Figure 1.
Obst-A and Gasp and their expression.
(a, b) Whole-mount in situ hybridization of wild-type embryos with obst-A (a) and gasp (b) probes. obst-A and gasp mRNA are detected in tracheal cells from stage 13 onward. Note the gaps in obst-A expression correspond to the position of the fusion cells (arrowheads). Scale bar: 25 µm (c) A phylogenetic tree of Obstructor proteins based on ClustalW method. The number in brackets indicates, which subgroup of the Obstructor family (1 or 2) each gene belongs to. (d) Schematic representation of the Obst-A and Gasp protein domains. N-terminal signal sequence is followed by three chitin-binding domains type 2 (CBD2).
Figure 2.
Gasp is the unknown antigen recognized by mAb 2A12.
(a) Wild-type embryo stained with antibodies against the luminal antigen 2A12. (b, c) gasp mutant embryo stained with 2A12 (b) and Gasp (c) antibodies. 2A12 and Gasp staining is not detected in gasp mutant. Scale bar: 25 µm (d–f) Confocal sections of embryonic hindgut of en>gasp embryos labeled with Gasp (d), 2A12 (e) and Verm (f) antibodies. The luminal staining is more pronounced with the Gasp antiserum. Gasp protein expressed in the dorsal part of the hindgut is recognized by Gasp and 2A12 antibodies, but not by the Verm antibody. Scale bar: 10 µm. (g) Western blots of bacterial extracts expressing recombinant Gasp-His and MTf-His. Gasp-His protein is detected by the anti Gasp, mAb2A12 and anti-His antibodies, MTf-His protein is only detected by the anti-His antibody. Expression of both proteins is visualized by Ponceau staining. Empty vector was used as a negative control.
Figure 3.
Gas filling and cuticle defects in obst-A; gasp mutant.
(a–d) Bright field photomicrographs of wild-type (a), obst-A (b), gasp (c) and obst-A; gasp (d) first instar larvae. Refracted light makes the gas filled trachea clearly visible in the wild-type and mutant larvae. The trachea is not gas filled in the double mutant. Arrows indicate collapsed regions in the tubes. Scale bar: 100 µm (e, f) Body size measurement of wild-type, obst-A, gasp and obst-A; gasp first instar larvae. Body length (e) and body width (f) (n = 10). * Indicates the statistical significance (p<0.0001) of the difference between wild-type and each mutant as estimated by a two-tailed distribution unpaired Student’s t-test. (g–j) Dark field of cuticle preparations of wild-type (g), obst-A (h), gasp (i) and obst-A; gasp (j) mutant embryos. obst-A; gasp double mutant shows dilated cuticle. Scale bar: 25 µm.
Figure 4.
obst-A and gasp mutants show defects in luminal tracheal matrix.
(a–d) Confocal microscopy projections of the trachea labeled with a FITC-conjugated chitin-binding probe (ChtB). The filamentous chitin is detected in the wild-type embryos at stage 16 (a). The chitin staining intensity is weaker in the obst-A (b), gasp (c) and obst-A; gasp (d) mutant embryos. (e–h) Optical sections of tracheal tubes stained for Verm. Verm is secreted to the tracheal lumen in wild-type embryos at stage 16 (e). The amount of secreted Verm is reduced in both obst-A (f) and gasp (g) single mutant embryos and obst-A; gasp (h) double mutant embryos. Scale bar: 10 µm.
Figure 5.
Ultrastructure of the tracheal lumen in obst-A; gasp mutants.
(a–c) Transmission electron micrographs of wild-type and obst-A; gasp dorsal trunks at the end of embryogenesis. The wild-type embryos (a) show uniformly distributed chitin rich procuticle. In obst-A; gasp double mutant (b, c) the taenidial folds are irregular and the procuticle is distorted with granular amorphous accumulations (arrow). epc = epicuticle (arrow heads), pro = procuticle layer (double arrows). Scale bar: 0.5 µm.
Figure 6.
Lumen diameter expansion requires Obst-A and Gasp.
(a–d) Confocal microscopy projections of the trachea of wild-type (a–a′′′), obst-A (b–b′′′), gasp (c–c′′′) and obst-A; gasp (d–d′′′) embryos labeled with the apical marker Uninflated (Uif) at stage 16.1. Inserts display y-z projections of the DT tubes from 3 different metameres. a′, b′, c′ and d′ inserts are from the middle of metamere 4, a′′, b′′, c′′ and d′′ from the middle of metamere 5 and a′′′, b′′′, c′′′ and d′′′ from the middle of metamere 6. Scale bar: 10 µm (e) Quantification of tracheal diameter of wild-type, obst-A, gasp and obst-A; gasp embryos at stage 16.1. The graph shows diameter measurements of three metameres: 4, 5 and 6. Number of embryos used for measurement of each genotype n = 6. The y-axis represents the diameter in micrometers. Tube diameter values in all mutant embryos were significantly different (p<0.05) from the wild-type by a two-tailed distribution unpaired Student’s t-test.
Figure 7.
Overexpression of Obst-family members causes tube dilation.
(a–c) Overexpression in wild-type embryos of btl>ANF-GFP (a), btl>Gasp-GFP (b) and btl>Gasp-GFP; Obst-A (c). The simultaneous overexpression of Gasp-GFP and Obst-A in trachea forms tube dilations in dorsal trunk (arrowheads) (c). (d) Quantifications showing the percentage (%) of embryos with dorsal trunk dilations in the following genotypes: btl>ANF-GFP (n = 16), btl>Gasp-GFP (n = 12) and btl>Gasp-GFP; Obst-A (n = 38). Bars show the means of three independent experiments and error bars show the ± standard error of the means. * and ** denote significant differences (p<0.01 and p<0.0001) between the means of btl>Gasp-GFP or btl>Gasp-GFP;Obst-A to the btl>ANF-GFP (control) by a two-tailed distribution unpaired Student’s t-test. Scale bar:10 µm.