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First published online 19 September 2007
doi: 10.1242/dev.007328

Development 134, 3665-3676 (2007)
Published by The Company of Biologists 2007
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Tramtrack regulates different morphogenetic events during Drosophila tracheal development

Sofia J. Araújo1,2, Carolina Cela1 and Marta Llimargas1,*

1 Institut de Biologia Molecular de Barcelona (IBMB-CSIC), Parc Cientific de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain.
2 Institut de Recerca Biomedica de Barcelona (IRB), Parc Cientific de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain.


Figure 1
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  		Fig. 1. Ttk tracheal expression and requirements. (A) Late-stage ttk mutant embryo showing an absence of branch fusion in the dorsal trunk (DT) (arrows). (B,C) Ttk69 protein accumulation in wild-type embryos at stage 11 (B) and 14 (C). Ttk (red) is expressed in all tracheal cells (green). (D-F) Embryos at late embryogenesis. Notice the rudimentary aspect of the tracheae in ttk mutants (E,F) as compared with wild type (WT; D). (G) Details of two tracheal metameres, showing the absence or reduction (arrows) of visceral branches (VBs) in a ttk mutant. (H) Schematic representation of wild-type bnl expression (dark grey) at stage 13. (I,J) Expression of bnl at stage 13. Notice the reduction in size and number of bnl spots in the ttk mutant (J). (K) A stage 12 embryo showing co-expression of bnl mRNA and Ttk69 protein (arrows). (L,M) Details of stage 15 embryos labelled to highlight DSRF-positive terminal cells (arrows). DSRF is expressed in several presumptive terminal cells in ttk mutants (arrows in L). All panels, except H, I and J, show projections of confocal sections of laterally viewed embryos. In this and all figures, dorsal is up and anterior to the left.

 

Figure 2
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  		Fig. 2. Regulated levels of Ttk specify fusion cell identity. (A,B) Stage 15 embryos labelled to highlight Esg-positive fusion cells (arrows in A). Tracheal overexpression of ttk (B) blocks fusion fate acquisition (arrows) and branch fusions. (C) Late-stage embryo overexpressing ttk specifically in fusion cells, which also leads to a lack of branch fusion. (D-F') Details of one or two tracheal metameres focused at the lateral trunk (LT; D,D'), dorsal trunk (DT; E,E') or dorsal branches (DBs; F,F'). Dys-positive fusion cells (arrows) show lower levels of Ttk69 (in greyscale in panels D',E' and F') when compared with the neighbouring tracheal cells. (G,G') Two tracheal metameres focused at the LT. All extra fusion cells (arrows) in N loss-of-function conditions show lower levels of Ttk69 (in greyscale in G'). (H,H') Four tracheal metameres focused at the DT. No cells show low levels of Ttk69 (in greyscale in H') when N is constitutively active. All panels show projections of confocal sections of laterally viewed embryos.

 

Figure 3
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  		Fig. 3. Ttk is required during tracheal cell intercalation. (A-D') Projections of confocal sections showing details of stage 13 (A,C) and 16 (B,D) embryos focused at the dorsal trunk (DT) and dorsal branches (DBs). No apparent differences are observed at the early stages (compare A with C). Accumulation of DE-cad (labelled with DCAD2 and shown in greyscale in B' and D') shows the presence of autocellular adherens junctions (AJs; arrow in B') in wild type (WT). (D') ttk embryos exhibit mainly intercellular AJs (arrowhead) and only occasional short stretches of autocellular ones (arrows). (E-F') Projections of confocal sections showing lateral trunk (LT) details of stage 16 embryos. Notice the presence of autocellular AJs in wild type (E'), and the elongated shape of the intercalated cells (E, cells have been coloured to facilitate observation of cell rearrangements and shape). Conversely, ttk mutants show mainly intercellular AJs (F') and cuboidal cells positioned side-by-side (F). (G) Projection of confocal sections showing a late-stage ttk mutant. The formation of autocellular AJs (arrows) is restored by tracheal expression of ttk. (H,I) Lateral views of ttk embryos at stage 13-14 showing normal accumulation of Sal and Kni proteins (arrows). (J,K) Projections of confocal sections showing details of stage 16 wild-type and ttk mutant embryos labelled to show Pio accumulation. (L-N) Expression pattern of pyd at the placode stage (arrows). Notice the absence of expression in ttk mutants (I) and increased levels in several structures (arrows in J) when ttk is generally overexpressed.

 

Figure 4
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  		Fig. 4. Ttk is required for tracheal cell rearrangements and cell shape changes. (A-L) Projections of confocal sections from a time-lapse experiment, showing the development of dorsal branches (DBs). Time is indicated in the top right corner of each panel. The cells of selected DBs have been coloured to facilitate observation of cell rearrangements. In ttk mutants (D-F,J-L), paired cells remain paired and cuboidal, and do not intercalate. Notice the formation of filopodia (arrows).

 

Figure 5
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  		Fig. 5. Ttk is involved in the formation of terminal and fusion branches. (A) Stage 15 embryo labelled to highlight Esg-positive cells (arrows). (B) Two adjacent dorsal branches (DBs), the tip cells (probably fusion cells) of which make contact. The lumen (highlighted in white) does not penetrate the contacting cells. (C,D) Contralateral (C) or adjacent (D) DBs, the tip cells of which make contact. An E-cad-rich structure (in greyscale in insets) forms at the contact point (arrows). (E-F') Details of DBs and lateral trunks (LTs). The lumen marker 2A12 (in greyscale in E',F') reveals the presence of abundant luminal vesicles at the tips of branches in the presumptive fusion (arrowhead) and terminal (arrows) cells in wild type (E'). ttk mutants (F,F') show a decreased density of luminal dots. (G,H) DBs showing DSRF-positive cells and terminal branches (arrow in G). Notice the absence of terminal branches (arrow in H) in spite of DSRF expression in ttk mutants (H). (I,J) Fragments of LTs of ttk mutants at stage 16. Terminal branch formation is rescued when ttk is expressed in tracheal cells (arrows in J), but not when the Btl pathway is constitutively activated (arrows in I). All panels except I and J show projections of confocal sections of laterally viewed embryos.

 

Figure 6
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  		Fig. 6. Ttk controls tracheal tube size. (A,B) Portions of dorsal trunk (DT) in longitudinal views (upper panels) or in cross-sections (lower panels) of wild type (WT; A) and ttk mutants (B). Thickness of the tubes is measured. (C,D) Stage 16 embryos showing accumulation of CBP in the DT. ttk mutants show an abnormal intraluminal chitin filament (D). (E-G) Expression pattern of mmy in stage 14 embryos. Notice the increased levels of expression in ttk mutants (F) and the absence of expression when ttk is generally overexpressed (G). (H,K) Stage 16 embryos. Verm is abnormally accumulated in ttk mutants (K). (I,J,L,M) Stage 16 embryos. Accumulation of septate junction (SJ) markers (FasIII and Cora; green) in ttk mutants (L,M) is only slightly affected (L) or is comparable to wild type (M). (N,O) Stage 16 embryos injected with a 10 kDa rhodamine-labelled dextran. ttk embryos (O) are permeable to the dye, which fills the tracheal lumen (arrows), whereas the wild-type trachea is impermeable (N). All panels except E-G,N and O show projections of confocal sections of laterally viewed embryos.

 

Figure 7
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  		Fig. 7. Ttk is required for proper luminal cuticle formation. Transmission electron microscopy (TEM) micrographs of late-stage embryos. (A,B) Longitudinal sections of the dorsal trunk (DT). Notice the taenidial folds (t) and the cuticle layers - envelope (env), epicuticle (epi) and procuticle (p) - within each taenidium. (B) ttk mutants show a disorganised taenidial structure and density differences at the level of the procuticle. (C,D) Detail of septate junctions (SJs; arrows) between adjacent DT cells. As in wild type (C), SJs are correctly positioned in ttk mutants. (E,F) Detail of the epidermal cuticle. In the procuticle (p) of wild-type embryos (E), the chitin laminae are arranged in a typical helicoidal pattern. In ttk mutants (F) the procuticle is very disorganised. Scale bars: 200 nm.

 

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© The Company of Biologists Ltd 2007