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The developmental expression of vasoactive intestinal peptide (VIP) in cholinergic sympathetic neurons depends on cytokines signaling through LIFRß-containing receptors

Max-Planck-Institut für Hirnforschung, Abteilung Neurochemie, Deutschordenstr. 46, 60528 Frankfurt / Main, Germany

View larger version (111K): [in a new window]   Fig. 1. Sequence comparison of predicted amino acid sequence of the chick LIFRß and its human/mouse ortholog. Identical amino acids are indicated in capital letters. Signal peptide (blue), transmembrane region (light red; amino acids 819-840), cysteine residues (orange) necessary for ligand binding and the two WS-X-WS motifs (green) are indicated. Potential asparagine-linked glycosylation sites are written in bold letters and the two regions with high homology to fn3 domains are in brackets. Tyrosine residues and YXXQ-motifs taking part in signal transduction via STAT molecules are shown in red. The JAK kinase-binding motif, box1 and box2 are boxed.

View larger version (77K): [in a new window]   Fig. 2. LIFRß mRNA is expressed in sympathetic ganglia, DRG and spinal cord motoneurons. (A) Thoracic sections of E14 chick embryos were analyzed by in situ hybridization. LIFRß mRNA is strongly detectable in spinal cord motoneurons (arrowhead), DRG (open arrow) and the sympathetic ganglia (arrow). SC, spinal cord; V, vertebra. Scale bar: 300 µm. (B) RT-PCR analysis indicates expression of LIFRß mRNA in the sympathetic ganglia between embryonic day (E) 7 and 14. No PCR products were obtained in the absence of RT (not shown).

View larger version (18K): [in a new window]   Fig. 3. Expression of antisense LIFRß mRNA in cultured E7 sympathetic neurons exhibit decreased responsiveness to CNTF. Primary cultures of sympathetic neurons co-transfected with pcDNA-lacZ in combination with either sense or antisense expression vectors or nontransfected control cultures were treated with CNTF and analyzed for VIP expression. Co-transfected cells were identified by immunostaining for ß-galactosidase, and the proportion of transfected cells immunoreactive for VIP was determined. Under control (dark gray) and sense (gray) treated conditions, CNTF induces VIP-immunoreactivity in about 50% of the neurons. Neurons transfected with LIFRß antisense mRNA exhibited a significantly reduced response to CNTF as compared with either non-transfected or sense-transfected cells. Data represent the mean±s.d. of several independent experiments as indicated by the numbers. P<0.001; (Student’s t-test, pairwise comparison between sense and antisense experiments).

View larger version (111K): [in a new window]   Fig. 4. Sympathetic ganglia infected with LIFRß antisense-RCAS retrovirus exhibit a decreased number of VIP-expressing neurons. Chick embryos were infected with LIFRß sense-RCAS (A-D) or LIFRß antisense-RCAS (E-H) at E2 and analyzed by in situ hybridization for viral infection (RT) (A,E), and the expression of VIP (B,F), TH (C,G) and SCG10 (D,H) on alternate thoracic sections of E13/14 embryos. Sympathetic ganglia expressing antisense LIFRß mRNA (F) displayed a strong reduction in the number of VIP-positive cells as compared to ganglia infected with sense-RCAS (B). By contrast, the expression of TH mRNA (C,G) and SCG10 mRNA (D,H) is not altered. Scale bar: 100 µm.

View larger version (88K): [in a new window]   Fig. 5. Quantification of VIP and ChAT expression in E13/14 sympathetic ganglia. The number of VIP- (A,D) and ChAT- (B,E) positive neurons was determined in cervical and thoracic ganglia infected by LIFRß antisense-RCAS and LIFRß sense-RCAS. The area of neuronal SCG10-positive cells in the sympathetic ganglia section was determined morphometrically, as described in Materials and Methods. The numbers of VIP- (G) and ChAT-positive (H) cells were referred to the SCG10-positive area. Scale bar: 100 µm. Data represent the mean±s.d. of several independent experiments as indicated by the numbers. P<0.0001 (Student’s t-test, pairwise comparison between sense and antisense experiments).

View larger version (118K): [in a new window]   Fig. 6. VIP and ChAT are co-expressed in sympathetic ganglion neurons at cervical and thoracic levels at E18. Double in situ hybridization on sections of wild-type E18 chick embryos, using cRNA probes against ChAT (A,C, red) and VIP (B,D, blue) demonstrates co-expression of ChAT and VIP at cervical (A,B) and thoracic (C,D) levels in sympathetic ganglion neurons. Please note that there are no ChAT-expressing cells (red) devoid of VIP expression. Scale bar: 50 µm (cervical); 100 µm (thoracic).

View larger version (121K): [in a new window]   Fig. 7. Expression of LIFRß antisense RNA in vivo interferes with the expression of endogenous LIFRß protein. Alternating sections from the thoracic region of E14 infected embryos were analyzed for virus infection (A,C) and LIFRß protein expression (B,D) on alternating sections. Expression of endogenous LIFRß protein (D) in LIFRß antisense-RCAS infected embryos should be compared with sense LIFRß-RCAS infected embryos (B). Note that the expression of antisense LIFRß mRNA in the sympathetic ganglia (arrow) results in strong decrease of endogenous LIFRß protein (D), whereas uninfected neurons in DRG (star) and spinal cord display normal expression levels of endogenous LIFRß. SC, spinal cord; V, vertebra. Scale bar: 200 µm.