Figure 1.
Design of tracheal mutant screen.
(A) Diagram of Drosophila tracheal system in third instar larva (dorsal view, anterior up unless noted otherwise). A close up of two hemisegments (Tr4 and Tr5) are shown at right, with some primary branches indicated. DT, dorsal trunk; DB, dorsal branch; LT, lateral trunk. (B) Schematic showing cellular structure of dorsal trunk and dorsal branch. Dashed lines indicate plane of section of cross-sections shown. DT is a multicellular tube with multiple cells and intercellular junctions seen in cross-section. DB stalk is an autocellular tube, a single cell wrapped around the luminal space and sealed by an autocellular junction. DB terminal cell (TC) forms multiple terminal branches, each of which is a “seamless” tube lacking junctions. The base of the terminal cell (*), from its junction with a stalk cell to the nucleus, is an autocellular tube. The fusion joint (FJ) is the position where two fusion cells, each of which forms a seamless tube, connect contralateral tracheal hemisegments. Lum, tracheal lumen (black); Jxn, intercellular junctions (red); Nuc, cell nuclei (black). (C) Fluorescence micrograph of two sibling F3 larvae from the F3 screen diagrammed in panel E. The GFP- larva at left is heterozygous for the mutagenized third chromosome; it is nearly invisible because it contains, in trans to the mutagenized chromosome, a Gal80-expressing balancer chromosome that prevents expression of btl-Gal4, UAS-GFP (btl>GFP). The GFP+ larva at right is homozygous for a mutagenized third chromosome; it lacks the Gal80 chromosome, so expresses GFP throughout the tracheal system. (D) Fluorescence micrograph of a segment (Tr5) of the tracheal system from a third instar larva generated by the genetic mosaic strategy shown in panel F. All tracheal cells express btl>DsRED (red); homozygous clones lack the UAS-GFP(RNAi), so express in addition btl>GFP (green). Dorsal trunk (DT), dorsal branch (DB) and terminal cell (TC) clones are marked (arrowheads). Dorsal branch fusion joint (FJ) connecting the left and right hemisegments is indicated. (E) Genetic scheme of F3 screen. EMS, ethyl methanesulfonate; Pr, Prickly; P[hs-hid], heat shock inducible hid transgene; TM3, third chromosome balancer; P[Gal80], transgene with ubiquitous tubulin promoter driving expression of Gal80, a Gal4 inhibitor; 2FRT, two Flp Recombinase Target (FRT) site transgenes (FRT2A on 3L and FRT82B on 3R) flanking the third chromosome centromere; Sb, Stubble; *, mutagenized chromosome. (F) Genetic scheme of the mosaic screen. hs-FLP, heat-inducible FLP recombinase transgene; UAS-GFP(RNAi), Gal4-inducible (Gal4 upstream activating sequence) GFP RNAi transgene.
Table 1.
Tracheal morphogenesis mutant collection.
Figure 2.
Tracheal cell selection/specification mutants.
(A, B) Lateral views (anterior left) of a portion of the lateral tracheal trunk (between two transverse connectives) of genetic mosaic third instar larvae with control wild-type clones (A) and homozygous no terminal cell clones-3L clones (B). All tracheal cells express DsRed (red) and tracheal clones also express GFP (green) so appear yellow. Terminal cell clones (arrowheads) are present in A but absent in B. (C–H) Portions of the tracheal system of wild type control and mutant third instar larvae homozygous for the mutations indicated. (C, D) Lateral views (anterior left) of wild type (C) and missing parts mutant (D). Tracheae are labeled with GFP (white). Positions of two normal terminal cells (arrows) and a lateral trunk (LT) fusion joint (arrowhead) are indicated in C. In D, the corresponding terminal cells and LT fusion joint are missing (*), with broken ends of LT indicated by white dots. (E, F) Dorsal views of distal ends of a pair of dorsal branches labeled with GFP (white) in wild type (E) and steeple mutant (F). Note terminal cells (arrowheads in E) are missing (*) in steeple mutant (F). (G, H) Dorsal view of posterior of wild-type (G) and loose caboose mutant (H) with tracheae labeled with GFP (white). Arrowhead, position where contralateral dorsal branches (Tr10) connect to form the DB10 fusion joint (G). DB10 fusion joint is missing (*) in H; in the absence of the fusion joint, the positions of the disconnected parts of the tracheal system are more variable. Open circles, posterior spiracles.
Figure 3.
Micrographs (top panels) and schematics (lower panels) of genetic mosaic third instar larva showing terminal cell (TC, A–E) and dorsal trunk (DT, A'–E') clones (GFP+, green; at right) of control wild type (A, A'), miracle-gro338 (B, B'), lotus312 (C, C'), sprout574 (D, D'), and cincher773 (E, E') cells. In A–E, a contralateral control heterozygous terminal cell (DsRED+, red; at left) is included for comparison. The maximal soma cross-sectional area of miracle-gro338 terminal cell clones (0.87±0.05 units in Image J (mean±SEM), n = 10 clones) was four-fold greater than that of wild type control terminal cell clones (0.22±0.03 units). Extra branches in the miraclo-gro clone are highlighted in Figure 4C/4C'. Bar, 50 µm (A–E), 10 µm (A'–E').
Figure 4.
Terminal cell branching mutants.
Fluorescence (A–F) and brightfield (A'–F') images of homozygous terminal cell clones (DsRED+, GFP+ so appear yellow in A–F) of the mutations indicated, with schematics of the phenotypes shown below. Open boxes, area enlarged in insets. (A, A') Control wild type clone. There are dozens of terminal branches (A), and each mature branch contains a single, continuous gas-filled lumen (A'). New terminal branches arise from filopodial growth cones (A, inset). (B, B') winded1508 clone. Note absence of terminal branches. (C, C') miracle-gro1483 clone. Note enlarged branches and multiple convoluted seamless tubes in enlarged soma (C', inset). (D, D') burs1139 clone. Note presence of first generation terminal branches but absence of most second and all subsequent generations. (E, E') oak gall696 clone. Note all but one terminal branch is missing, and remaining branch is short and stout (arrowheads). Another phenotype is the tiny gap in the gas-filled lumen at or near the position where autocellular and subcellular tubes connect in terminal cell (E', inset; compare to inset in A'). (F, F') spikes773 clone. Note excess filopodia arising from terminal branches (F, inset) but normal or slightly reduced numbers of mature terminal branches (F'). Bar, 20 µm.
Figure 5.
Fluorescence photomicrographs of control wild type (A, G, I) and homozygous mutant (B–F, H, J, K) clones in seamless, autocellular, and multicellular tracheal tubes in third instar larvae. Schematics of the phenotypes are diagrammed below. Clones are marked with GFP (white in A–F, green in G–K) and all tracheal cells with DsRED (red in G–K); brightfield images in I'–K' show air-filled lumens of multicellular tubes. (A) Wild type control clone in seamless tube. (B) whacked220 clone. Note most of the lumen is missing and the terminus of the residual lumen (arrowhead) is dilated and irregularly shaped. (C) moon cheese1524 clone. (D) wavy lumens894 clone. (E) cystic lumens1243 clone. (F) black hole538 clone. The regions where the lumen appears to be dilated (e.g., boxed area, upper inset) are actually regions in which a vacuole, which can be distinguished from the lumen by its accumulation of lumGFP (not shown), intimately surrounds a lumen of normal diameter (lower inset, brightfield view of boxed area). The vacuole is outlined in red in schematic. (G) Wild type control clone in autocellular tube. The single marked cell (GFP+, green) surrounds the lumen, sealed by an autocellular junction. (H) conjoined356 clone. The mutant cell (GFP+, green) does not form an autocellular junction but instead forms the lumen by making intercellular junctions with a heterozygous cell (DsRED+, red). (I) Wild type control clone in dorsal trunk, a multicellular tube. (J) bulgy636 clone. Lumen bulges outward into mutant cell, forming a local dilatation. (K) constricted960 clone. Lumen constricts inward at site of mutant cell by ∼7% relative to the neighboring, fully wild type dorsal trunk segments. Bar, 5 µm (A–F), 10 µm (G,H), 10 µm (I–K).
Figure 6.
Lumen clearance and gas-filling mutants.
Fluorescence (A–G) and bright field micrographs (A'–G') of control wild type (A, F) and homozygous mutant clones (B–E, G) in seamless and autocellular tracheal tubes as indicated. Clones are labeled with cytoplasmic DsRed (red) and also express lumGFP (green), a secreted form of GFP; the fluorescence micrographs (A–G) are DsRed/lumGFP merged images, except for E, which shows only the lumGFP channel (white). Lumen defects are diagrammed below, with air-filled lumens in white and matrix-filled lumens and tracheal cell cytoplasm in grey. (A, A') Wild type control terminal cell. lumGFP has been cleared from the mature, gas-filled lumen (A'). The only lum-GFP visible is small puncta in the cytoplasm at the tip (A, arrowhead). (B, B') impatent1757 clone. This is a mutant, like those described in Figure 5, in which the seamless lumen is missing (B'): lumGFP is detected only in puncta (B, arrowheads), presumably aberrant intermediates in lumen formation, distributed in the soma and along the lumenless terminal branch. (C, C') ichorous206 clone. Although no mature, gas-filled lumen is detected by brightfield optics (C') as in impatent mutant cells, a lumen has formed–just not cleared–as shown by luminal lumGFP staining (C). (D, D') littoral762 clone. A specialized clearance defect: the central terminal branch forms a normal gas-filled lumen but the tips of growing side branches (brackets) contain a lumen that has not cleared (D') and remains loaded with lumGFP (green, D). (E, E') lotus312 clone. Another specialized clearance defect, restricted to the junction between (arrowheads) the base of the branch (connection with stalk cell) and the seamless tube. The fluorescence signal in E above and below the arrowheads is autofluorescence of the cuticle, not lumGFP. (F, F') Control wild type autocellular tube. (H, H') asthmatic1530 clone. Lumen is difficult to detect (G') because it remains filled with luminal matrix and lumGFP (green, G). Bar, 5 µm (in C, A–E), 10 µm (F,G).
Table 2.
Molecular identification of tracheal genes.
Figure 7.
Genetic analysis of terminal cell growth control pathway.
(A–D) Close-ups of the soma of third instar larva terminal cell clones of the indicated genotypes. Terminal cell cytoplasm is marked with GFP (green) and nuclei in A–C are marked with nuclear DsRed2 (red). Note that the cell body and nucleus of the miracle-gro(warts)388/388 clone (B) and the clone expressing λ-Breathless (C), a constitutively active form of Breathless FGFR, are enlarged with ectopic lumens coursing through the soma. By contrast, the soma of the miracle-gro(warts)388/388 clone in a larva homozygous for blistered l(2)3267, a downstream transcription factor in the Breathless pathway (D), is smaller and there are no ectopic lumens (black asterisk). However, the single, truncated lumen of the clone is dilated compared to the truncated lumen of the contralateral control terminal cell (white asterisk). (E) Genetic pathway of terminal cell growth control. Bar, 20 µm.
Figure 8.
Genetic dissection of terminal branch morphogenesis.
The major, genetically separable processes in the terminal branching program are illustrated, in the order in which they occur, along with representative mutations that disrupt them. There is an initial patterning step (Selection/Specification) that selects and specifies the terminal cell, followed by five morphogenesis (Branching, Growth, Tubulogenesis) and maturation (Clearance/Gas-Filling, Maintenance) steps. The steps can be functionally subdivided further by the more specific phenotypes of the mutants shown. Where the molecular identities of the genes are known, the protein products are given (in parentheses) to indicate some of the molecular functions involved in each step. The SRF transcription factor Blistered (Pruned), a key regulator of terminal branching and the last gene in the Selection/Specification step, presumably controls expression of at least some of the downstream morphogenesis and maturation genes including ones involved in growth and tubulogenesis (Figure 7E).