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First published online 10 July 2006
doi: 10.1242/dev.02482

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Egfr is essential for maintaining epithelial integrity during tracheal remodelling in Drosophila

Institut de Biologia Molecular de Barcelona (IBMB), CSIC, C/Josep Samitier 1-5, 08028 Barcelona, Spain.

View larger version (85K): [in a new window]   Fig. 1. Characterisation of btlGal4UAS-tauGFP-801 tracheal phenotypes. (A) Wild-type stage 14 embryo. (B) Stage 14 embryo showing defects in the formation of some DBs and GBs (arrowheads), and branch breaks (arrows). (C,D) Detail of five tracheal metameres focused on the DBs (C) or the GBs (D) of late stage 15 embryos. Note the presence of broken branches leaving a few cells completely isolated from the rest of the tree (arrowheads), and of branches with cells connected by cytoplasmic extensions (arrows).

View larger version (59K): [in a new window]   Fig. 2. Identification of the gene responsible for the overexpression phenotype of line 801. (A) Genomic organisation of the region showing the relative positions of different GS and EP elements with respect to Mkp3 and MESR6. EP3142 should only drive MESR6 expression. (B-D) Details of four to five DBs of late stage embryos. Note the defects in continuity of some branches (arrows) when lines GS10283 (B) and Mkp3M76 (C) are crossed to btlGal4. By contrast, a mutation in the Mkp3 gene prevents the GS element of the Mkp3M76 line from producing a branch integrity phenotype (D). (E-G) In situ hybridisation with an antisense Mkp3 probe detects accumulation of the transcript in the tracheal placodes at stage 11 (arrow in E) and in the tracheal branches at later stages (arrow in F) in the wild type. Higher levels are detected when line 801 is crossed to btlGal4 (G).

View larger version (74K): [in a new window]   Fig. 3. Requirement of Egfr, btl and the MAPK pathway in branch integrity maintenance. Panels A-I and K-P show lateral views of two to five posterior tracheal metameres of stage 15 or 16 embryos focused on the DT and DBs. (A,B) The downregulation of the MAPK pathway leads to defects in the continuity of the tracheal tissue (arrows). (C) Constitutive activation of the pathway is able to rescue the branch integrity defects produced by line 801 tracheal expression. (D) Loss of pnt activity does not result in branch integrity defects. (E-G) When the Egfr signal is downregulated, branch interruptions and branches with cells only connected by cytoplasmic extensions are commonly observed (arrows). (H,I) By contrast, the downregulation of Btl does not result in a reproducible branch integrity phenotype. (J) Expressivity of the phenotypes of branch formation and branch integrity of the indicated genotypes represented as percentage of DBs affected. n is the number of DBs analysed. (K-M) Constitutive activation of the pathway results in delays in branch extension (arows in L,M) and defects in cell rearrangements (arrowheads in K,L) when visualised with different markers. (N-P) Projections of confocal sections of embryos stained with DCAD2 antibody. Some DBs show incomplete or impaired cell intercalation events as visualised, respectively, by the presence of abnormal intercellular AJs (arrows in N,O) or by stretches of intercellular AJ in regions where autocellular AJs are expected (arrow in P).

View larger version (89K): [in a new window]   Fig. 4. Branch integrity defects are not due to cell death or tracheal misspecification. (A) Single confocal section of a stage 14 embryo showing a few tracheal cells stained for caspase-3 (in red) and displaying an apoptotic shape (arrow). (B-D) When cell death is prevented under Egfr downregulation conditions, branch breaks (arrowheads) and cells attached by cytoplasmic extensions (arrows) are still detected. (E) Projection of several confocal sections showing the expression of DSRF at the tip of primary branches (arrows) and the visceral branch. (F) Under Egfr downregulation conditions, kni is normally expressed.

View larger version (140K): [in a new window]   Fig. 5. DE-cad and actin accumulation in the tracheal system are affected by the Egfr pathway. (A-E') Projections of confocal sections showing a portion of DT and two to three DBs of the posterior region of stage 16 embryos. The embryos are labelled with DCAD2 (red in A-E) to detect DE-cad and anti-GFP (A,C-E) or anti-Trh (B) to detect the tracheal cells (green). Greyscale images of DCAD2 are shown separately in panels A'-E'. Note that under conditions of Egfr downregulation (B-D), the levels of DCAD2 are lower than in wild type (A), and the staining in the DBs (arrows) is less conspicuous. By contrast, high levels are detected when Egfr is constitutively activated (E), and some DBs remain multicellular, as visualised by the presence of intercellular AJs (arrow). Note that DE-cad is properly localised. (F-J') Single confocal sections showing details of the DT of the posterior region of stage 16 embryos. The embryos are labelled with anti-actin (red in F-J) and anti-GFP (green in G,I,J) or anti-Trh (green in F,H). Greyscale images of anti-actin are shown alongside. Note the presence of a thick cortical staining in the wild-type (F) compared with staining in conditions of Egfr downregulation (G-I). A thicker accumulation of cortical actin is detected when Egfr is constitutively activated (J).

View larger version (95K): [in a new window]   Fig. 6. DE-cad accumulation is modulated upon Egfr activity. (A-G) Projections of confocal sections showing small groups of tracheal cells expressing actinGFP and EgfrDN (A-D) or top (E-G) under the control of btlGal4. The embryos are labelled with DCAD2 (red) and anti-GFP (green). Greyscale images of DCAD2 are shown separately in panels A'-G'. Note a decrease in DE-cad in cells marked with actinGFP compared with the surrounding cells in A-D, and an increase in E-G (arrows).

View larger version (130K): [in a new window]   Fig. 7. Newly synthesized DE-cad in Egfr downregulation conditions. Late stage embryos labelled with mAb2A12 (red) and anti-GFP to detect the DE-cadGFP protein (green in A-E; greyscale in A'-E') under the control of heterologous promoters. (A-C') Projections of confocal sections of embryos carrying btlGal4 UAS-DE-cadGFP. The GFP signal in B and C is weaker and not as clearly detected in some thin tracheal branches (arrows) as in the wild type (A). (D,E,G,H) Single confocal sections showing tracheal branches (D,E) or salivary glands (G,H) of embryos carrying 69B UAS-DE-cadGFP. Note the lower levels of GFP staining in embryos overexpressing 801, especially in the tracheal branches (arrow in E'), and the reduced accumulation of the chimaeric protein in the apical part of the salivary glands (arrows in G,H). (F) Western blot analysis of GFP to detect the DE-cadGFP protein level and -tubulin (as a protein loading control) in embryos with indicated genotypes. After densitometric quantification of GFP and -tubulin levels, we detected a 30% decrease in GFP levels in line 801 (lane 5 compared with lane 3) and top showed a >3-fold increase (lane 1 compared with lane 3).

View larger version (86K): [in a new window]   Fig. 8. Branch integrity phenotypes of cell adhesion downregulation. (A,B) Posterior tracheal metameres of shg2 (A) and cv-c7 (B) stage 15 or 16 embryos focused on the DT and DBs. Note the presence of branch interruptions and branches with cells only connected by cytoplasmic extensions (arrows). (C) Single confocal section of a stage 16 embryo labelled with anti-actin (red in C; greyscale in C') and anti-Trh (green). In shg mutants, cortical actin is greatly reduced compared with the wild type (Fig. 5F). (D) Projection of confocal sections of a stage 16 embryo labelled with anti-DCAD2 (red in D; greyscale in D') and anti-Trh (green). Note that in cv-c mutants the levels of DCAD2 are lower than in the wild type (see Fig. 5A), and that staining in the DBs is less conspicuous (arrow).