Regulation of the Quail Tyrosine Hydroxylase Gene in Neural Crest Cells by cAMP and β-Adrenergic Ligands

Abstract

Expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine (CA) synthesis, was studied in quail neural crest (NC) cultures to investigate the role of environmental factors in the development of the adrenergic phenotype. First, the activity of the quail TH gene promoter was analyzed in immortalized quail NC cells by transient transfection assay. We found that chick embryo extract (CEE), which is known to trigger adrenergic differentiation of NC cells in vitro, strongly enhanced reporter gene transcription. A sequence of only 77 nucleotides, including a cAMP-responsive element, was sufficient to elicit this response. Implication of cAMP in the CEE effect was further supported by the fact that CEE produced a stimulation of adenylate cyclase activity that was sensitive to β-adrenoreceptor antagonists. Moreover, stimulation of TH gene expression by CEE could be reduced by β-adrenergic antagonists or CA-depleted extract. We have subsequently investigated the possibility that TH expression during differentiation of nontransformed NC cells could also be influenced by CA via the cAMP pathway. In dissociated cultures of 3-day-old quail sclerotomes and of 2-day-old quail trunk NC, differentiation of TH + cells was induced in the presence of forskolin, an activator of adenylate cyclase, and modulated by β-adrenoreceptor ligands. In particular, the adrenergic-promoting effect of 10% CEE on trunk NC cultures was inhibited by β-adrenoreceptor antagonists or by eliminating CA from the extract. In conclusion, these data suggest that CA (through activation of β-adrenergic receptors and cAMP production) can stimulate the initial expression of TH in cultured NC cells and enhance TH transcription in immortalized NC cells. This supports the notion that exogenous CA, which is present in the early embryo, may promote the differentiation of sympathoadrenal precursors in vivo by stimulating TH gene activity.