Fig. 5: TNFR-Wgn forms a complex with FGFR-Btl and regulates its activity.
From: The TNFR Wengen regulates the FGF pathway by an unconventional mechanism
a–c Dorso-lateral views of stage 14/15 embryos stained with dpERK (magenta), DSRF (cyan) and a tracheal marker (green). dpERK accumulates in the nuclei of the tip cell and activates DSRF. In TNFR-wgn mutants more cells accumulate nuclear dpERK, while in TNFR-wgn overexpression no dpERK accumulates in tip tracheal cells. d, e Dorso-lateral views of stage 14/15 embryos stained with DSRF (green) and CBP (trachea, magenta). f, g Dorso-lateral views of two dorsal branches of stage 14 embryos stained to detect FGFR-Btl (green) and DSRF or TNFR-Wgn (magenta). Note the accumulation of FGFR-Btl at the basal membrane (blue arrows in (f)) and in intracellular vesicles (green arrows in (f)). Many FGFR-Btl containing vesicles contain also TNFR-Wgn (white arrows in (g)), but TNFR-Wgn and FGFR-Btl single vesicles are also detected (magenta and green arrows respectively). h, i Co-immunoprecipitation experiments. Western blot using αBtl (upper panels) and αFlag (lower panels) of third-instar larvae salivary gland extracts expressing either TNFR-wgn-Flag and FGFR-btl-GFP (h) or TNFR-wgn-Flag and FGFR-λbtl (i). Extracts were immunoprecipitated using αFlag or a control antibody (αAbd-B). Input corresponds to 5% of the immunoprecipitated material. Note that αBtl recognises two specific bands. j, k In situ PLA experiments. k Tracheal metamere 6 of larval DTs. Note the presence of PLA interactions (magenta) when FGFR-Btl and TNFR-Wgn are expressed in the trachea. j Quantification of PLA signal (number of PLA spots per 400 μm2 region) indicate a significant presence of interactions in the experimental condition (αGFP and αFLAG in btlGal4>btl-GFP; wgn-flag) compared to control conditions (αGFP and αFLAG in btlGal4> wgn-flag; αTrh and αFLAG in btlGal4>btl-GFP;wgn-flag). n, number of DT regions analysed (metameres 5 and 6), in brackets number of larvae analysed. Bars show mean ± SD. ****p < 0.0001, Kruskal–Wallis by ranks followed by Dunn’s multiple comparisons test. l, m Representative example of lateral views of stage 14 embryos showing accumulation of FGFR-Btl (green) and TNFR-Wgn (magenta). n Quantification of the percentage of FGFR-Btl vesicles that contain TNFR-Wgn. n, number of DBs analysed, in brackets number of embryos analysed. Bars show SD of mean. ****p < 0.0001, non-parametric Mann-Whitney two-tailed test. Scale bar: (a–c, f, g) 10 μm; (d, e, k) 20 μm, l, m 5 μm. Source data and details of statistical tests used and p values are provided as a Source Data file.
