Supplementary Figure 4: staccato mutations affect a late step of the tube fusion process.
(a) Immunostaining of stage 15 embryos labeled with anti-Dys (green in merge) and a probe to detect chitin (magenta). Dys protein is present in FC nuclei in wild-type and stac3B20 mutant embryos. Note the interrupted tracheal lumen in the stac3B20 embryo. Images are representative of six embryos. (b) During contact formation between FCs, E-cadherin (E-cad, cyan), F-actin (magenta) and Short Stop (Shot, yellow) accumulate at the FC-FC interface. All three markers are present and correctly localised in wild-type and stac3B20 mutant embryos. Images are representative of >10 embryos from two experiments. (c) A cytoskeletal track (visualised by α-Tubulin staining, green) extends in the central region of each FC along the luminal axis in wild-type and in stac3B20 mutant embryos. Images are representative of >10 embryos. (d,e) The FC-specific zona pellucida protein CG13196 (green) is localised to FC apical membranes during late fusion. After fusion, in wild-type DT (d) and DB (e) FCs, CG13196 is detectable as rings surrounding the newly connected lumen. In stac3B20 embryos CG13196 is present on the primary apical membranes and surrounding isolated pockets of luminal material at FC-FC interfaces (white arrowheads), indicating that apical trafficking of CG13196 (and of Uif, see Fig. 1) is not perturbed. Images are representative of 10 embryos. (f) Arf-like 3 (Arl3, green) is specifically expressed in FCs and localises to puncta in wild-type and in stac3B20 mutant FCs. Images are representative of nine embryos from two experiments. In panels b,c and e the FC-FC interface is indicated by a white arrowhead. In panels c and f deconvolution was applied. Scale bars, 10 μm.
