Figure 3: Adherens junctions and role of the MBD of Btsz in terminal cells.

(a,b) E-cadherin (E-cad) in control and in btsz^K13-4 mutant (c) dorsal terminal cells in third instar larvae. Junctions are seen, both in control and mutant larvae, at the site where the terminal cell is joined to the dorsal branch (red arrowheads) and between the two dorsal fusion cells (asterisk) as well as along the length of the unicellular junction in the dorsal branch cell. Compare (a) and (b) for variability in expression of E-cad in control junctions. Nuclei of the terminal cells in the low-magnification panels (a–c) are marked with green arrowheads. E-cad-stained control (d) and btsz^K13-4 mutant (e,f) fusion cells that connect the segmental units of the dorsal trunk in the third instar larva. 4,6-diamidino-2-phenylindole staining to visualize nuclei confirms that two cells are present (see Supplementary Movie 8). (g) Schematic of the btsz transcription unit and two of the alternative transcripts (adapted from Serrano et al.²⁶). The wild type and the btsz^K13-4 genomic regions are represented as lines with the dashed region indicating the deletion in the btsz^K13-4 allele. Boxes below represent the 15 exons of btsz (black) and the organization of exons in btsz transcripts 1 (green) and 2 (blue). (h–j) Ability of btsz transgenes to suppress defects in btsz^K13-4 mutants. The indicated transgenic constructs were expressed in the tracheal system of btsz mutant animals and various aspects of the btsz mutant phenotype were scored. The colours of the bars correspond to those in the diagram above. Genotypes of crosses and number of larvae scored: red: btl-GAL4, UAS-GFP; btsz^K13-4/TM6B, Tb (n=17). Green: btl-GAL4, UAS-GFP; btsz^K13-4/TM6B, Tb X UAS-Btsz1; btsz^K13-4/TM6B, Tb (n=18). Blue: btl-GAL4, UAS-GFP; btsz^K13-4/TM6B, Tb X UAS-Btsz2; btsz^K13-4/TM6B, Tb (n=15). Error bars in h and i represent s.d.. Btsz2, but not Btsz1, suppresses the defects in branching (h) and the multi-lumen phenotype (i) of btsz^K13-4 homozygous mutants (P=5.5 × 10⁻⁷ and 5.6 × 10⁻⁵ respectively, by two-tailed t-test). (j) Ability of the same transgenes to suppress lethality of homozygous btsz^K13-4 adults. If lethality of btsz^K13-4 were completely suppressed, the expected proportion of homozygous mutant flies among the offspring of the cross would be 33% (the remaining 66% are heterozygous for the balancer chromosome; homozygous balancer animals die in early larval instars). Thus, about half of the mutants were rescued to viability by the transgene. Images (a–f) are projections of confocal image stacks. Scale bars: (a–c left) 75 μm and (a–c middle and right, d–f) 20 μm. C2, region encoding the tandem C2 domains; GFP, green fluorescent protein; MBD, region encoding the Moesin binding domain; SHD, region encoding a Synaptotagmin-like-protein homology domain.