Fig. 2
NK2R-HA induced N signalling is mediated ligand and Kuzbanian independent. A–C’ Expression of N-HA and NK2R-HA in Dl rev10 Ser RX82 double mutant cell clones in late third instar wing imaginal discs. Double mutant cell clones are indicated by the absence of RFP. N-HA and NK2R-HA were expressed under control of ptc-GAL4 at the A/P boundary. N signalling is detected by the expression of NRE-GFP. The clone boundaries in (A’, B’, C’) are outlined by the stippled yellow lines. A, A’ Endogenous N signalling along the D/V boundary of the wing is abolished in double mutant cell clones (arrow). Due to relief of cis-inhibition, N signalling is induced in the Dl Ser double mutant cells at the clone boundary (arrowhead). B, B’ Expression of N-HA is not sufficient to rescue the loss of N signalling in double mutant cell clones at the D/V boundary (arrow). C, C’ In contrast to N-HA, NK2R-HA induces strong ectopic N signalling in Ser Dl double mutant cell clones, indicating that it acts in a ligand-independent manner (arrows). D–F’ MARCM clones expressing N-HA or NK2R-HA in kuz ES24 mutant cells. The kuz ES24 clones are indicated by the absence of arm-lacZ (see also M&M) and the clone boundaries in D’, E’, F’ are outlined by the stippled yellow lines. Expression of the constructs is indicated by RFP. Area magnified in E’ and F’ are highlighted by the white boxes in E, F. D, D’ N signalling at the D/V boundary is abolished in kuz ES24 mutant cell clones (arrow). E, E’ N-HA was unable to induce ectopic N signalling in mutant cell clones (arrow), indicating that N-HA activation depends on the function of kuz. F, F’ In contrast, expression of NK2R-HA induced strong expression of NRE-GFP in in the absence of kuz function. G–H’ Expression of N-HA and NK2R-HA in Psn C1 mutant wing imaginal discs. The variants were expressed under control of ptc-GAL4. G, G’ Psn C1 mutant wing imaginal discs display a loss of expression of the target Wg along the D/V boundary (arrow in G). Moreover, the inner ring-like domain of Wg expression (arrowhead in G) is reduced to a spot or is even absent (arrowhead in G’). Ectopic expression of N-HA (H, H’) or NK2R-HA (I, I’) did not induce Wg expression at the D/V boundary in the absence of Psn function, indicating the requirement of S3 cleavage for their activation of the Notch pathway