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First published online 30 November 2005
doi: 10.1242/dev.02189

Development 133, 141-150 (2006)
Published by The Company of Biologists 2006
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Target-dependent specification of the neurotransmitter phenotype: cholinergic differentiation of sympathetic neurons is mediated in vivo by gp130 signaling

Matthias Stanke1, Chi Vinh Duong1, Manuela Pape1, Markus Geissen1,*, Guido Burbach2, Thomas Deller2, Hugues Gascan3, Rosanna Parlato4, Günther Schütz4 and Hermann Rohrer1,{dagger}

1 Research Group Developmental Neurobiology, Max-Planck-Institute for Brain Research, Deutschordenstrasse 46, 60528 Frankfurt/M, Germany.
2 Institute of Clinical Neuroanatomy, J.W.-Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt/M, Germany.
3 INSERM U564, CHU d'Angers, 4 rue Larrey, 49033 Angers Cedex, France.
4 Deptartment of Molecular Biology of the Cell I, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.



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  		Fig. 1. Analysis of Cre expression in DBH-Cre mice. (A-C) Using the ROSA26 reporter mouse line, Cre-mediated lacZ expression was detected by ß-galactosidase staining in sympathetic ganglia of E16.5 mouse embryos. lacZ expression is demonstrated for the sympathetic chain (A,B) and is absent in sweat gland tissue (C). (D) Cre-mediated elimination of exon 16 in gp130DBHcre mice is revealed by the amplification of an 800 bp band from sympathetic ganglia (1), adrenal gland (2) and the absence in non-adrenergic tissue (tail-cut, 3). (A) Whole-mount staining; (B,C) ß-galactosidase staining of tissue sections.

 


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  		Fig. 2. Sweat gland innervation in gp130DBHcre mice lacks cholinergic properties. Frozen sections from P60 mouse footpads of control gp130fl/fl and mutant gp130DBHcre mice were co-stained for (A-F) TUJ1 and VIP, (G-M) TUJ1 and VAChT, (N-S) and TUJ1 and ChT1. Nuclei were stained with DAPI. The expression of VIP-IR, VAChT-IR and ChT1-IR is virtually absent in sweat gland innervation of gp130DBHcre mice, as is evident from the individual stainings (F,M,S) and the quantitative analysis (bar graphs, left). By contrast, TUJ1-IR fibers are not affected, as shown by the individual stainings (E,L,R) and the overlays (D,K,Q). For the quantitative analysis, sections were stained individually for VIP-IR, VAChT-IR and ChT1-IR (no co-staining with TUJ1). Quantitative data are expressed as mean±s.e.m. (n=3-8 for each group).

 


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  		Fig. 3. ß-III-tubulin (TUJ1) and TH expression in gp130DBHcre sweat gland innervation. Sections from P60 mouse footpads of control gp130fl/fl and mutant gp130DBHcre mice were stained for TUJ1 and TH. (A-C) The expression of TUJ1-IR is not reduced in gp130DBHcre (B) sweat glands compared with gp130fl/fl (A). Quantitative analysis of TuJ1-IR is shown in (C). (D-F) Sweat glands in mutant gp130DBHcre mice (E) display a slightly stronger TH-IR signal than control tissues do (D), which is also reflected in the quantitative analysis (F). Data are expressed as the mean±s.e.m. (n=8-9 for TUJ1; n=3-4 for TH). The differences between gp130fl/fl and gp130DBHcre are not significant.

 


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  		Fig. 4. The number of VIP-IR neurons is reduced in gp130DBHcre stellate ganglia. (A,B) VIP-staining of sections from P60 stellate ganglia of gp130fl/fl (A) and gp130DBHcre (B) mice demonstrate a strong reduction in the density of VIP-IR neurons. (C) Quantitative analysis of VIP-IR neurons per ganglion. Data are expressed as mean±sem (n=3-5). The differences between gp130fl/fl and gp130DBHcre mice are significant (P<0.05, unpaired, two-tailed t-test with Welch-correction). (D) VIP-staining of sections from P2 stellate ganglia of gp130fl/fl and gp130DBHcre mice show no significant difference.

 


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  		Fig. 5. IL6 cytokine expression in mouse sweat gland tissue. (A,B) Individual sweat gland coils were dissected from P4 Toluidine-stained sections by LCM. (A) Before and (B) after dissection of sweat gland coil. (C) RT-PCR detected the expression of CNTF, CLC, CT1 and NP, but not of OSM and LIF, in P4 sweat glands, whereas all cytokines could be detected in total RNA from E15 mouse embryos. (D-F) In situ hybridisation of P10 sweat glands revealed expression of CLC (D), CLF (E) and CT1 (F). The in situ hybridization signal is mostly localized lateral to the secretory cells, and is most pronounced for CLF (E). (G) No signal was detected for NP, correlating with the minor band in the RT-PCR (see C). (H) GPA (2 ng/ml), CNTF (1.5 ng/ml), CLC/CLF (100 ng/ml) and NP (500 ng/ml) induce the expression of VIP in cultures of chick sympathetic neurons.

 


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  		Fig. 6. Secretory responsiveness develops in the absence of cholinergic innervation. Sweating response in P60 mice after intraperitoneal injection of pilocarpine. (A,B) The glands in both control gp130fl/fl (A) and gp130DBHcre (B) footpads respond robustly to the pilocarpine treatment. Each pore, which appears as dark spot, represents the activity of a single gland. (C) Quantification of the number of active glands/per interdigital footpad. Data are presented as mean±s.e.m. of 12 control and six gp130DBHcre mice. The difference between gp130fl/fl and gp130DBHcre mice is significant (P<0.05; unpaired, two-tailed t-test).

 

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