Cholinergic differentiation occurs early in mouse sympathetic neurons and requires Phox2b

The generation of neurotransmitter identity in the autonomic nervous system is a classical model system to study the development of neuronal diversity. Analysis of the expression of genes coding for enzymes of noradrenaline biosynthesis in the sympathoadrenal system allowed the characterization of factors involved in the differentiation of the noradrenergic transmitter phenotype. The development of cholinergic properties in the autonomic system is less well understood. Here we show that expression of mRNAs for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT), both encoded by the cholinergic gene locus, is induced in mouse sympathetic ganglia at embryonic day 11 (E11). Positive cells amount to more than 50% of Phox2b-positive sympathetic cells at cervical levels. The proportion declines caudally, decreasing to approximately 20% of Phox2b-positive cells at lower thoracic levels. In the adrenal anlage, ChAT and VAChT mRNA are largely undetectable at E11 and E13. In mice homozygous for a mutational inactivation of the transcription factor Phox2b, ChAT and VAChT mRNA expression is absent from sympathetic ganglia. The data show that expression from the cholinergic gene locus is regulated differently in sympathetic neurons and adrenal chromaffin cells. Phox2b is required for development of cholinergic neurons but does not suffice to support cholinergic properties in chromaffin cells.