spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 6 February 2003
doi: 10.1242/jcs.00298

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in JCS
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Roch, F.
Right arrow Articles by Akam, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Roch, F.
Right arrow Articles by Akam, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Drosophila miniature and dusky encode ZP proteins required for cytoskeletal reorganisation during wing morphogenesis

Fernando Roch1,2,*, Claudio R. Alonso1 and Michael Akam1

1 Laboratory for Development and Evolution, University Museum of Zoology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
2 Centre de Biologie du Développement, CNRS UMR5547, Université Paul Sabatier, Bât. 4R3, 118 Route de Narbonne 31062, Toulouse, France



View larger version (107K):

[in a new window]
  		Fig. 1. Cuticle morphology is disrupted in Df(1)MR adult wings. Wild-type and Df(1)MR wings dissected 2 hours after adult eclosion. (A-D) Light microscopy. The mutant wing in B is reduced in size, although veins (L2-L5) and wing margin (WM) are correctly patterned. (C,D) A closer view of the same wings reveals that hairs are much closer in the mutant than in the wildtype (arrowheads). (D) The picture of the mutant corresponds to a slightly lower focus plane, showing the cell outlines. Note the ring of cuticle surrounding each hair in the mutant. (E,F) SEM micrographs. The mutant hairs appear twisted and branched (open arrowheads), in contrast to the long and slender wild-type epidermal hairs (E). Some cuticle blobs appear bulging out of the main wing surface in the mutant (arrow). (G-J) TEM sections. The two wing surfaces (dorsal and ventral) are separated in the mutant (H,J), and the resulting space contains cuticle invaginations (small arrows) and cell debris (arrows). The presence of cuticle defects is indicated by arrowheads and at a higher magnification in the lower panels (I,J). The position of some epidermal hairs is also indicated (open arrowheads in G,H).

 


View larger version (79K):

[in a new window]
  		Fig. 2. Apical membrane reorganisation fails to occur in Df(1)MR mutants. Confocal images of wild-type and Df(1)MR pupal wings of different stages stained as indicated. Cartoons show cell outlines (grey) and the position of the apical junctions (black) in a transverse section of the epithelium at each stage. At 28-32 hours APF, wild-type and mutant epithelia are indistinguishable. By 46-50 hours APF, the formation of hair pedestals accumulating large amounts of actin (arrowheads) is hindered in the mutant. Note also the presence of an actin-ring around each cell in the mutant, visible also in the transverse sections (open arrowheads). The mutant cells also fail to acquire a star-like cell contour at the level of their apical junctions. At 62-66 hours APF, the rhodamin-phalloidin binds the apical membrane that appears folded inside the wing. Note that in the mutant a space between the apical membrane folds is present, where cuticle is secreted (open arrowheads). The aberrant apical junctions of the mutants also accumulate large amounts of {alpha}-catenin—GFP. Bar, 5 µm.

 


View larger version (48K):

[in a new window]
  		Fig. 3. m and dy encode transmembrane proteins with a ZP domain. (A) Genomic organisation of the m/dy region. The position of exons is indicated by black boxes. The regions deleted in the different deficiencies are shown below. The position of the small rearrangement present in the m1 mutant is indicated (arrow). (B) Similarity diagrams based on a ClustalW multiple sequence alignment of the ZP domains from the indicated Drosophila, nematode and mammal proteins. (C) Predicted modular structure of Min, Dy and Dyl proteins (protein accession numbers are AAF48088, AAF48089 and AAF47884, respectively). The position of the transmembrane domain is highlighted by a vertical box. Tetrabasic RRR/AR motifs are indicated by triangles. Signal peptides are indicated by black boxes (SP). (D) Alignment of Dy, Dyl, Min and Cut-1 ZP domains. Black and shaded boxes indicate identical and similar residues, respectively. The position of the ZP structural domain is shown between brackets, and the eight conserved cysteines characteristic of this domain are marked with asterisks (see text for details).

 


View larger version (66K):

[in a new window]
  		Fig. 4. dy, Min and dyl are expressed in cuticle-forming tissues. dy (A,D) and dyl (C,F) RNA in situ hybridisation and anti-Min immunostaining (B,E) in wild-type embryos of stage 16-17. (A-C) A view of the embryonic epidermis. By stage 16, the three genes are upregulated in regions corresponding to the dorsal and ventral denticle belts (arrowheads). (D-F) View of embryonic internal structures. dy and Min are expressed in the pharynx (ph), oesophagus (es), the duct of the salivary glands (sgd) and in the hindgut (hg). dyl is present in the epidermis but also in the pharynx and oesophagus (arrowheads in F). (G-I) Pupal wings dissected at 28 hours APF and stained as above. At this stage all wing cells express both dy (G) and Min (H), but not dyl (I).

 


View larger version (92K):

[in a new window]
  		Fig. 5. The Min protein localises to the apical membrane at the time of cuticulin envelope assembly. (A,B) Confocal image of a transverse section taken from wild-type pupal legs stained for Min protein. (A) At 6 hours APF, Min protein accumulates in the cytoplasm (n, cell nuclei), whereas the staining accumulates in the cells apical surface 1 hour later (B). The position of the basal and apical surfaces is indicated. (C,D) Confocal images corresponding to an apical plane of a slightly tilted pupal wild-type wings. (C) At 32 hours APF, Min (green) accumulates in the cytoplasm, both apically (arrowheads, upper part of the picture) or in a more basolateral focal plane (arrows, bottom of the picture). (D) At 34 hours APF, a uniform layer stained with the anti-Min antibody covers the whole wing and the epidermal hairs (arrowheads). Anti E-cadherin antibodies (red) mark the position of apical junctions. (E-F) Transverse section of similar wings to those shown in B. Strong expression of {alpha}-catenin—GFP (red) marks both apical junctions and the basal part of the cells (white line). (E) At 32 hours APF, Min protein accumulates in the apical side of the cell and in the cytoplasm (green). Nuclei position is marked by the TOPRO dye (blue). The dorsal and ventral cell layers composing the wing are evident; the basal membrane is now in the middle of the wing (white line). (F) At 34 hours APF, Min protein is detected only in the apical side of the cells. (H,I) Confocal view of the wing margin row of stout bristles stained with anti-Min. (H) By 32 hours APF, most of the staining accumulates in the cell body of the trichogen cells (tr), whereas the bristle shafts are not stained (arrowheads). (I) 2 hours later the staining has disappeared from the cytoplasm of the trichogen cells (tr) and accumulates in the periphery of the growing shaft (arrowheads). The tormogen cells (to) of each bristle still accumulate Min in their cytoplasm at this stage.

 


View larger version (79K):

[in a new window]
  		Fig. 6. TEM sections of wing epidermis of wildtype (A,C) and Df(1)MR (B,D) at 36 hours APF (A,B) and 44 hours APF (C,D) (bar, 500 nm). The insets correspond to micrographs of thick sections of the same specimens, stained with toloudine blue and showing the whole wing (bar, 50 µm). (A) At 36 hours APF, the small patches of cuticulin envelope (arrowheads) appear at the tip of microvilli (mv) in the wildtype. (B) In the mutant, microvilli are shorter, and the process of cuticle deposition is less advanced (arrowheads). (C) At 44 hours APF, the cuticulin envelope forms an almost continuous layer over the epidermis in both wild-type and mutant wings. In some regions (arrowheads), the characteristic trilayer can be observed, whereas in other regions (brackets) the cuticulin layer seems less organised. (D) The structure of the apical microvilli is disorganised in the mutant wings (mv). The process of wing extension is prevented in the Df(1)MR mutant (compare insets in C and D).

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003