Fig. 3. knk and rtv are required for tracheal luminal chitin filament assembly. (A-E) Early stage 15 embryos double labelled with luminal antibody 2A12 (red; top panel) and FITC-conjugated chitin-binding probe (CBP; green, bottom panel), where analogous DT segments from each genotype are shown. All embryos were fixed and labelled in parallel and the confocal images taken in the same session with identical settings to display comparative levels of luminal chitin. (In the middle merged panel, colour levels are adjusted to enable visualization of both 2A12 and CBP labelling.) (A) In wild-type embryos, CBP labels a luminal chitinous fibre with `threads' running parallel to tube length. The merged image shows that the CBP-labelled fibre is confined to only a part of the lumen, leaving narrow gaps to the surrounding epithelium. (B) The trachea of wild-type embryos laid by Nikkomycin-fed parentsdisplay kkv phenotype and barely detectable luminal CBP levels. (C,D) In both knk^7A69 (C) and rtv¹¹ (D) mutant trachea, CBP labelling reveals a broad chitinous matrix, which fills the entire lumen. (E) The intensity of this CBP labelling is weaker than in the wild type, but stronger than in embryos with reduced CS-1 activity upon treatment with a low Nikkomycin dose (0.5 mg/ml) to reproduce the kkv and rtv mutant phenotypes. Scale bars: 5 µm in A-E.