Cell proliferation and programmed cell death in Tr2 trachea during L3. (A) Scaled drawings of the tracheal branches in Tr2 depicting the distribution of cells. (B–K) Wing discs and associated Tr2 tracheal branches. Anti-activated Drice (23) (B–F) and TUNEL (24) (G–K) staining identified cells undergoing programmed cell death in the wing disc (B–D, H, and I) at all time points tested (hours after L3 molt), but in the transverse connective (B–E, G–J; green nuclei) and dorsal trunk (F and K), signal was detected in only a few cells at 48–50 h. Arrowheads, stained cells in wing disc; arrows, stained tracheal cells.

DNA and centriole content of larval tracheal cells. (A and B) Cells that constitute Tr2 trachea did not incorporate BrdU during L2 (A) but did incorporate BrdU during L3 (B). Cells of the other tracheal metameres that are thought to undergo endoreplication through the larval instars incorporated BrdU in L2 (A), but incorporation during L3 was infrequent (B). Cells of the wing disc (arrows, A and B) incorporated BrdU at all stages that were examined. DT, dorsal trunk; DB, dorsal branch. (C and D) Bright-field images of portions of the dorsal trunk of Tr2 and Tr5 dissected from larva at the L2 to L3 molt. Striations of the taenidial cuticle can be seen in both the smaller L2 (arrowheads) and larger L3 (arrows) tubes. (E) Histograms showing DNA content of L2 (blue) and wandering L3 (red) nuclei in Tr2, Tr3, and Tr5. DNA content in Tr3 and Tr5 increased (Middle and Right) whereas the DNA content of cells in Tr2 did not (Left). The numbers of nuclei examined were 53 (n = 7 animals) for Tr2 L2 and 129 (n = 11 animals) for Tr2 L3, 51 (n = 7 animals) for Tr3 L2, 58 (n = 8 animals) for Tr3 L3, 47 (n = 9 animals) Tr5 L2, and 42 (n = 6 animals) for Tr5 L3. (F–I) Cells in Tr5 lacked Sas4-GFP, a marker for centrioles, both in L2 (G), and L3 (I) animals, whereas cells in Tr2 had Sas4-GFP-containing structures (arrowheads) in L2 and L3 (F and H).

Proliferation of the cells that constitute Tr2 during L3. (A–C) Lineage tracing of tracheal cells in a single larva imaged at three times during L3 reveals that a single larval cell in Tr2 (A, arrow) that had been marked during embryogenesis (see Methods) divided during L3 (B and C, arrows). A larval cell in Tr3 (A–C, arrowhead) that was also marked during embryogenesis did not divide during the same period. Distribution of cells and the marked cells are diagrammed below.