Figure 7. Interaction with glypicans is required for cytoneme stabilization by Ihog in Hh-receiving cells.

(A, B, C) Ihog-RFP labelled cytonemes arising from the A compartment cells (A, dpp.LexA>LexAop.ihog-RFP / tub.Gal80^ts) are dependent on the glypicans Dlp (B, dpp.LexA>LexAop.ihog-RFP / tub.Gal80^ts, hh.Gal4>UAS.dlp-RNAi) and Dally (C, dpp.LexA>LexAop.ihog-RFP / tub.Gal80^ts, hh.Gal4>UAS.dally-RNAi) levels in the P compartment (arrows). All larvae were grown 30 hr at the restrictive temperature before dissection. (D) A dally³² dlp²⁰ FRT2A double mutant clone (absence of GFP) induced in the P compartment and touching the A/P compartment border in a wing disc that expresses ihog-RFP in the receiving cells (ptc.Gal4, tub.Gal80^ts>UAS.ihog-RFP) to visualize cytonemes. Note the loss of cytoneme visualization crossing the clone (arrowhead). (E) Endogenous Dlp is accumulated in A compartment cytonemes expressing Ihog-RFP (ptc.Gal4, tub.Gal80^ts>UAS.ihog-RFP) after 30 hr at the restrictive temperature (arrows). (F) Wing disc showing that the endogenous Dlp accumulated in A compartment cytonemes belongs to the P compartment cells (arrows), since Dlp has been knocked down in the A compartment (ptc.Gal4, tub.Gal80^ts>UAS.ihog-RFP>UAS-dlp-RNAi) after 30 hr at the restrictive temperature before dissection. The data shown were consistent in at least three independent experiments with an average of 5–10 discs in each experiment. Bars, 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.24045.023

Figure 7—figure supplement 1. Trans interaction with glypicans is required for cytoneme stabilization by Ihog in Hh-producing cells.

(A, B, C) Ihog-RFP labelled cytonemes arising from the P compartment cells (A, hh.LexA>LexAop.ihog-RFP / tub.Gal80^ts) are dependent on the glypicans Dlp (B, ptc.Gal4>UAS.dlp-RNAi, hh.LexA>LexAop.ihog-RFP / tub.Gal80^ts,) and Dally (C, ptc.Gal4>UAS.dally-RNAi hh.LexA>LexAop.ihog-RFP / tub.Gal80^ts) levels in the A compartment (arrows). All larvae were grown 30 hr at the restrictive temperature before dissection. (D) Graph representing the maximum extent of cytonemes emanating from the P compartment when A compartment cells are mutant either for Dlp or Dally compared with the control wing disc (same genotypes as those show in A, B and C, respectively) (average of 5 discs in 3 independent experiments; error bars represent SDs). Note that the effect on cytoneme abrogation in the P compartment is weaker than in case of cytoneme abrogation in the A compartment (Figure 7). This result is because in this case Ihog is induced during the whole development (hh.LexA is not repressed by Gal80) and the expression of the RNAi to knock down Dlp and Dally is under ptc.Gal4 that is target of the Hh pathway, which is compromised when Ihog is overexpressed in the P compartment (Bilioni et al., 2013). (E) A hh.LexA>LexAop.ihog-RFP wing disc shows the accumulation of the endogenous Dlp in cytonemes extending from P to A compartment (arrows). (F) A hh.Gal4 tub.Gal80^ts >UAS.ihog-RFP>UAS.dlp-RNAi wing disc shows that Dlp accumulated in the P compartment cytonemes belongs to the A compartment cells (arrows), because the endogenous Dlp of the P compartment has been knocked down. Larvae in E and F were for 30 hr at restrictive temperature before dissection. The data shown were consistent in at least three independent experiments with an average of 5–10 discs in each experiment. Bars, 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.24045.024