Tyrosine hydroxylase in chick embryo brain development

Tyrosine hydroxylase (TH) catalyses the first, rate limiting step in catecholamine biosynthesis. In vertebrates the catecholamines dopamine, noradrenaline and adrenaline play important roles in many physiological functions in the central and peripheral nervous systems as well as in the endocrine system. Thus the regulation of TH expression and activity is crucial for neuronal and hormonal functions that involve catecholamines. 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, ubiquitination. The importance of TH expression during embryonic development have been demonstrated by the targeted inactivation of the TH gene that determines, in mice, midgestional lethality. In most species is present a single TH mRNA, that following translation produces a single form of the protein. In some species, however, alternative splicing of the primary transcript can result in different forms of TH mRNA that encode for different protein isoforms with specific regulative properties. Considering the importance of the TH expression during embryonic development and the presence in many species of multiple TH protein/mRNA forms led us to study the enzyme expression during chick embryo brain development. By means of western blotting we found that in the chick embryo brain, together with the expected form of ~65 kDa, is present a TH protein showing a molecular weight of ~75 kDa from E (embryonic day) 8 to E14. The analysis of the cDNA sequence of TH obtained from chick embryo brains at E8-E14 revealed the possible presence of a mRNA splice variant that presents a nucleotide insertion which encodes for an additional aminoacidic sequence in the regulative N-terminal domain of the protein. We also found that TH immunoreactive bands showing an higher molecular weight were immunoreactive for ubiquitin, too. These results suggest that during chick embryo brain development is transiently present a TH protein of ~75 kDa; the possible mechanisms involved in this process are alternative splicing and/or ubiquitination.