Journal: eLife

Article Title: The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

doi: 10.7554/eLife.05491

Figure Lengend Snippet: ( A ) Analysis in third instar larval fillet preparations expressing Ret-mCherry (anti-DsRed immunostaining) in class IV da neurons with ppk-Gal4 > CD8-GFP (anti-GFP). Ret was fairly evenly distributed in low and high order dendrites and the axon (yellow arrow). Scale bar 25 μm. ( B ) S2 cells were co-transfected with mys and Ret and imunostained with specific anti-mys (green) and anti-Ret antibodies (magenta). Mys and Ret colocalization in S2 cells is particularly strong in filopodial tips (arrows). Scale bar 1 μm. ( C ) S2 cells were transfected with V5-tagged Ret and flag-tagged mys with or without mew co-transfection. Mys-flag was immunoprecipitated and Ret interaction was probed by Western blotting with an anti-V5 antibody. The asterisks indicate secondary antibody cross-reactivity with the IP antibody. DOI: http://dx.doi.org/10.7554/eLife.05491.011

Article Snippet: After extensive washing with lysis buffer, the samples were denatured and analyzed on Tris-Acetate gels (Life Technologies) and Western blotting against Ret-mCherry (anti-DsRed, 1:1000, BD Clontech, Mountain Viev, CA) and mys (anti-flag M2, 1:10.000, Sigma).

Techniques: Expressing, Immunostaining, Transfection, Cotransfection, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

doi: 10.7554/eLife.05491

Figure Lengend Snippet: ( A ) Cell surface immunostaining of pHluorin-Ret-mCherry expressed in C4da neurons using ppk-Gal4 . Surface exposed Ret labeled by pHluorin (surface-stained with anti-GFP antibody) showed even labeling of the entire dendritic tree, while the mCherry signal displayed a more granular distribution of cellular Ret. Scale bar: 30 μm. ( B ) Cell surface immunostaining of pHluorin-Ret-mCherry co-expressing mys and mew in C4da neurons using ppk-Gal4 . Cell surface and cellular Ret were partially co-distributed with Mew in dendrites, as evident from fluorescence intensity plots along ( B′ ) low order and ( B′′ ) terminal dendrites. Scale bar 20 μm. ( C ) Quantitative colocalization analysis of Ret and integrins coexpressed in C4da neurons ( ppk-Gal4,CD8-GFP > Ret-mCherry,mys,mew ). Pearson coefficients were calculated for C4da neuron soma and dendrite regions showing a positive correlation of Ret and integrin signals (see ‘Materials and methods’ for details, mean ± SD, n = 5 per genotype). ( D and E ) Colocalization analysis of endogenous Ret and integrins overexpressed in C4da neurons ( ppk-Gal4,CD4-tdGFP > mys,mew ). Endogenous Ret signal was colocalized with ( D ) anti-mys or ( E ) anti-mew immunoreactivity in C4da neurons and colocalized pixels visualized in false color representations (coloc). ( D′ and E′ ) Stretches of terminal dendrites resliced in Z direction showing partial colocalization of ( D′ ) Ret and mys or ( E′ ) Ret and mew along the basal dendrite facing the ECM as indicated by arrows (see schematic drawing). Line intensity plots of the same dendrite portion show signal distribution of endogenous Ret and integrins together with the colocalized signals (coloc) and the CD4-tdGFP membrane marker. ( D′′ and E′′ ) Line intensity plots for a primary dendrite portion (indicated in D and E ). ( F ) Quantitative colocalization analysis of endogenous Ret and integrins expressed in C4da neurons ( ppk-Gal4,CD4-tdGFP > mys,mew ). Pearson coefficients calculated for C4da neuron soma and dendrites showing a positive correlation of endogenous Ret and integrin signals (see ‘Materials and methods’ for details, mean ± SD, n = 5 per genotype). DOI: http://dx.doi.org/10.7554/eLife.05491.010

Article Snippet: After extensive washing with lysis buffer, the samples were denatured and analyzed on Tris-Acetate gels (Life Technologies) and Western blotting against Ret-mCherry (anti-DsRed, 1:1000, BD Clontech, Mountain Viev, CA) and mys (anti-flag M2, 1:10.000, Sigma).

Techniques: Immunostaining, Labeling, Staining, Expressing, Fluorescence, Marker

Journal: eLife

Article Title: The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

doi: 10.7554/eLife.05491

Figure Lengend Snippet: ( A – D ) Maximum projections of C4da neurons visualized with ppk-Gal4 > CD4-tdGFP are shown for ( A ) wildtype and ( B – D ) Ret mutant animals. C4da neuron specific expression of ( C ) UAS-Ret-mCherry or ( D ) UAS-mys/UAS-mew in Ret mutant larvae rescues dendrite crossing defects. However, only re-expression of Ret fully restores dendritic field coverage to wildtype levels. Scale bar 100 μm. ( A′ – D′ ) Magnified view of the indicated dendrite area for the different genotypes. Dendrite crossing points are indicated by arrowheads. Note that Ret-mCherry or integrin expression in Ret mutant C4da neurons strongly reduced dendrite crossing events. Scale bar 30 μm. ( E ) Quantitative analysis of out of plane dendrite crossing for the indicated genotypes. Overexpression of Ret or integrins specifically in C4da neurons of Ret mutant animals fully rescues dendrite crossing defects (p < 0.001, n = 5) ( F ) Dendrite coverage index (ratio of dendrite field area and segment area ) is shown for the indicated genotypes. Defects in receptive field coverage of Ret mutant C4da neurons are rescued by C4da specific expression of the Ret-mCherry transgene, but not integrin overexpression (mean ± SD, p < 0.001, n = 5, Mann–Whitney U-test). DOI: http://dx.doi.org/10.7554/eLife.05491.007

Article Snippet: After extensive washing with lysis buffer, the samples were denatured and analyzed on Tris-Acetate gels (Life Technologies) and Western blotting against Ret-mCherry (anti-DsRed, 1:1000, BD Clontech, Mountain Viev, CA) and mys (anti-flag M2, 1:10.000, Sigma).

Techniques: Mutagenesis, Expressing, Over Expression, MANN-WHITNEY

Journal: eLife

Article Title: The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

doi: 10.7554/eLife.05491

Figure Lengend Snippet: Integrin co-expression suppresses Ret induced photoreceptor degeneration. Overexpression of Ret with GMR-Gal4 caused a rough eye phenotype and pigmentation defects, which can be fully rescued by co-expression of integrins. Co-expression of Ret-mCherry and Mys-GFP could be readily detected by their fluorescent tag confirming the presence of both proteins in the rescued eye. Note that although Ret dependent photoreceptor phenotype was rescued, pigmentation defects observed upon Ret overexpression were not. DOI: http://dx.doi.org/10.7554/eLife.05491.008

Article Snippet: After extensive washing with lysis buffer, the samples were denatured and analyzed on Tris-Acetate gels (Life Technologies) and Western blotting against Ret-mCherry (anti-DsRed, 1:1000, BD Clontech, Mountain Viev, CA) and mys (anti-flag M2, 1:10.000, Sigma).

Techniques: Expressing, Over Expression

Journal: eLife

Article Title: The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

doi: 10.7554/eLife.05491

Figure Lengend Snippet: ( A ) Analysis in third instar larval fillet preparations expressing Ret-mCherry (anti-DsRed immunostaining) in class IV da neurons with ppk-Gal4 > CD8-GFP (anti-GFP). Ret was fairly evenly distributed in low and high order dendrites and the axon (yellow arrow). Scale bar 25 μm. ( B ) S2 cells were co-transfected with mys and Ret and imunostained with specific anti-mys (green) and anti-Ret antibodies (magenta). Mys and Ret colocalization in S2 cells is particularly strong in filopodial tips (arrows). Scale bar 1 μm. ( C ) S2 cells were transfected with V5-tagged Ret and flag-tagged mys with or without mew co-transfection. Mys-flag was immunoprecipitated and Ret interaction was probed by Western blotting with an anti-V5 antibody. The asterisks indicate secondary antibody cross-reactivity with the IP antibody. DOI: http://dx.doi.org/10.7554/eLife.05491.011

Article Snippet: Primary antibodies used were: rabbit anti-DsRed (1:500, BD Clontech), mouse anti-mew (1:100, DSHB), mouse anti-mys (1:100, DSHB).

Techniques: Expressing, Immunostaining, Transfection, Cotransfection, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

doi: 10.7554/eLife.05491

Figure Lengend Snippet: ( A ) Cell surface immunostaining of pHluorin-Ret-mCherry expressed in C4da neurons using ppk-Gal4 . Surface exposed Ret labeled by pHluorin (surface-stained with anti-GFP antibody) showed even labeling of the entire dendritic tree, while the mCherry signal displayed a more granular distribution of cellular Ret. Scale bar: 30 μm. ( B ) Cell surface immunostaining of pHluorin-Ret-mCherry co-expressing mys and mew in C4da neurons using ppk-Gal4 . Cell surface and cellular Ret were partially co-distributed with Mew in dendrites, as evident from fluorescence intensity plots along ( B′ ) low order and ( B′′ ) terminal dendrites. Scale bar 20 μm. ( C ) Quantitative colocalization analysis of Ret and integrins coexpressed in C4da neurons ( ppk-Gal4,CD8-GFP > Ret-mCherry,mys,mew ). Pearson coefficients were calculated for C4da neuron soma and dendrite regions showing a positive correlation of Ret and integrin signals (see ‘Materials and methods’ for details, mean ± SD, n = 5 per genotype). ( D and E ) Colocalization analysis of endogenous Ret and integrins overexpressed in C4da neurons ( ppk-Gal4,CD4-tdGFP > mys,mew ). Endogenous Ret signal was colocalized with ( D ) anti-mys or ( E ) anti-mew immunoreactivity in C4da neurons and colocalized pixels visualized in false color representations (coloc). ( D′ and E′ ) Stretches of terminal dendrites resliced in Z direction showing partial colocalization of ( D′ ) Ret and mys or ( E′ ) Ret and mew along the basal dendrite facing the ECM as indicated by arrows (see schematic drawing). Line intensity plots of the same dendrite portion show signal distribution of endogenous Ret and integrins together with the colocalized signals (coloc) and the CD4-tdGFP membrane marker. ( D′′ and E′′ ) Line intensity plots for a primary dendrite portion (indicated in D and E ). ( F ) Quantitative colocalization analysis of endogenous Ret and integrins expressed in C4da neurons ( ppk-Gal4,CD4-tdGFP > mys,mew ). Pearson coefficients calculated for C4da neuron soma and dendrites showing a positive correlation of endogenous Ret and integrin signals (see ‘Materials and methods’ for details, mean ± SD, n = 5 per genotype). DOI: http://dx.doi.org/10.7554/eLife.05491.010

Article Snippet: Primary antibodies used were: rabbit anti-DsRed (1:500, BD Clontech), mouse anti-mew (1:100, DSHB), mouse anti-mys (1:100, DSHB).

Techniques: Immunostaining, Labeling, Staining, Expressing, Fluorescence, Marker