FIB/SEM visualization reveals an extensive flat interface between DLM myotubes and associated myoblasts. (A) Schematics of an early pupa (left) and an adult fly (right) illustrating the position and relative size of the IFMs. A swarm of wing disc–derived myoblasts (green) fuses with a set of three persistent larval muscles in each pupal hemithorax, which go on to split and mature into a set of six DLMs. (B) A set of six nascent DLMs (asterisks) at 20 h APF visualized using mef2-GAL4>UAS-mCD8-GFP (green). DAPI-stained nuclei (blue) fill the syncytial muscles and also mark the positions of the mononucleated myoblasts (m) surrounding the muscle fibers. (C) Low magnification TEM of DLMs at 20 h APF. Mononucleated myoblasts (MBs) surround a syncytial myotube (MT). (D–D”) Reconstruction of a FIB/SEM dataset (see Video 1) of a DLM myotube and associated myoblasts. D displays the semitransparent myotube (green) and neighboring myoblasts (individually colored), whereas D’ shows the same view of the myoblasts alone, where the flattened surfaces of the myoblasts are readily apparent. D” displays the same cells as in D, at the same magnification, but from a different (tilted) angle, revealing a single myoblast (red, marked by an asterisk, and shown on its own in the inset) extending protrusions toward the myotube. (E and E’) Low (E) and high (E’) magnification views of the interface between a DLM myotube (MT) and an associated myoblast (MB), prepared for TEM using the hybrid CF and HPF/FS protocol (see Materials and methods section TEM), which allows for high quality preservation of smooth cell membranes and a rich cytoplasm. n, nucleus. Bars: (B) 20 µm; (C and D”) 10 µm; and (E and E’) 500 nm.