The expression of tyrosine hydroxylase and the transcription factors cPhox-2 and Cash-1: evidence for distinct inductive steps in the differentiation of chick sympathetic precursor cells.

The adrenergic differentiation of sympathetic neurons is controlled by complex interactions with the embryonic environment. To provide a basis for the experimental analysis of these interactions, the expression of the adrenergic marker enzyme tyrosine hydroxylase (TH) was analyzed by immunohistochemistry and in situ hybridization in sympathetic ganglia and the adrenal medulla of chick embryos. In parallel, the developmental expression of the transcription factors cPhox-2 and Cash-1 was analyzed by in situ hybridization. TH protein was first detectable during the third day of development (stage 19) in cells of the primary sympathetic strands. A few hours earlier (stage 18), TH mRNA could be found by in situ hybridization. At the very same time and location, mRNA for the transcription factor cPhox-2 was first observed. In contrast, mRNA for the transcription factor Cash-1 was detected much earlier, at stage 15, dorsal to the aorta where the primary sympathetic ganglia form. High TH mRNA levels are maintained during later embryonic development (stage 35) in both sympathetic ganglia and adrenal chromaffin cells. In contrast, cPhox-2 and Cash-1 mRNA are selectively reduced in chromaffin cells and sympathetic ganglia, respectively. The results show that TH and cPhox-2 are early markers expressed in sympathetic ganglia. Their coordinated expression points towards an inductive event possibly occurring close to the aorta and leading to the expression of an adrenergic phenotype. Cash-1 is detected significantly earlier, suggesting that its expression is induced by a separate event.