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First published online June 8, 2005
doi: 10.1242/10.1242/dev.01886

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Dynamics of the basement membrane in invasive epithelial clusters in Drosophila

Institute of Signaling, Developmental Biology and Cancer, UMR 6543 CNRS, University of Nice Sophia-Antipolis, Parc Valrose, 06108 Nice, cedex 2, France

View larger version (97K): [in a new window]   Fig. 1. Apical capping of anterior polar cells with basement membrane material. (A) Formation and migration of the BC cluster. (a) A stage 8 egg chamber made of a monolayer epithelium (follicle cells, light gray) surrounding the germline (blue, nurse cells; yellow, oocyte). (b) Anterior polar cells (orange) express the Upd ligand activating the JAK/STAT pathway in follicle cells (arrows). (c) Cells receiving the highest levels of Upd are committed into the outer BC fate (beige). (d) A mature cluster delaminates and starts migration. (B) GFP-Vkg (green) accumulates in the BM. At stage 8, an apical cap forms over anterior polar cells (arrowhead). (C,D) Close up of the framed regions shown in B. Polar cells are marked by Fas2 expression (B-D; red). (E-G) Co-localization of GFP-Vkg and Coll.IV 1 (F,G; red), at the BM and in the apical cap (G). (H) Antibodies are specific to Coll.IV 1, as they do not recognize GFP-Vkg. The lower band (asterisk) is non-specific. w, white. (I-K) Co-localization of GFP-Vkg and Laminin A (red) in the apical cap. (L-N) Apical cap accumulation of BM protein-trap lines G205 (Vkg), 2840 and 2867 (Perlecan; see Fig. S1 in the supplementary material). Anterior is to the left. Scale bars: in B, 10 µM; in C, 5 µM for C,D; in E, 5 µM for E-G; in I, 5 µM for I-K; in L, 5 µM for L-M.

View larger version (119K): [in a new window]   Fig. 2. Apical capping is specific to anterior polar cells. (A) Overexpression of the polar cell fate repressor eya. The polar cell marker Fas2 is shown in red. (B,C) Enlarged views of the framed regions shown in A. Note the absence of anterior polar cells and an apical cap (compare with Fig. 1B-D). Egg chambers show Laminin A (green) and nuclei (blue). (D-I) Mosaic egg chambers containing eya mutant follicle cells making ectopic polar cells (Fas2, in red). (D-F) Anterior stage 8 egg chamber with two normal polar cells and their apical cap (D, arrowheads 1, 2). (E) A ectopic polar cell close by shows a nascent apical cap (arrowhead 3). (F) Merged image of D and E. (G-I) A group of five to six ectopic polar cells have assembled two separate and large apical caps (arrowheads 1, 2). (I) Merged image of G and H. Scale bars: in A, 10 µM; in B, 10 µM for B,C; in D, 5 µM for D-F.

View larger version (40K): [in a new window]   Fig. 3. The apical cap is asymmetric and dynamic. (A-F) Three-dimensional imaging and reconstruction of the anterior region of stage 8 egg chambers, showing GFP-Vkg (green), Fas2 (red) and Hoeschst (blue, in A). (A) The original stack is a projection of 20 z sections (total depth is 7.6 µm). (B) Intermediate processing showing BM (green), polar cells (white) and apical cap (blue). (C) The resulting 3D-reconstructed polar cells with the apical cap. (D-F) Three other examples of reconstructed polar cells with their apical cap. Insets in C-F show the apical side of polar cells. The red line is the boundary between the two polar cells. (G-O) GFP-Vkg (green; G-0) egg chambers showing Fas2 (G-L), Crumbs (M,O) or Fas3 (N) in red. The process of apical capping is transient and can be described in four discrete phases (1-4; see text for details). After shedding (L), the BC delaminate and invade the nurse cell compartment (M). During migration (phase 5), a BM containing GFP-Vkg is present in polar cells specifically (M-O), opposite to the apical side (marked with Crumbs in M and O). The polar cell BM is maintained when BCs reach the oocyte (O). Anterior is to the left. Scale bars: in A, 5 µM for A-F; in G, 5 µm for G-O.

View larger version (81K): [in a new window]   Fig. 4. Drab5-dependent basal to apical transcytosis of basement membrane. (A-C) GFP-Vkg non-expressing cells in a GFP-Vkg/+ background. The Myc clonal marker is in red; GFP-Vkg non-expressing cells are outlined (dashed lines). When polar cells only (A), or polar and outer BCs (B,C), do not express GFP-Vkg, the apical cap forms normally. (D) Wild type; (E,F) polar cells expressing Drab5S43N (E) do not form an apical cap or (F) show a `microcap' phenotype. Frequency of caps (G) and microcaps (H) of stage 8 egg chambers in wild-type (n=625) flies, or flies expressing Drab5S43N (n=298), Drab5WT (n=201) or ShiK44A (strong; n=183). Data are presented as mean±s.e.m. (see Materials and methods). Anterior is to the left. Scale bar: in A, 5 µM for A-F.

View larger version (77K): [in a new window]   Fig. 5. GFP-Vkg basement membrane is assembled cell non-autonomously. (A) Confocal image showing the anterior region of an ovariole. Note that BM is present at all stages of egg chamber development. (B) Scheme of the FLP-FRT-mediated clonal analysis used to generate mosaic egg chambers containing clones of cells expressing either no copies (0; blue color code), one copy (gray color code) or two copies (red color code) of GFP-Vkg. In this scheme, the clonal marker is a nuclear GFP (nls-GFP, green). (C) A mosaic egg chamber containing several clones of cells expressing 0, 1 or 2 copies of GFP-Vkg. Note the uniform basement membrane. In particular, cells that do not express GFP-Vkg (blue lines) have a normal BM, indicating that GFP-Vkg is not contributed autonomously to the BM by follicle cells. (D) Another example in which a large clone of cells expressing two copies of GFP-Vkg (red line) abut cells expressing no copies of GFP-Vkg (blue line). In this case, as well, there is no change in the apparent thickness or staining of the BM. (E) The same egg chamber double labelled with phalloidin-TRITC (red) to outline the cell boundaries. (F-G') Two egg chambers in which a big clone of cells that do not express GFP-Vkg (blue line) is adjacent to cells expressing 2 copies of GFP-Vkg (red lines). In these mosaic egg chambers, the GFP-Vkg BM is uniform. (C-E) The clonal marker is a nuclear GFP; (F-G') the clonal marker is a Myc tag (anti-c-Myc shown in red). Scale bars: in A, 10 µM; in C 10 µM for C-E; in F 10 µM for F-G'.

View larger version (42K): [in a new window]   Fig. 6. BC migration defects of Drab5S43N and ShiK44A egg chambers. Expression of Drab5S43N, Drab5WT and ShiK44A in egg chambers was driven by slbo-GAL4 at 29°C. To measure the extent of migration, the nurse cell compartment was divided into four equivalent regions (1 to 4: clusters in region 1 have almost completely migrated whereas clusters found in region 4 have not migrated at all; see inset) and the position of the BC relative to this coordinate system monitored. Drab5S43N and ShiK44A led to similar and strong migration defects.

View larger version (42K): [in a new window]   Fig. 7. Apical cap shedding requires JAK/STAT signaling and the recruitment of outer BCs. GFP-Vkg (green) egg chambers expressing Domecyt (A,B) or Drac1N17 (C,D). Follicle cells are marked with phalloidin (A,C,D; red). In B, polar cells are marked with slbo-lacZ (red). (A) Blocking outer BC recruitment does not affect the formation of the apical cap at stage 9. However, shedding does not take place and a cap is still present in stage 10 Domecyt egg chambers (B; phenotype schematized in the upper left panel). Expression of Drac1N17 blocks migration (D) but has no effect on outer BC recruitment and apical cap formation (C) (phenotype schematized in the lower left panel). (E) A four-step model for BC formation and migration: (1) formation of an asymmetrical apical cap (green; containing Collagen IV 1 and 2 chains, Laminin A, Perlecan) over the anterior polar cells (orange) through Drab5-dependent transcytosis; (2) polar cells send a JAK/STAT signal to recruit outer BCs (beige) from the surrounding follicle cells (light gray); (3) shedding of the apical cap is dependent on the presence of outer BCs; (4) migration of the mixed BC cluster starts, with polar cells maintaining a BM. Anterior is to the left. Scale bar: in A, 5 µM for A-D.