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First published online December 20, 2005
doi: 10.1242/10.1242/dev.02206

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Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in Drosophila

¹ Department of Medical Biochemistry, Göteborg University, Sweden.
² Department of Genetics, Max-Planck-Institute for Developmental Biology, Tübingen, Germany.
³ Microscopy Section, Max-Planck-Institute for Developmental Biology, Tübingen, Germany.

View larger version (140K): [in a new window]   Fig. 1. Defective differentiation of mmy mutant cuticles. (A-G) Light microscopy of wild-type and mmy mutant larvae. (A) The wild-type larva has a colourless cuticle with tanned ventral denticles and head skeleton that is visible through the vitelline membrane. In larvae homozygous for mmyIK63 (B) or for the P-element insertion KG08617 (C), no ventral denticles, head skeleton or cuticle are visible, and transheterozygous mmyIK63/Df(2L)BSC6 larvae (D) display similar cuticle defects to homozygous mmyIK63 mutant larvae. In all these mutant genotypes, the salivary glands accumulate abnormal debris (arrows in B-D). (E-H) Removal of the vitelline membrane reveals that larvae mutant for mmyIL07 (F) have a distended cuticle compared with the wild type (E). Moreover, the mmyIL07 mutant head skeleton (H) is darker and appears discontinuous compared with the wild type (G).

View larger version (161K): [in a new window]   Fig. 2. Chitin is absent in the mmy mutant cuticle. (A-C) Comparison of wild-type and mmy mutant larval (end of stage 17) epidermis by transmission electron microscopy (TEM). The wild-type epidermis (A) consists of an epithelial monolayer of flattened cells (inset) beneath a stratified cuticle. The cuticle has three distinct layers: the outermost envelope (env), the middle protein-rich epicuticle (epi) and the underlying chitin-rich lamellar procuticle (pro) attached to the apical plasma membrane (pm). The epidermis of mmyIK63 mutant larvae (B) is an epithelium with cuboidal cells (inset) covered by a disorganised cuticle. Arrows in the insets of A and B indicate the lateral membrane that is undulated in the wild-type but not in the mmyIK63 mutant epidermal cell. The mmyIK63 procuticle varies in thickness and is devoid of lamellar chitin microfibrils, the overlying epicuticle is irregular, and the outermost envelope is reduced to three, instead of five, alternating electron-dense sheets. The envelope and the epidermis of larvae homozygous for the weaker mmyIL07 allele (C) appear to be normal, the epicuticle is, however, discontinuous and the procuticle harbours electron dense inclusions, probably orphan proteins. (D-F) Detection of chitin in wild-type, mmyIK63 and mmyIL07 mutant cuticles with gold labelled WGA. Chitin is detected in the wild-type and mmyIL07 mutant procuticle (D,F; black dots), but is absent in the mmyIK63 cuticle (E). Scale bars: 0.5 µm (bar in A applies to A-C; bar in D applies to D-F); 1 µm for insets.

View larger version (134K): [in a new window]   Fig. 3. Septate junctions (SJs) are defective in mmy mutant epithelia. (A-C) TEM-analysis of septate junctions (SJs) of wild-type, mmyIL07 and mmyIK63 mutant larval (end of stage 17) epidermal cells. The SJs of the wild-type (A) and mmyIL07 mutant (C) epidermal cells are seen as ladder-like structures. In mmyIK63 mutant epidermal cells (B), this ladder-like assembly is absent, but electron-dense intercellular material at the position usually occupied by the SJ is present. (D-G) Fas3 (red) is occasionally mislocalised in mmyIK63 mutant epithelia. In wild-type (D) hindgut epithelia, Fas3 concentrates within the apical-most region of the lateral membrane, whereas in mmyIK63 mutants (E) Fas3 is found along the entire lateral cell surface. Fas3 localisation is similarly affected in the mmyIK63 mutant salivary gland (G) when compared with the wild type (F). Scale bars: 0.125 µm in A-C; 5 µm in D-G.

View larger version (90K): [in a new window]   Fig. 4. Mmy is required for uniform tracheal tube growth. (A-C) Lateral view of wild-type and mmy mutant embryos labelled with the tracheal lumen-specific antibody 2A12. The 2A12 antibody marks the lumen of wild-type (A) and mmyIL07 mutant (B) tracheae. The dorsal trunks (DTs) of mmyIL07 embryos are slightly convoluted compared with wild type. The tracheal lumen of mmyIK63 mutant embryos fails to label with 2A12 (C). (D) Expression of the pan-tracheal lacZ marker 1-eve-1 in mmyIK63 mutants and labelling with anti-ß-gal reveal a correctly patterned tracheal network in these mutants, but some of the narrower ganglionic and dorsal branches are discontinuous (arrow indicates a dorsal branch gap). (E-G) Confocal analysis of two or three DT segments in embryos that carry the pan-tracheal lacZ marker 1-eve-1, doubly labelled with 2A12 (red) and ß-gal (green). In stage 15 mmyIK63 mutants (F), the DT fusion branches fail to expand (arrowheads); at stage 16 (G), the DTs develop severe constrictions and dilations, when compared with the wild type (E). Scale bars: 50 µm in A-D; 10 µm in E-G.

View larger version (131K): [in a new window]   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 mmyIL07 mutants has a slightly grainy appearance (B), and is absent in mmyIK63 mutants (C). The mmyIK63 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 mmyIK63 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) mmyIK63 mutants display normal apicobasal polarity and can secrete the luminal Pio protein. Stage 15 wild-type (H) and mmyIK63 (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 mmyIK63 mutant tracheae (L). Wild-type (G) and mmyIK63 mutant embryos (J) stained with the Pio antibody (red) reveals that the Pio filament is present in mmyIK63 mutant tracheae. One segment of the DT is shown in G-L. (M-T) Double labelling with WGA and Crumbs in wild-type and mmyIK63 mutant embryos. In wild-type stage 14 embryos (M,N), WGA labels the tracheal lumen as well as the developing epidermis (M), whereas mmyIK63 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 mmyIK63 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.

View larger version (74K): [in a new window]   Fig. 6. Mmy encodes the Drosophila UDP-N-acetylglucosamine pyrophosphorylase. (A) Genome organisation of mmy, showing the exons (grey boxes) used in each of the two mmy transcripts RA and RB. Start and stop codons are marked in red and green, respectively. The two EMS-induced mmy mutations mmyIK63 and mmyIL07 introduce single mis-sense mutations in the third common exon, whereas P-element KG04349 is inserted 5' to the ATG of RA, and P-element KG08617 disrupts the second intron of both RA and RB. (B) Sequence alignment of the Mmy protein with its orthologues in yeast, human and Aedes. The mis-sense mutations in mmyIK63 and mmyIL07 cause substitution of conserved amino acids as indicated above the sequence. The extra 37 amino acids in isoform A are underlined and do not show homology to Mmy orthologues. (C) Illustration of the biochemical reaction catalysed by Mmy and the main biological processes that require the Mmy product.

View larger version (33K): [in a new window]   Fig. 7. Post-translational modification of Knk depends on Mmy activity. (A) Western blot with protein extracts from wild-type and different mutant larvae labelled with a specific antibody against the extracellular cuticle protein Knk. In the wild type, Knk migrates as a single band (ExB and EndoH-), and EndoH treatment to cleave N-glycosylated sugar-residues generates smaller co-migrating Knk species (EndoH+). In mmyIK63 and mmyKG08617, several Knk proteins with different sizes are present. No Knk protein is detected in knk14D79 mutants, and Knk protein migration is not affected in the remaining mutants tested. (B) Western blot with the same extracts as in A labelled with antisera against the membrane-attached Syntaxin1A (Syx1A), the transmembrane protein Tout-velu (Ttv) and cytosolic -Tubulin (Tub) as loading control. Ttv also migrates as a smaller protein after EndoH-treatment, and in mmyIK63 and mmyKG08617 mutant larvae differently sized Ttv proteins are present (stars). (ExB, extraction buffer PLC). (C-E) Co-labelling of stage 16 wild-type, knk mutant and mmyIK63 mutant epidermis with anti-Knk (green) and anti-Fas3 (red). Knk localises to the apical plasma membrane in the wild type (C) and is absent in knk mutants (E). In the mmyIK63 mutant epidermis, the amount of Knk is severely reduced in the apical membrane, and some signal is detected within the cell (D).

View larger version (129K): [in a new window]   Fig. 8. Mmy expression is temporally upregulated in developing epithelia and altered in mutants required for 20-hydroxyecdysone biosynthesis. (A-I) In situ hybridisation of wild-type embryos with mmy antisense probe. Embryos at early blastoderm stage display high levels of maternal mmy RNA (A), but no mmy transcript is detected in embryos between stages 5-10 (B; stage 8). Zygotic mmy expression is observed from stage 11 in tracheal cells as they invaginate from the ectoderm (C) and persists in the developing tracheal system until stage 15 (D,E,G) after which it decreases. At stage 16 mmy is strongly expressed in the salivary glands (F,H) and weakly detected in the epidermis (F,I). (J-O) In shade (shd) mutant embryos labelled with the mmy antisense probe, tracheal expression of mmy is not detected (J), instead mmy is prematurely expressed in the epidermis (J, black arrowhead; L,O), salivary gland (J,K; white arrowhead) and proventriculus (J,K; white arrow) during stage 15. A similar pattern of mmy expression is also found in mutants for shadow (sad; M and N). Scale bars: 50 µm in A-F,J,K,M,N; 25 µm in G; 10 µm in H; 25 µm in I,L,O.