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Fig. 5. GlcNAc-groups, including tracheal chitin, are severely reduced in mmy mutants. (A-C) Detection of chitin with a FITC-conjugated chitin-binding probe (CBP) in wild-type and mmy mutant embryos. CBP labelling reveals a broad intraluminal chitin cable with filamentous appearance in stage 15 wild-type tracheae (A). The chitin cable in mmy^IL07 mutants has a slightly grainy appearance (B), and is absent in mmy^IK63 mutants (C). The mmy^IK63 embryo in C is also labelled for the 1-eve-1 marker with ß-gal (red) to visualise the tracheal epithelium. (D-F) Detection of GlcNAc-group in tracheae with FITC-conjugated WGA. In wild-type tracheae, WGA labels the lumen and the apical surface of tracheal cells (D). In mmy^IK63 mutant tracheae, neither the lumen nor the apical plasma membrane is labelled with WGA (E). By comparison, in chitin-deficient kkv¹ embryos, the luminal detection is reduced to a thread of non-chitinous filament, whereas labelling of the apical plasma membrane appears to be normal (F). (G-L) mmy^IK63 mutants display normal apicobasal polarity and can secrete the luminal Pio protein. Stage 15 wild-type (H) and mmy^IK63 (K) embryos stained with Crb (red) reveal a correct localisation of this apical determinant. The microtubuli minus-end reporter Nod-ß-gal localises to the apical cortex in wild-type tracheal cells (I) as well as in mmy^IK63 mutant tracheae (L). Wild-type (G) and mmy^IK63 mutant embryos (J) stained with the Pio antibody (red) reveals that the Pio filament is present in mmy^IK63 mutant tracheae. One segment of the DT is shown in G-L. (M-T) Double labelling with WGA and Crumbs in wild-type and mmy^IK63 mutant embryos. In wild-type stage 14 embryos (M,N), WGA labels the tracheal lumen as well as the developing epidermis (M), whereas mmy^IK63 mutants at the same stage (O,P) show severely reduced WGA-labelling in both tissues (P). WGA also detects intracellular spots in wild-type stage 15 salivary glands (Q,R), presumably secretory vesicles, but not in mmy^IK63 mutants at the same stage (S,T). Scale bars: 3 µm in A-C; 4 µm in D-L; 30 µm in M-P; 10 µm in Q-T). Scale bars: 3 µm in A-C; 4 µm in D-F; 30 µm in G-J; 10 µm in K-N; 4 µm in O-T.

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