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First published online December 17, 2003
doi: 10.1242/10.1242/dev.00917

Development 131, 181-190 (2004)
Published by The Company of Biologists 2004
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Lachesin is a component of a septate junction-based mechanism that controls tube size and epithelial integrity in the Drosophila tracheal system

Marta Llimargas1,*,{dagger}, Maura Strigini2,*, Markella Katidou3, Domna Karagogeos2,4 and Jordi Casanova1,{dagger}

1 Institut de Biologia Molecular de Barcelona (IBMB), CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
2 IMBB/FORTH, Vassilika Vouton, Iraklio, Crete GR-71110, Greece
3 Department of Biology, University of Crete, Iraklio, Crete GR-71110, Greece
4 Medical School, University of Crete, Iraklio, Crete GR-71110, Greece



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  		Fig. 1. Organization and expression of the Lac gene, and structure of the Lac protein. (A) The Lac gene is uncovered by Df(2R)CB1 and Df(2R)BSC3, but not by Df(2R)vg135. (B) The Lac gene encompasses around 12 kb and its cDNA is around 2.1 kb. BG1462 is inserted at position 46 of the Lac cDNA, 270 bp before the ATG. BG1462 is late embryonic lethal both when homozygous, and over the deficiencies that uncover the Lac gene, but not over Df(2R)vg135. Lac2 is an imprecise excision of BG1462 that removes the ATG. (C) The Lac protein contains three Ig domains and a C-terminal hydrophobic domain, characteristic of GPI-linked proteins. (D-G) In situ hybridization with an antisense Lac probe does not detect any signal in oocytes (not shown). (D) At stage 5, the Lac transcript is detected in the cellularized blastoderm but it is excluded from the ventral side. (E) At germ band extension, a striped pattern of Lac transcript can be detected in the ectoderm. Later on, the Lac transcript accumulates in some cells of the nervous system (F) and in the tracheal cells (G).

 


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  		Fig. 2. Characterization of the tracheal phenotype of Lac mutants. (A-D,H,I) Projections of several confocal sections showing details of 2-3 tracheal metameres of wild type (A,H), Lac2 (B) or Lac1 (C,D,I) mutants at stage 16, stained only with mAb2A12 to show the lumen (red), or with mAb2A12 and an anti-GFP antibody to detect the expression of tauGFP in tracheal cells (green), which highlights cell shape. Note the defects in mAb2A12 accumulation (arrowhead in C), branch sinuosity (arrows in B,C), and branch breaks (arrows in D,I). (E) Lateral view of a stage 15 wild-type embryo stained with mAb2A12. (F) Lateral view of a stage 15 Lac1 embryo, in which Lac and tauGFP are expressed in the tracheal cells stained with mAb2A12 (black) and anti-GFP (brown). Note the rescue of the tracheal phenotype. (G) Lateral view of a Lac1/bulb embryo at stage 16 stained with mAb2A12 (black), showing the same tracheal phenotype as Lac1 or Lac2 mutants. (J-O) Details of the dorsal trunk between tracheal metameres 7 and 8 at stage 16 of wild type (J,L,N) and Lac1 mutants (K,M,O). Embryos are labelled with anti-GFP (J,K) to show the increased length of the tracheal cells (highlighted by expression of tauGFP) and, as a consequence of the dorsal trunk, with a Crb antibody (L,M), and with a GFP antibody (N,O) to detect nodGFP accumulation in tracheal cells.

 


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  		Fig. 3. Lac behaves as a homophilic cell adhesion protein. Confocal microscopy analysis of the bead aggregation assay. (A) Control or (B) Lachesin-Fc-coupled beads were incubated in solution for 1 hour at room temperature and observed under the confocal microscope for formation of bead clusters. Whereas control beads do not form aggregates, Lachesin-Fc beads form large aggregates. Inset (B) shows one aggregate at higher magnification.

 


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  		Fig. 4. Lac co-localizes with SJs proteins. (A,B) Stage 14 wild-type embryo labelled with an antibody against Lac; two different focal planes are shown, trachea and epithelium (arrow and arrowhead, respectively, in A) and the CNS (arrow in B). (C-F) Details of the tracheal dorsal trunk (C,E) or the hindgut (D,F) of LacGFP stage 16 embryos labelled with anti-GFP to detect Lac accumulation (green), and an anti-Crb (C,D; red) or an anti-Arm antibody (E,F; red). (G,H) Detail of a stage 16 tracheal dorsal trunk (G-G2) or the hindgut (H-H2) of wild-type embryos, labelled with anti-Lac (G1,H1; green) and anti-Cora (G2,H2; red). Note, co-localization of the two proteins. (I,J) Detail of a stage 16 tracheal dorsal trunk (I-I2) or the hindgut (J-J2) of wild-type embryos labelled with an anti-Lac antibody (I1,J1; green) and a Nrx antibody (I2,J2; red).

 


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  		Fig. 5. Lac accumulates at the SJs. Electron micrographs of the ectoderm of wild-type embryos at the end of embryogenesis, labelled with an anti-Lac antibody (A-D). The labelling is visible as gold particles (arrows). (A) Lac specific accumulation along the lateral membrane. (B,C) Lac in the SJs, visible as stretches of electron-dense membrane. (D) Tangentially sectioned SJs with Lac accumulation. (E) Shows a control sample treated equally, but not incubated with the primary antibody (see Materials and methods).

 


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  		Fig. 6. Lac is required for the distribution of SJs proteins, which are also required for tracheal morphogenesis. (A,B) Detail of the posterior part of stage 16 embryos, in dorsal view, 40 minutes to 1 hour after injecting dye. In the wild type (A), the tracheae (visible with transmission) remains non-permeable, whereas in Lac1 mutants (B) the tracheae completely fills with dye. (C,D) Salivary glands of wild type (C) and Lac2 mutants (D) labelled with anti-Cora antibody. (E,F) Detail of the dorsal trunk of wild type (E) or Lac2 mutants (F) labelled with anti-Cora antibody. (G,H) Salivary glands of wild type (G) and Lac2 mutants (H) labelled with anti-Dlg antibody. (I,J) Salivary glands of wild type (I) and cora5 mutants (J) labelled with anti-Lac antibody. (K,L) Detail of the dorsal trunk of wild type (K) or cora5 mutants (L) labelled with anti-Lac antibody. (M-N) Detail of 3-4 tracheal metameres of cora5 (M) or Nrx4304 (N) mutants stained with mAb2A12. Compare with Fig. 2A-D.

 




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