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First published online February 9, 2006
doi: 10.1242/10.1242/dev.02266

Development 133, 957-966 (2006)
Published by The Company of Biologists 2006
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The Drosophila formin DAAM regulates the tracheal cuticle pattern through organizing the actin cytoskeleton

Tamás Matusek1, Alexandre Djiane2, Ferenc Jankovics3, Damian Brunner3, Marek Mlodzik2,* and József Mihály1,*

1 Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62, Hungary.
2 Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
3 European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.


Figure 1
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  		Fig. 1. The organization of the DAAM locus and the isolation of DAAM mutations. (A) The 1F2-3 cytological region includes the predicted gene CG14622 that we named DAAM. Position of two P-element insertion are shown, EP(1)1336 and EP(1)1542, which have been used to generate DAAM loss-of-function alleles including large 5' deficiencies. The full-length cDNA clone RE67944 consists of 12 exons, translation starts in exon 4. (B) The full-length DAAM cDNA clone carries a 1165 bp 5' UTR and a 731 bp 3' UTR, and encodes a predicted protein of 1153 amino acids, which contains several homology domains, including GBD (GTPase binding domain), DID (Diaphanous inhibitory domain), DD (dimerization domain), CC (coiled-coil region), FH1 (formin homology domain 1), FH2 (formin homology domain 2) and DAD (Diaphanous autoinhibitory domain). The activated form of DAAM (C-DAAM) includes the C-terminal 637 amino acids of the protein. The position of the 3' deficiency alleles is shown at the bottom.

 

Figure 2
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  		Fig. 2. The cuticle structure of wild-type and DAAM mutant Drosophila tracheal tubes. Schematic drawing of a wild-type main airway (A) shows that tracheal cuticle is laid down on the inner apical surface of tracheal cells. The cuticle is characterized by taenidial folds, running perpendicular to the tube axis, that are clearly visible on a native tracheal tube dissected out from a third instar larvae (B). The tracheal tubes of a DAAMEx68 homozygous mutant larvae exhibit a strongly impaired cuticle pattern in both the main airways (C) and the side branches (D), often leading to the collapse of the tubes. (E) The trachea phenotype of DAAMEx68 over Df(1)AD11 (a deficiency that uncovers DAAM) is as strong as that of the homozygous DAAMEx68 phenotype (compare E with C). (F) btl-Gal4-driven overexpression of the full-length DAAM protein (FL-DAAM13.59) partly rescues the tracheal cuticle defects induced by DAAMEx68. Scale bars: 50 µm.

 

Figure 3
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  		Fig. 3. DAAM expression in the embryonic tracheal system and the DAAM antibody. RNA in situ hybridization of DAAM (A) and immunohistochemical staining with a polyclonal anti-DAAM serum (B) revealed a strong expression in the developing embryonic tracheal system. Brown staining in A and red staining in B show DAAM expression in a stage 16 embryo. The anti-DAAM serum barely detects any DAAM protein in a DAAMEx68 homozygous mutant embryonic trachea (C,D) and in mitotic clones induced in DAAMEx68, w, FRT19A/w, arm-lacZ, FRT19A; ey-flp/+ larval eye imaginal disc [E-H; homozygous mutant clones (white arrows) lack DAAM staining]. Clones are marked by absence of ß-gal (green). Elav (blue) is a neuronal marker. H is the merge of E-G. Anterior is towards the left, and dorsal is upwards in all panels.

 

Figure 4
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  		Fig. 4. DAAM is required to organize apical actin into parallel running bundles in tracheal cells. In a wild-type (wt) tracheal tube, actin is organized into parallel running bundles that are perpendicular to the tube axis (A). The number and phasing of these actin bundles correspond to the taenidial fold pattern displayed on the tracheal cuticle (B). Actin bundles formed in the tracheal cells are located at the level of the adherens junctions (C,D). (C) A confocal projection of a wild-type tracheal tube where actin is visualized in red, while the adherens junctional marker, DE-cadherin is shown in green. (D) An optical xz section along the white line in C, apical is at the top. (E-G) The DAAM protein is largely colocalized with actin in the embryonic tracheal cells. Confocal sections have been collected from a one-segment wide region of the dorsal trunk of a stage 16 embryo. The cytoplasm of tracheal cells is labeled with GFP in green, actin is shown in red, DAAM is in blue. Arrow in F indicates the fusion cells located at the segmental boundary. DAAM is not expressed in these cells. (H-J) 3D projections of the same confocal sections shown in E-G. Sections were rotated within the XZ plane by 90°. There is strong colocalization of actin and DAAM at apical membranes of the tracheal tubes (J). In DAAMEx68 mutant tracheal tubes, not only is the cuticle pattern impaired (K), but actin organization is also severely altered in both the larval (L) and embryonic (M) tracheal cells. Actin cables formed are thinner and shorter than their wild-type counterparts (compare A with L, and E with M), and fail to organize into regularly aligned bundles. The formation of the apical actin bundles in the embryonic tracheal cells (marked by actin::GFP) is first detected at approximately 13 hours AEL (N). Scale bars: 20 µm in A for A,B; 50 µm in C for C,D; 50 µm in L for K,L; 10 µm in E-G for E-J; 10 µm in M for M.

 

Figure 5
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  		Fig. 5. The trachea specific overexpression of activated forms of DAAM and Dia. Btl-Gal4 driven expression of C-DAAM (activated DAAM) affects the cuticle pattern (A) and impairs actin organization in the tracheal system (B). Fusion cells are narrow doughnut-shaped cells that are easily distinguished from the regular tracheal cells by their spotted cuticle pattern (C). Actin in fusion cells (red) is also concentrated into a spotted pattern (D). When C-DAAM is ectopically expressed in the fusion cells, it often induces a change in their shape, and leads to a partial transformation of the spotted cuticle towards the taenidial fold pattern (E). Additionally, we detected a strong actin accumulation in the fusion cells where short actin bundles are often visible (F). DE-cadherin labels the cell boundaries on B,D,F; arrows indicate the fusion cells on D and F. C and D are the same wild type larval trachea, E and F are from the same DAAM overexpressing trachea. Whereas, C-DAAM expression in the tracheal network does not significantly alter the general structure of the tubular system (G), the presence of DiaCA (an activated form of diaphanous) leads to fusion defects in the dorsal trunk (black arrow), constrictions at the fusion cells (green arrowhead), widening at other areas of the dorsal trunk (black arrowhead) and impairments of terminal branch differentiation (green arrow) (H). The tracheal system in G and H is visualized by the luminal antibody 2A12. Scale bars: 50 µm in B for A,B;10 µm in D for C,D; 10 µm in F for E,F; 10 µm in G,H.

 

Figure 6
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  		Fig. 6. The genetic interaction partners of DAAM. Trachea dissected out from third instar DAAMEx1 mutant larvae display a moderate cuticle phenotype (A), which is strongly enhanced by removal of one copy of RhoA (B), Tec29 (C) and Src42A (D). The DAAM trachea phenotypes are already exhibited in first instar larvae (E,F). Homozygous mutant Tec29 (G) and Src42A (H) first instar larvae display a moderately strong taenidial phenotype similar to DAAMEx1 (compare G and H with E). Consistent with this, apical actin organization is partly impaired in Tec29 (I) and Src42A (J) mutant embryonic tracheal tubes (compare I and J with Fig. 4E). Scale bars: 50 µm in A-H; 10 µm in I,J.

 

Figure 7
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  		Fig. 7. Epistasis experiments with C-DAAM. Cuticle defects induced by C-DAAM expression in tracheal cells (A) are not modified by RhoA (B), but are strongly suppressed by Src42A (C), Tec29 (D) and co-expression with the full-length form of DAAM (FL-DAAM13.59) (E). Scale bars: 50 µm.

 

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© The Company of Biologists Ltd 2006