Org-1–dependent derepression of Yki is needed for AM lineage reprogramming. (A) Schematic drawings of Hpo and Yki constructs used for forced expression. Asterisks indicate positions of amino acid exchanges. (B) Frequencies of observed VLM differentiation in pharate adult stages P15 in the respective genetic backgrounds. n, number of animals phenotypically classified. Coexpression of Yki^S168A can significantly rescue the phenotypes provoked by forced expression of Hpo^ΔC (n = 62, ***, P ≤ 0.001) as well as the phenotypes induced by RNAi against Org-1 (n = 22, **, P ≤ 0.01) and Tup (n = 52, ***, P ≤ 0.001). (C) Forced expression of phosphorylation-resistant Yki^S168A (org-1>>yki.S168A) with org-1-GAL4 does not provoke a VLM phenotype. (D) Forced expression of the N-terminal part of Hpo containing a functional STE20 kinase domain (org-1>>hpo.ΔC) disrupts VLM formation and AM fragmentation. (E–G) Forced expression with org-1-GAL4 of the N-terminal part of Hpo containing a dead STE20 kinase domain (org-1>>hpo.ΔC.K71R; E), full-length Hpo (org-1>>hpo; F), or an N-terminally truncated version of Hpo (org-1>>hpo.ΔN; G) does not provoke a significant VLM phenotype. (H) VLM formation and AM lineage reprogramming can be partially rescued by coexpression of Yki^S168A in a Hpo^ΔC background (org-1>>hpo.ΔC; yki.S168A). (I and K) Induction of RNAi with org-1-GAL4 against org-1 (org-1>>dsRNA-org-1; I) or tailup (tup; org-1>>dsRNA-tup; K) disrupts VLM formation and AM lineage reprogramming. (J and L) Coexpression of Yki^S168A leads to the partial rescue of the phenotypes induced by the RNAi against either org-1 (org-1>>dsRNA-org-1; yki.S168A; J) or tup (org-1>>dsRNA-tup; yki.S168A; L). Actin is visualized with phalloidin. Scale bars: 100 µm.