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GDNF and neurturin are target-derived factors essential for cranial parasympathetic neuron development

Eri Hashino1,2,*, Marlene Shero1, Dirk Junghans3, Hermann Rohrer3, Jeffrey Milbrandt4 and Eugene M. Johnson, Jr.5

1 Center for Hearing and Deafness and
2 Department of Anatomy and Cell Biology, State University of New York at Buffalo, Buffalo, NY 14214, USA
3 Max-Planck-Institut für Hirnforschung, Deutschordenstrasse 46, D-60628 Frankfurt, Germany
4 Departments of Pathology and Internal Medicine,
5 Departments of Neurology, Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA



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  		Fig. 1. GDNF and NRTN promote the neurite outgrowth and survival of ciliary ganglion neurons in vitro in a stage-specific manner. (A,B) E9 ciliary ganglion explant with (A) or without (B) NRTN at 50 ng/ml. (C,D) E16 ciliary ganglion explant with NRTN (C) or CNTF (D) at 50 ng/ml. Note that NRTN induces an extensive outgrowth from E9, but not from E16 ciliary ganglia. In contrast, CNTF has positive effects on E16 ciliary ganglion neurite outgrowth. A and D were immunostained with a TUJ1 antibody and B and C were unstained phase-contrast micrographs. Scar bar, 500 µm. (E,F) Quantitative analysis of neurite outgrowth from E9 (E) or E16 (F) ciliary ganglion explants in the presence of GDNF family ligands, CNTF or BDNF. Graphs show the average neurite length (± s.e.) for ciliary ganglion explants grown in medium alone or medium containing either GDNF, NRTN, ARTN, PSPN, CNTF or BDNF (50 ng/ml, for each factor) for 3 days. (G,H) Survival of E9 (G) or E16 (H) ciliary ganglion neurons in the presence of GDNF family ligands, CNTF or BDNF (50 ng/ml for each factor). Results are expressed as a percentage of the neurons that survived after 48 hours in culture (± s.e.). Note that the majority of E9 ciliary ganglion neurons are dependent on GDNF or NRTN (G), whereas only a sub-population of E16 ciliary ganglion neurons are supported by these factors (H). Each bar represents data from 7-14 explants. **, Significantly different from the control group (P<0.01).

 


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  		Fig. 2. Expression of GFR{alpha}1 and Ret proteins declines in ciliary ganglion neurons after target innervation. (A) Western blot characterization of anti-GFR{alpha}1, GFR{alpha}2 and Ret antibodies used in the present study. The GFR{alpha}2 antiserum generated in this study recognizes an approx. 55 kDa protein, equivalent to the estimated size of GFR{alpha}2, in E9 ciliary ganglion neurons. The anti-GFR{alpha}1 antibody recognizes two protein species of approx. 55 and 65 kDa, whereas the anti-Ret antibody recognizes a single protein of 170 kDa in ciliary ganglion neurons. (B) Western blot analysis for GFR{alpha}1, GFR{alpha}2, Ret, CNTFR{alpha} and gp130 in ciliary ganglion lysates (left) or whole brain lysates (right) at different embryonic ages.

 


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  		Fig. 3. Expression of GFR{alpha}1 and Ret mRNAs, but not of GFR{alpha}2 mRNA, is down-regulated in ciliary ganglion neurons during development. (A) Calibration PCR reaction profile with E5 ciliary ganglion neuron cDNA. To check the linearity of the detection system, various dilutions (1, 1:10, 1:100, 1:1000) of E5 ciliary ganglion neuron cDNA as well as water (control) were amplified with a primer pair and a TaqMan probe for L27 housekeeping gene. The correlation coefficient was calculated from the standard curve that displays threshold cycles (Ct) as a function of log10 cDNA concentrations (inset). (B,C) Real-time PCR reactions for GFR{alpha}1, GFR{alpha}2 and Ret mRNA expression in E5 and E15 ciliary ganglion neurons. Real-time PCR for each of the three receptors was run using E5 (B) or E15 (C) ciliary ganglion neuron cDNA samples. For an internal control, the L27 mRNA level was measured with each cDNA sample. Each solid line in the graphs shows the average of quadruplicate PCR reaction profiles. Note that the Y axis in the graphs is in a log scale. A dotted horizontal line in each graph indicates a threshold for that experiment. (D-F) Comparison of the expression of GFR{alpha}1, GFR{alpha}2 and Ret mRNAs between E5 and E15 ciliary ganglion neurons. Based on Ct values obtained from 2 separate experiments, the mRNA level for each probe relative to L27 mRNA level (internal control) was calculated. The GFR{alpha}1 and Ret mRNA levels in E5 ciliary ganglion neurons are approximately 19 times and 1.5 times higher, respectively, than those in E15 ciliary ganglion neurons. **, Significantly different from the E5 group (P<0.01); *, P<0.05.

 


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  		Fig. 4. GDNF and NRTN are present in the eye during and after target innervation. (A) Western blot analysis for GDNF and NRTN in eye lysates collected from different embryonic ages. The GDNF-immunoreactive band at a similar molecular mass to recombinant human GDNF (rhGDNF) is present in E6 and E9 eye lysates, but is absent in E12 and E16 eye lysates. The anti-GDNF antibody used in this study does not cross react with recombinant human NRTN (rhNRTN) or ARTN (rhARTN). Each recombinant protein (rhGDNF, rhNRTN or rhARTN) was loaded at 1 ng/lane. In contrast to the temporal changes in GDNF, a constant level of NRTN is detected in eye lysates at all developmental stages tested. This anti-NRTN antibody does not cross react with rhGDNF or rhARTN. (B,C) Immunohistochemical localization of GDNF in E7 eye tissues. Strong immunoreactivity was observed in ciliary muscle (B) as well as lateral and medial rectus muscle located along the lateral sides of the eye (C). (Inset in C) A higher magnification photograph of immunoreactive rectus muscle. Scale bars, 200 µm (B), 500 µm (C).

 


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  		Fig. 5. GDNF and NRTN are secreted from eye tissues during and after target innervation. (A-D) E9 ciliary ganglion explants grown for 2 days in the presence of GDNF (A), GDNF + anti-GDNF antibody (B), NRTN + anti-GDNF antibody (C), or E5 ECM + anti-GDNF antibody (D). Note that anti-GDNF suppresses GDNF-induced neurite outgrowth, but not NRTN-induced neurite outgrowth. Anti-GDNF is able to suppress E5 ECM-induced neurite outgrowth. A and C were immunostained with a TUJ1 antibody and B and D were unstained phase-contrast micrographs. Scale bar, 200 µm. (E,F) Quantitative analysis of in vitro function-blocking effects by anti-GDNF antibody. (E) Anti-GDNF antibody specifically reduces GDNF-induced neurite outgrowth in a dose-dependent manner. E9 ciliary ganglion explants were grown for 2 days in medium containing 10 ng/ml GDNF and different concentrations of anti-GDNF antibody. The length of neurites (± s.e.) extending from treated and untreated ciliary ganglion explants was measured. In another group, explants were grown in medium containing NRTN or CNTF (10 ng/ml, each) with or without anti-GDNF antibody (0.5 µg/ml). The function-blocking effect by the GDNF antibody is specific to GDNF, and this antibody has no effect on NRTN- or CNTF-induced neurite outgrowth. (F) E5 ECM-induced neurite outgrowth is suppressed by co-incubation with anti-GDNF antibody. In contrast, the GDNF antibody has no effect on E12 ECM-induced neurite outgrowth. (G,H) Quantitative analysis of in vitro function-blocking effects by anti-NRTN antibody. (G) Anti-NRTN antibody reduces NRTN-induced neurite outgrowth in a specific and a dose-dependent manner. The graph shows the average length of neurite (± s.e.) extended from ciliary ganglion explants that were cultured for 2 days in medium containing 10 ng/ml NRTN and different concentrations of anti-NRTN antibody. The graph also shows the effects of anti-NRTN antibody (0.5 µg/ml) on neurite outgrowth induced by GDNF or CNTF (10 ng/ml, each). (H) Anti-NRTN antibody has little effect on E5 ECM-induced neurite outgrowth, but decreases E12 ECM-induced neurite outgrowth. E9 ciliary ganglion explants were grown in medium containing E5 ECM or E12 ECM with or without anti-NRTN antibody. Each bar represents data from 8-14 explants. **, Significantly different from the 0 µg/ml group (P<0.01).

 


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  		Fig. 6. GDNF is required for target innervation of ciliary ganglion neurons. (A,B) An E7 chick ciliary ganglion stained with Toluidine Blue (A) or anti-Ret antibody (B). The ciliary ganglion is located near the optic nerve and extends its axons in the distal direction. The majority of neurons in the ciliary ganglion at this stage are Ret positive. (C,D) DiI labeling of ciliary ganglion pioneer axons in an E8 chick embryo that was grown for 4 days in vivo in the absence (C) or presence (D) of a function-blocking antibody to GDNF. Growing axons and branches usually seen in control embryos (arrowheads in C) were totally suppressed in embryos that were grown with the exogenously applied GDNF antibody (D). (E,F) Transverse sections of part of the head of chick embryos grown in the presence of the GDNF antibody. (E) Several neurons in the ciliary ganglion exhibit shrinkage of their cell body (arrowheads). (F) Vestibular ganglion axons (arrows) show normal innervation patterns and extend their processes into the vestibular epithelium. Scale bar (shown in A): A,B, 200 µm; C,D, 400 µm; E, 40 µm; F, 250 µm. CG, ciliary ganglion; VG, vestibular ganglion; SE, sensory epithelium.

 


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  		Fig. 7. Schematic diagram showing developmental changes in the synthesis of GDNF, NRTN and CNTF proteins in the chick embryonic eye. The darkest area indicates the highest protein level. The data on GDNF and NRTN are from the present study, whereas the data on CNTF are from Leung et al. (Leung et al., 1992) and Finn and Nishi (Finn and Nishi, 1996).

 

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