Characterization of tyrosine hydroxylase expression during chick embryo brain development

INTRODUCTION: Tyrosine hydroxylase (TH) is a monooxygenase which catalyzes the first and rate-limiting step of the catecholamines biosynthesis, such as dopamine, noradrenaline and adrenaline. Similarly to the mammals, in the chick brain catecholamine containing neurons are localized mainly in the brain stem: noradrenaline producing cells are almost exclusively confined to the locus coeruleus and subcoeruleus nuclei, while the majority of dopaminergic neurons are localized in the nucleus tegmenti pedunculopontinus (substantia nigra) and the ventral tegmental area. Thus the regulation of the TH levels and enzymatic activity directly influences the levels of those neurotransmitters. TH is regulated by many ways: among them, gene expression modulation, alternative RNA processing, allosteric modulation by polianions (mainly RNA), site-specific phosphorilation, feedback inhibition. The importance of TH expression regulation during embryonic development have been demonstrated by the targeted inactivation of the TH gene that determines, in mices, midgestional lethality (1). METHODS: Protein lysates were obtained from chick embryo brains from E (embryonic day) 8 to E19. Proteins were separated by SDS gel electrophoresis, blotted on nitrocellulose membrane and incubated with an anti-TH antibody. TH positive bands were quantified by densitometric analysis. Molecular weights (MW) were calculated comparing TH bands with molecular markers with known MW. 10 μm thick paraffin serial sections were obtained from embryo brains at E13, E15, E17 and E19 and then incubated with the anti-TH antibody according to ABC technique. RESULTS AND DISCUSSION: First, following the immunohistochemical staining of the sections with the same anti-TH antibody used for the immunoblotting we observed, as expected, most of the TH positive neuroblasts in the brain stem, belonging mainly to the locus coeruleus and subcoeruleus nuclei, the nucleus tegmenti pedunculopontinus and the ventral tegmental area. The TH expression analysis revealed that the enzyme amount changes significantly during the chick embryonic development; more surprisingly, we also found that this enzyme showed different MW depending on the embryonic day considered. In particular, we observed the maximum expression of the enzyme already at E8-E10. From E11 to E15 the enzyme was expressed at a rather high rate, while from E16 it resulted slightly reduced. From E8 to E14 TH showed a MW of 74-78 kD; at E15-E16 we observed another band of 63-67 kD, that corresponded to the MW of the adult rat brain TH. From E17 the higher MW band disappeared, while the 63-67 kD one reached its maximum. These results suggest that during first days of chick embryo brain development, TH mRNA can undergo a differential splicing, giving a TH protein that is expressed massively.