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Fig. 1. Tracheal luminal phenotype of tendrils mutations. (A,B) Dorsal and lateral views of third instar larval tracheal system (Rühle, 1932). Positions of lateral trunk (LT), dorsal trunk (DT), dorsal branch (DB), fat body branch (FB) and visceral branch (VB) are indicated. Anterior is leftwards. Dorsal is upwards in B. (C) Fluorescence micrograph montage of two DBs and their terminal cells (corresponding to boxed region in A) in mosaic third instar larva. Tracheal system is labeled with DsRed; DB terminal cell clone on the right is marked with GFP. Each terminal cell forms over a dozen terminal branches that attach tightly to the underlying muscles. s, DB stalk connecting DB to DT. f, fusion branch formed by individual cell connecting DB to contralateral DB. Arrowhead, DB terminal cell nucleus. Broken line indicates continuation of DB outside focal plane. (D) Schematic of terminal cell with multiple terminal branches (cellular projections), each with a membrane-bound lumen coursing through it. (E) Wild-type (tendrils⁺) DB terminal cell clone induced in 2- to 4-hour-old embryo marked with cytoplasmic GFP and examined in wandering third instar larvae. (F) Fluorescent (left; `cytoplasm'), bright-field (middle; `lumen') and merged (right) images of boxed region in E. There is a single lumen (GFP-excluded region in left panel, refractile region in middle panel) in the center of each branch. (G) Similar view of a homozygous tendrils^6-66 DB terminal cell clone. Multiple convoluted lumens of different sizes are present in a single thick branch. (H-J) TEM analysis of tendrils⁺ DB terminal cell clone induced as above and marked with GFP and CD2-HRP. (H) Clone visualized by DAB staining prior to sectioning. Broken line indicates approximate sectioning plane. (I) Section through terminal branch of the clone. An electron dense layer of DAB staining is present at the plasma membrane. A single lumen (red dot) is present in cross section through branch. tc, terminal cell; m, muscle. (J) Higher magnification of the lumen in I. (K-M) Similar analysis of a tendrils^- (rhea⁷⁹) clone. More than 25 lumen cross-sections are visible in L, all in the same terminal branch (as shown by absence of basal plasma membrane between lumens in M). (N) Fluorescence (left) and bright-field (right) images of tendrils^13-8 DB clones (marked with GFP) that include one of the two terminal cells shown (arrow) as well as neighboring fusion (f) and stalk (s) cells. Only the mutant terminal cell (arrow) is affected. Mutant stalk and fusion cells, and neighboring tendrils⁺ terminal cell (arrowhead) are indistinguishable from wild type. (O) tendrils^13-8 fat body (FB) terminal cell clone as in N. Multiple convoluted lumens are present in single terminal branch, similar to DB terminal cell clones in G and N. Scale bars: 100 µm in C; 25 µm in E; in G, 25 µm for F,G; 25 µm in N,O; in I, 2 µm for I,L; in J, 0.5 µm for J,M.

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