurons (0.26% +/- 0.07%) from the ventral midbrain of Nurr1 -/- pups were TH-immunoreactive (TH-IR). No change in TH expression was observed in the presence of glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), or DA alone or in combination. Treatment with forskolin (Fsk), however, significantly increased the percentage of TH-IR neurons (1.36% +/- 0.15%). Combination of Fsk, BNDF, and DA further increased the percentage of TH-IR neurons (2.58% +/- 0.50%). Therefore, these data suggest that dopaminergic neuron precursors, which develop in vivo without Nuur1, remain in an undifferentiated condition that is permissive to the induction of TH in vitro. Published 2001 Wiley-Liss, Inc.(dagger).