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First published online 6 July 2005
doi: 10.1242/dev.01910

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Requirement for JAK/STAT signaling throughout border cell migration in Drosophila

Debra L. Silver^1,*, Erika R. Geisbrecht^1, and Denise J. Montell^1,

¹ Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 212052, USA

View larger version (80K): [in a new window]   Fig. 1. STAT expression in wild-type egg chambers. (A) Schematic drawing of a Drosophila ovariole, with stages (S) indicated above. Pre-migratory and migratory border cells are indicated in blue and polar cells are indicated in red. (B-H) Confocal micrographs of egg chambers stained with anti-STAT (green) and anti-FasIII (red) antibody. (B) The germarium through stage 2 of egg chamber development; (C-F) egg chambers at stages (C) 4-6, (D) 8, (E) 9 and (F) 10. (G,H) High magnification images of the stage 9 border cell cluster shown in E, with and without FasIII staining to mark the interface between the two polar cells. Border cell clusters are indicated by the white arrowheads, stalk cells are indicated by the open arrowhead, and the outer follicle cells are indicated by the arrow. nc, nurse cells; o, oocyte. Scale bars: in B, 50 µm for B-F; in G, 10 µm for G,H.

View larger version (95K): [in a new window]   Fig. 2. Overexpression of HOPTUM increases STAT levels in border cells. (A) An ovariole from a c306-GAL4; UAS-mCD8-GFP fly stained with DAPI (blue), showing the pattern of expression of the GAL4 driver (green). (B) An ovariole from a slbo-GAL4; UAS-mCD8-GFP fly stained with an antibody against Armadillo (red), showing the GAL4 pattern of expression (green). Arrow indicates the normal border cell cluster; arrowheads indicate the anterior cells that express the GAL4 driver but do not normally migrate. (C-H) Egg chambers from c306-GAL4; UAS-hopTUM flies stained with antibodies against STAT (green). (C-E) Antibody staining for FASIII (red); (G,H) phalloidin staining of F-actin (red). Arrowheads indicate some of the ectopic border cells that formed. In all examples, anterior is to the left. Scale bars: 50 µm in A,B; in C, 50 µm for C-F; in D, 10 µm for D,E,G,H.

View larger version (95K): [in a new window]   Fig. 3. STAT expression is reduced in stat mutants. (A-C) Outer follicle cells of stat397 mosaic egg chambers. (A) Bright GFP (green) indicates homozygous wild-type cells, intermediate GFP staining indicates heterozygous cells, whereas lack of GFP depicts stat mutant cells. (B) STAT staining (red). (C) Co-localization of GFP and STAT (merged image of A and B). (D) STAT (green) and rhodamine phalloidin (red) staining in slboLY6/slboE7b mutant egg chambers. (E,F) STAT (green) and FASIII (red) staining of stat1681/statts egg chambers, after (E) 0 hours and (F) 2 hours at 29°C. (G,H) STAT (green) and rhodamine phalloidin (red) staining in stat3391/statts egg chambers kept at 29°C for (G) 0 hours and (H) 6 hours. Arrowheads indicate border cells with reduced expression of STAT. Scale bars: in A, 50 µm for A-C; in D, 10 µm for D-H.

View larger version (47K): [in a new window]   Fig. 4. stat is required throughout border cell migration. (A-F) Graphs depicting the percentage of egg chambers of stat mutants of the genotypes indicated that exhibit border cell migration defects. Flies were shifted to 29°C for (A) 0 hrs, (B) 30 minutes, (C) 1 hour, (D) 2 hours, (E) 4 hours and (F) 6 hours. For each genotype, the percentage of egg chambers is shown in which the border cells migrated 0%, partially, or 100% with respect to the extent of migration observed in wild-type egg chambers. For each time point at least 50 egg chambers were examined.

View larger version (96K): [in a new window]   Fig. 5. stat migration defects are evident prior to cell identity changes. (A-D) Confocal immunoflourescence images of two representative statts/stat397; MA33 egg chambers kept at 18°C (A,B) or at non-permissive temperature for 1 hour (C,D). Singed staining (red) indicates the migrating border cells; the enhancer trap MA33 (green) is a marker for squamous follicle cell fate. Arrow indicates the border cell cluster. (E-H) Armadillo staining (red) indicates the border cells, and phalloidin (green) indicates all cells in wild-type (E,F) and statts/stat1681 (G,H) egg chambers; anterior at the top. Scale bars: in A, 50 µm for A-D; in E, 10 µm for E-H.

View larger version (66K): [in a new window]   Fig. 6. Ectopic expression of UPD and UPDTM induces extra migratory border cells. (A-G) Confocal immunofluorescence images of egg chambers stained with anti-GFP to detect those cells expressing UAS-UPD (green) and those expressing STAT (red). (A-C) HS-FLP-GFP; UAS-upd egg chamber and (D-F) HS-FLP-GFP; UAS-updTM egg chamber. (G) An example of the ectopic border cells that surround the cell expressing UAS-updTM. Arrows indicate normal border cell clusters; arrowheads indicate ectopic border cell clusters. Scale bars: in A, 50 µM for A-F; in G, 10 µM.

View larger version (76K): [in a new window]   Fig. 7. Overexpression of SOCS and Domeless causes defective border cell migration and organization of the cluster. (A-C) Confocal micrographs of egg chambers from c306-GAL4; UAS-SOCS flies kept at 29°C, stained with STAT (green) and Armadillo (red). The border cell clusters appear to be less cohesive than normal and can exhibit reduced STAT staining (arrows). (D-F) Nomarski images of (D,E) wild-type and (F) slbo-GAL4,PZ1310;UAS-DomeCYT egg chambers. Egg chambers were stained for ß-galactosidase activity (blue). PZ1310 is an enhancer trap insertion into the slbo locus. Arrow indicates the location of the border cell cluster. (G) Graph depicting the extent of border cell migration in the indicated genotypes. For each genotype at least 50 egg chambers were examined. Scale bars: in A, 50 µm for A-C; in D, 50 µm for D-F.

View larger version (69K): [in a new window]   Fig. 8. Overexpression of DN-Shibire inhibits border cell migration and alters Dome and STAT protein distribution. (A-J) Confocal immunofluorescence images showing egg chambers of wild type (A,C,G,I) and slbo-GAL4;DN-shibire (B,E,H,J) stained with the following antibodies: (A-C,E) Singed (red); (C,E) Domeless (green); (G,H) Cadherin (green) and FasIII (red); or (I,J) STAT (green) and Singed (red). (D,F) Egg chambers expressing slbo-GAL4; UAS-dome-GFP (D) or slbo-GAL4; DN-shibire; UAS-dome-GFP (F) with GFP marking the border cells (green). Scale bars: in A, 50 µm for A,B; in C, 10 µm for C-F; in G, 10 µm for G-J.