Fig. 3: 20HE induces Wnt/Wg ligand expression in EC and consequently Wnt/Wg signalling activity in ISC/EB.

A–D’ Confocal images of GFP-tagged Wingless ligand (wg::GFP) in R5 regions of midguts from (A–A’) MF fed with MeOH Control compared to (B–B’) MF fed with RH5849, and (C–C’) w¹¹¹⁸ control flies compared to (D–D’) dominant ovo^D1 MF. A–D Sole Wg::GFP signal is shown in greyscale and (A’–D’) in green colour combined with αArm and αPros to identify ISC/EB,EE and EC. Exemplary measured EC are outlined by white dashed lines. Scale bar is 20 µm. E–H’ R5 regions of MF midguts with RFP expressed under control of the fz3 promotor (fz3-RFP) showing Wnt/Wg activity in (E–E’) MF fed with MeOH Control compared to (F–F’) MF fed with RH5849, and (G–G’) w¹¹¹⁸ control flies compared to (H–H’) dominant ovo^D1 MF. Exemplary ISC/EB are marked by white arrows. Scale bar is 20 µm. I–J Wg::GFP intensities in EC and fz3-RFP intensities in ISC/EB upon (I) RH5849 and (J) ovo^D1. I–J Scatter dot plots show individual values with indication of means ± SD. N and n values represent number of cells and number of biological replicas, respectively. Asterisks denote significances from two-sided Mann-Whitney U tests (****p < 0.0001). Fold changes are shown in percentages. Source data are provided as a Source Data file. K Schematic of paracrine Wnt/Wg activation in ISC/EB upon 20HE increment. RH5849 is incorporated into EC from the midgut lumen, whereas 20HE from surrounding haemolymph is imported into EC by EcI. Within EC 20HE hormone and RH5849 activate expression of Crol and Wg. Wg ligand then non-autonomously activates Wnt/Wg signalling in ISC/EB visualized by fz3-RFP activity.