The early expression of VAChT and VIP in mouse sympathetic ganglia is not induced by cytokines acting through LIFRbeta or CNTFRalpha.

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 LIFRbeta-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 LIFRbeta or CNTFRalpha. The density of VAChT- and VIP-immunoreactive cells in stellate ganglia of new-born animals was not different in LIFRbeta(-/-) and CNTFRalpha(-/-) 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 LIFRbeta- or CNTFRalpha-containing receptors.

The Phox2 homeodomain proteins are sufficient to promote the development of sympathetic neurons.

The development of sympathetic neurons is controlled by a network of transcriptional regulators, including the paired homeodomain proteins Phox2a and Phox2b. To understand the role of Phox2 proteins in more detail, the effect of Phox2 overexpression was analysed in the avian peripheral nervous system. Phox2a expression in neural crest cultures elicited a strong increase in the number of sympathoadrenergic cells. Expression of Phox2a in the chick embryo promoted the generation of additional neurons expressing the noradrenergic marker genes DBH and TH, pan-neuronal genes SCG10 and NF160 and cholinergic genes ChAT and VAChT. Phox2a-induced neurons were found in ectopic locations such as dorsal root ganglia and peripheral nerve. Sympathoadrenergic development could be elicited in cultures of E5 dorsal root ganglia, demonstrating the presence of Phox2a-responsive cells in non-autonomic peripheral ganglia. Phox2b induced ectopic neurons in the chick embryo in the same way as Phox2a. These results show that Phox2 proteins are sufficient to promote sympathetic neuron generation and control, directly or indirectly, the expression of a large number of genes characteristic for sympathetic neurons.

The specification of sympathetic neurotransmitter phenotype depends on gp130 cytokine receptor signaling.

Sympathetic ganglia are composed of noradrenergic and cholinergic neurons. The differentiation of cholinergic sympathetic neurons is characterized by the expression of choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP), induced in vitro by a subfamily of cytokines, including LIF, CNTF, GPA, OSM and cardiotrophin-1 (CT-1). To interfere with the function of these neuropoietic cytokines in vivo, antisense RNA for gp130, the common signal-transducing receptor subunit for neuropoietic cytokines, was expressed in chick sympathetic neurons, using retroviral vectors. A strong reduction in the number of VIP-expressing cells, but not of cells expressing ChAT or the adrenergic marker tyrosine hydroxylase (TH), was observed. These results reveal a physiological role of neuropoietic cytokines for the control of VIP expression during the development of cholinergic sympathetic neurons.

Onset of CNTFRalpha expression and signal transduction during neurogenesis in chick sensory dorsal root ganglia.

The expression of ciliary neurotrophic factor receptor alpha (CNTFRalpha) was investigated in the developing chick dorsal root ganglion (DRG) using affinity-purified anti-CNTFRalpha antibodies. At thoracic levels, CNTFRalpha-immunoreactivity (CNTFRalpha-IR) was first observed at stage 19 (E3) in cells with neuronal morphology. CNTFRalpha-IR is restricted to the neuronal lineage in the DRG throughout development. CNTFRalpha expression precedes that of neuron-specific beta tubulin, Hu antigen, and Q211 antigen, which are markers expressed in developing sensory neurons. [3H]Thymidine-labeling studies showed the onset of CNTFRalpha expression during terminal mitosis of sensory neuron precursors, making CNTFRalpha the earliest known neuronal marker in the DRG. CNTFRalpha-mediated signal transduction was demonstrated in E7 and E11 DRG neuron cultures by CNTF-induced STAT3 phosphorylation. Although low ligand concentrations (5 pM) elicit STAT3 phosphorylation in E7 and E11 DRG neurons, a survival response is only observed in neurons from E11 DRG. This implicates a complex readout mechanism downstream of STAT3 phosphorylation leading to different cellular responses that depend on the age of the DRG neuron. These results argue against a role of CNTFRalpha ligands in the control of early neuron survival but are compatible with other functions in neurogenesis and sensory neuron development.

The Drosophila melanogaster tumor suppressor gene lethal(3)malignant brain tumor encodes a proline-rich protein with a novel zinc finger.

The lethal(3)malignant brain tumor [t(3)mbt] gene causes, when mutated, malignant growth of the adult optic neuroblasts and ganglion mother cells in the larval brain and imaginal disc overgrowth. Via overlapping deficiencies a genomic region of approximately 6.0 kb was identified, containing l(3)mbt+ gene sequences. The l(3)mbt+ gene encodes seven transcripts of 5.8 kb, 5.65 kb, 5.35 kb, 5.25 kb, 5.0 kb, 4.4 kb and 1.8 kb. The putative MBT163 protein, encompassing 1477 amino acids, is proline-rich and contains a novel zinc finger. In situ hybridizations of whole mount embryos and larval tissues revealed l(3)mbt+ RNA ubiquitously present in stage 1 embryos and throughout embryonic development in most tissues. In third instar larvae l(3)mbt+ RNA is detected in the adult optic anlagen and the imaginal discs, the tissues directly affected by l(3)mbt mutations, but also in tissues, showing normal development in the mutant, such as the gut, the goblet cells and the hematopoietic organs.

Analysis of function and expression of the chick GPA receptor (GPAR alpha) suggests multiple roles in neuronal development.

Growth promoting activity (GPA) is a chick growth factor with low homology to mammalian ciliary neurotrophic factor (CNTF) (47% sequence identity with rat CNTF) but displays similar biological effects on neuronal development. We have isolated a chick cDNA coding for GPA receptor (GPAR alpha), a GPI-anchored protein that is 70% identical to hCNTFR alpha. Functional analysis revealed that GPAR alpha mediates several biological effects of both GPA and CNTF. Soluble GPAR alpha supports GPA- and CNTF-dependent survival of human TF-1 cells. In sympathetic neurons, GPAR alpha mediates effects of both GPA and CNTF on the expression of vasoactive intestinal peptide (VIP) as shown by the inhibition of GPA- and CNTF-mediated VIP induction upon GPAR alpha antisense RNA expression. These results demonstrate that GPAR alpha is able to mediate effects of two neurokines that are only distantly related. GPAR alpha mRNA expression is largely restricted to the nervous system and was detected in all neurons that have been shown to respond to GPA or CNTF by increased survival or differentiation, i.e. ciliary, sympathetic, sensory dorsal root, motoneurons, retinal ganglion cells and amacrine cells. Interestingly, GPAR alpha mRNA was additionally found in neuronal populations and at developmental periods not known to be influenced by GPA or CNTF, suggesting novel functions for GPAR alpha and its ligands during neurogenesis and neuron differentiation.