Figure 2. IFM innervation precedes tracheal invasion.

(A,B) DLMs at 48 hr APF (A) and 61 hr APF (B). Tracheal cell membranes are labeled by palmitoylated mNeonGreen (green) driven by btl-Gal4. F-actin is labeled with Phalloidin (blue) and neurons with HRP (magenta). Note that at 48 hr APF (A–A’’), when tracheal invasion has just started, the muscle is already innervated with motor neurons. At 61 hr APF tracheae have invaded the myotube (B–B’’). Cross-sections to the right show that most tracheal branches reside on the surface of the myotube at 48 hr APF, whereas tracheal branches are inside the myotube at 61 hr APF. Scale bars: 10 μm (A,B).

Figure 2—figure supplement 1. Mitochondria change their morphology during IFM development and partially enwrap tracheoles.

(A–H) Transmission electron micrographs of sagittal (A–C’,G) and cross-sections (D–F’,H) of DLMs at 32 hr APF (A–D’), 48 hr APF (B–E’) and in adult flies (C–F’). Note that mitochondrial morphology changes from elongated-tubular (48 hr APF) to globular shape in the adult. Cristae become increasingly elaborated as IFM development proceeds (compare A’,B’ to C’). (G,H) Tracheoles are closely associated with mitochondria. The region marked by a dashed line in H) is shown as a close-up (inset). (I) Analysis of the cross-sectional area (in µm²) of mitochondria (black bars) and tracheal lumen (gray bars), respectively, at 32 hr APF, 48 hr APF, and in adult flies. (J,J’) show two consecutive confocal sections demonstrating partial enclosure of a tracheole (autofluorescence in green) by a mitochondrium labeled with anti-ATP5A antibody (magenta). Images were acquired using the Airyscan detector (LSM880) followed by Airyscan processing. (K,K’) Segmentation of tracheal branches (white) and mitochondria (color-coded for volume) in a myotube. (J) shows a projection of the entire z-stack, (J’) shows a projection starting from the plane with the tracheole. Note that mitochondria partially enwrap tracheoles. (L,M) Color-coding of mitochondria for volume (L) or sphericity (M) shows that mitochondria in proximity to tracheal branches do not differ in volume or morphology from mitochondria more distant to tracheal branches. Scale bars: 1 μm (A,B,C’,D,E,F’,G,H), 200 nm (A’,B’D’,E’), 2 μm (C,F), 0.4 μm (J,J’), 2 µm (K,K’), 10 µm (L,M).