Matthias Stanke

Target-dependent specification of the neurotransmitter phenotype: cholinergic differentiation of sympathetic neurons is mediated in vivo by gp130 signaling

Sympathetic neurons are generated through a succession of differentiation steps that initially lead to noradrenergic neurons innervating different peripheral target tissues. Specific targets, like sweat glands in rodent footpads, induce a change from noradrenergic to cholinergic transmitter phenotype. Here, we show that cytokines acting through the gp130 receptor are present in sweat glands. Selective elimination of the gp130 receptor in sympathetic neurons prevents the acquisition of cholinergic and peptidergic features (VAChT, ChT1, VIP) without affecting other properties of sweat gland innervation. The vast majority of cholinergic neurons in the stellate ganglion, generated postnatally, are absent in gp130-deficient mice. These results demonstrate an essential role of gp130-signaling in the target-dependent specification of the cholinergic neurotransmitter phenotype.

The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons

The basic helix-loop-helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.

Interaction of Mash1 and Phox2b in sympathetic neuron development.

The transcription factors Mash1 and Phox2b are both essential for sympathetic neuron development. To understand in more detail their function and interaction, Phox2b and Mash1 were ectopically expressed in vivo, in peripheral nerve precursors. Here, we demonstrate that the Phox2b-induced generation of ectopic noradrenergic neurons in chick peripheral nerve involves the induction of Cash1, the chick homolog of Mash1. All Phox2-induced neurons coexpress the noradrenergic marker genes TH and DBH. Conversely, Mash1 induces neuronal differentiation characterized by the expression of generic neuronal genes SCG10, Hu and NF160; however, only a subpopulation of these neurons also displays an autonomic, noradrenergic phenotype. This context-dependent action of Mash1 implicates autonomic codeterminants, required for noradrenergic differentiation in response to Mash1. In contrast, Phox2b coordinates generic and noradrenergic gene expression, recruiting Mash1/Cash1, which may have a major function in the control of pan-neuronal gene expression during noradrenergic neuron development.

BMP growth factors and Phox2 transcription factors can induce synaptotagmin I and neurexin I during sympathetic neuron development

Synaptotagmin I and neurexin I mRNAs, coding for proteins involved in neurotransmitter secretion, become detectable in primary sympathetic ganglia shortly after initial induction of the noradrenergic transmitter phenotype. To test whether the induction of these more general neuronal genes is mediated by signals known to initiate noradrenergic differentiation in a neuronal subpopulation, we examined their expression in noradrenergic neurons induced by ectopic overexpression of growth and transcription factors. Overexpression of BMP4 or Phox2a in vivo results in synaptotagmin I and neurexin I expression in ectopically located noradrenergic cells. In vitro, BMP4 initiates synaptotagmin I and neurexin I expression in addition to tyrosine hydroxylase induction. Thus, the induction of synaptotagmin I and neurexin I, which are expressed in a large number of different neuron populations, can be accomplished by growth and transcription factors available only to a subset of neurons. These findings suggest that the initial expression of proteins involved in neurotransmitter secretion is regulated by different signals in different neuron populations.

The early expression of VAChT and VIP in mouse sympathetic ganglia is not induced by cytokines acting through LIFRβ or CNTFRα

Abstract Sympathetic ganglia consist of noradrenergic and cholinergic neurons. The cholinergic marker protein vesicular acetylcholine transporter (VAChT) and the neuropeptide vasoactive intestinal peptide (VIP), co-expressed in mature cholinergic sympathetic neurons, are first detectable during embryonic development of rat sympathetic ganglia. However, the subpopulation of cholinergic sympathetic neurons which innervates sweat glands in mammalian footpads starts to express VAChT and VIP during the first postnatal weeks, under the influence of sweat gland-derived signals. In vitro evidence suggests that the sweat gland-derived cholinergic differentiation factor belongs to a group of neuropoietic cytokines, including LIF, CNTF and CT-1, that act through a LIFRβ-containing cytokine receptor. To investigate whether the embryonic expression of cholinergic properties is elicited by a related cytokine, the expression of VAChT and VIP was analyzed in stellate ganglia of mice deficient for the cytokine receptor subunits LIFRβ or CNTFRα. The density of VAChT- and VIP-immunoreactive cells in stellate ganglia of new-born animals was not different in LIFRβ−/− and CNTFRα−/− ganglia as compared to ganglia from wild-type mice. These results demonstrate that the early, embryonic expression of VAChT and VIP is not induced by cytokines acting through LIFRβ- or CNTFRα-containing receptors.