Sd and Yki are required for AM dedifferentiation. (A) Schematic depiction of AM lineage reprogramming during metamorphosis. After onset of metamorphosis, Org-1 and Tup expression is initiated specifically in the three anterior pairs of AMs, thus triggering dedifferentiation of the syncytial AMs into mononucleate AMDCs. Each of the mononucleate AMDCs serves as a muscle founder cell, fuses with fusion-competent myoblasts (FCMs), and forms a syncytial muscle cell. The newly formed syncytia migrate to the ventral anterior part of the heart tube and differentiate into the VLM. (B and C) Ex vivo live imaging of dissected pupae carrying the org-1-HN18-RFP (org-1-RFP) and hand-GFP reporter constructs. In larval stage L3, org-1-RFP expression is seen in the first three pairs of AMs (B; arrows) that initiate dedifferentiation in pupal stage P4 (C) and fragment into mononucleated progenitor-like cells, AMDCs (arrows). (D) org-1-RFP drives reporter expression in the VLM attached to the heart of a pharate adult stage P15. (E and F) Induction of CRISPR in the AMs with HN39-org-1-GAL4 (org-1-GAL4) against sd (org-1>>Cas9; t::gRNA-sd^4x; E) or yki (org-1>>Cas9; t::gRNA-yki^4x; F) blocks VLM formation and prevents AM fragmentation during metamorphosis into pharate adult stage P15 (arrows). (G and H) Visualization of the org-1-RFP lineage marker and GFP-tagged versions of Sd (Sd::GFP) and Yki (Yki::GFP). Sd::GFP can be detected at pupal stage P3 in the nuclei (arrowheads) of the syncytial AM (G and G′; arrow) whereas Yki::GFP can be detected during induction of AM fragmentation in the nuclei (H and H′; arrowheads) of the forming AMDCs (arrows) during pupal stage P4. Scale bars in A–E: 100 µm; F and G: 10 µm. Actin is visualized with phalloidin; DNA is visualized with DAPI.