Figure 1: Phenotype of terminal cells in btszK13-4 mutant larvae and subcellular localization of Btsz.
(a–d) Overview and details of control (a,c) and btszK13-4 mutant (b,d) terminal cells in third instar larvae. (a,b) The terminal cell nuclei are visualized by immunostaining with antibodies against SRF (red) and the cells are labelled with cytoplasmic GFP (green). btszK13-4 mutant terminal cells have fewer or no branches compared with control. (c,d) High magnification details of terminal cells shown in inverted colour or grey scale for better clarity. (c) In the control cell, a single lumen is present in each branch. (d) Example of a mutant cell containing many parallel, thin lumens emanating from a larger lumen. (e,f) Subcellular localization of Btsz. Single focal plane details of terminal branches expressing cytoplasmic GFP and tagged Btsz proteins, visualized by staining with antibodies against the tags. The connections of the part of the cell shown in the image to the rest of the cell is in a different focal plane. Btsz2-Glu (e) has the C2 domains and localizes to the apical membrane. Btsz2-ΔC2-HA (f) lacks the exons encoding the C2 domains and the encoded protein is distributed throughout the cytoplasm. The staining seen outside the terminal branch in e is background staining by the Anti-Glu antiserum, which is also seen in the absence of the Btsz2-Glu transgene. Images (a–d) are projections of confocal image stacks and (e,f) are single focal planes. Scale bars: (a,b) 50 μm and (c–f) 10 μm. (g) Terminal cell branching during larval development. Larvae expressing UAS-GFP btl-Gal4 were analysed at the indicated ages. The number of branches in dorsal terminal cells in tracheal segments tr2 to tr9 were counted (n>10 for each genotype and time point, error bars represent s.d.). GFP, green fluorescent protein.
