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Fused-dependent Hedgehog signal transduction is required for somatic cell differentiation during Drosophila egg chamber formation

Institut Jacques Monod, UMR 7592 – CNRS/Université Pierre et Marie Curie/Université Denis Diderot, Laboratoire de Génétique du Développement et Evolution, 2-4 place Jussieu, 75251 Paris Cedex 05, France

View larger version (70K): [in a new window]   Fig. 1. fu mutations generate ovarioles with egg chambers containing abnormal numbers of germ cells. All ovarioles are oriented with anterior towards the top or right. (A-F) DAPI staining of wild-type (A), fuJB3 (B-D,F) and fu1 (E) ovarioles. Arrowheads indicate compound egg chambers (B,C,E,F) or chambers with less than 16 germline cells (D). (B) Arrow indicates a degenerating egg chamber. (C) Arrow indicates apposed egg chambers. (F) Arrows indicate two vitellogenic oocytes in the same egg chamber. (G-H) Wild-type (G) and fuJB3 (H) egg chambers stained with DAPI (G,H), anti-Orb antibodies (G',H') and phalloidin (G'',H''). Arrowheads indicate Orb-expressing oocytes. Scale bars: 10 µm.

View larger version (68K): [in a new window]   Fig. 2. fu is required for correct encapsulation of germline cysts by prefollicular cells in the germarium. All germaria are oriented with the anterior towards the top or top right-hand corner. (A-B'') Double labeling of wild-type (A-A'') and fuJB3 (B-B'') germaria with anti-Fas III (A,B) and anti--Spectrin (A',B') antibodies, both of which stain prefollicular cells and their descendants (anti--Spectrin also stains fusomes, the branched cytoplasmic structures that link all the cystocytes of a developing cyst). FIII denotes Fas III. Spec denotes -Spectrin. Basal membranes are defined by contacts with the peripheral basal lamina. Arrowheads in A indicate prefollicular cells extending cell processes centripetally and the arrow indicates prefollicular cells that have begun the intercalation process (germarial region 2b/3). (C,D) fuJB3 germaria stained with anti-Fas III antibodies. (C',D') Corresponding DAPI nuclear staining. Arrows in C,D indicate irregular intercalation between prefollicular cells. Arrowheads in C,D indicate somatic cells, within egg chambers, weakly staining for Fas III. Asterisks in C,C',D,D' highlight peripheral aggregates of somatic cells. (D') In the focal plane shown here, 15 nurse cell nuclei are visible in the budding egg chamber, whereas additional nurse cell nuclei are visible in other focal planes of this chamber (data not shown). Scale bars: 10 µm.

View larger version (106K): [in a new window]   Fig. 3. fu mutations affect stalk morphogenesis. All ovarioles are oriented with the germaria towards the right. (A,B) 93-F/+ (A) and fuJB3; 93F/+ (B) ovarioles double-stained with anti-Fas III antibodies (A,A',B,B', red) and anti-ß-galactosidase antibodies (A',B', green). FIII denotes Fas III. Arrow in B and insert in B' indicate Fas IIIlow/93F+ peripheral somatic cells. (C',D') Expression of the 93F enhancer-trap line after X-gal detection of ß-galactosidase activity in wild-type (C') and fuJB3 (D') ovarioles. (C,D) Corresponding DAPI nuclear staining. (E-F') A101/+ (E) and fuJB3; A101/+ (F) ovarioles triple-stained with DAPI (E,F), anti-Fas III antibodies (E',F', red) and anti-ß-galactosidase antibodies (E'-F', green). Arrows in F,F' indicate interfollicular cells arranged in a double row. (G-J) Wild-type (G,I) and fuJB3 (H,J) ovarioles stained with DAPI. (G'-J') Magnification of the boxed area of G-J germaria, double-stained with DAPI (red) and anti DE-Cadherin antibodies (green). Ovarioles were dissected either from 7- to 8-day-old females (A-D) or from younger 3-4 day old females (E-J). Scale bars: 10 µm.

View larger version (70K): [in a new window]   Fig. 4. fu is expressed in both the germline and somatic cells of the ovary. All ovarioles are oriented with the germaria towards the top or top right-hand corner. (A) Distribution of fu transcripts in a wild-type germarium and stage 2 and 3 egg chambers. (B,B') Two different confocal sections of a wild-type germarium and stage 2-3 egg chambers stained with anti-Fu antibodies. Confocal section in B corresponds to follicular somatic cells (revealed by their ‘honey comb-like’ staining, arrow) and confocal section in B' to germline cells and anterior-most somatic cells. (C,D) Confocal sections of wild-type (C) and fu1 (D) stage 4 egg chambers stained with anti-Fu antibodies, showing reduced levels of Fu protein accumulation in fu mutant follicular cells compared with wild type. Identical confocal settings and image processing with Adobe Photoshop were used for C,D.

View larger version (57K): [in a new window]   Fig. 5. fu function is required in prefollicular cells for centripetal migration and stalk formation. All ovarioles are oriented with the anterior-most region towards the top or top right-hand corner. (A,A') Mosaic ovariole in which fumH63 mutant follicle cell clones lacking an arm-lacZ reporter construct have been induced. Ovariole is triple-stained with DAPI (A), anti-ß-galactosidase (A', green) and anti-Fas III (A', red) antibodies. fu+ somatic cells are found only in the most anterior part of the ovariole (prefollicular cells, bracket). Follicular cells exhibit weak Fas III, but no ß-galactosidase staining. White arrowheads point to pairs of polar cells belonging to the second egg chamber, whereas the open arrowhead indicates anterior polar cells belonging to the more posterior egg chamber (as determined after superposition of DAPI and ß-galactosidase images, data not shown). Polar cells presented in insert are not in the same focal plane as follicular and germline cells shown in A and A'. (B-E') Mosaic ovarioles in which fu mutant follicle cell clones lacking a tub-lacZ reporter construct have been induced. Ovarioles are double-stained with DAPI (B-E) and anti-ß-galactosidase antibodies (B'-E'). (B,B',E,E') fuG3 mosaic ovarioles. (C-D') fuJB3 mosaic ovarioles. (B) Arrows indicate the two small oocyte nuclei present in the multicyst egg chamber. (C,C') Arrows indicate lateral accumulation of fu+ cells in stalk-like structures. (D-E') Arrows indicate fu mutant somatic cells and asterisks indicate fu+ cells.

View larger version (77K): [in a new window]   Fig. 6. fu function is required in prefollicular cells for posterior oocyte positioning. All ovarioles are oriented with the anterior-most region towards the top or top right-hand corner. (A-A'') fuG3 mosaic ovariole double-stained with anti-Orb (A, red) and anti-ß-galactosidase (A', green) antibodies. (A'') Merge. fu+-expressing follicle cells are delimited by broken lines. (B,C) Wild-type (B) and fuG3 mosaic germaria (C) triple-stained with DAPI (B,C), anti-Orb (B,B',C,C', red) and anti-ß-galactosidase (C', green) antibodies. fu+-expressing follicle cells in C' are delimited by broken lines and correspond primarily to stalk cells. Arrows in B' indicate posteriorly localized Orb-expressing oocytes and arrows in A, A'',C' to mislocalized Orb-expressing oocytes. Note that, in addition, the normal posterior subcellular localization of the Orb crescent within the oocyte (arrows in B') is perturbed in fu mosaic ovarioles (arrows in C').

View larger version (55K): [in a new window]   Fig. 7. fu interacts genetically with components of the Hedgehog pathway in the ovary. (A-C) Expression of a ptc-lacZ enhancer-trap in wild-type (A), hs-hh (B) or fu1/fuJB3; hs-hh/+ germaria. Ovarioles are oriented with anterior towards the left. (D,E) Removing one copy of cos2 (D) or two copies of Sufu (E) restores the fu ovarian phenotype. Class II fu alleles were not tested in these experiments because fuII; cos2WI/+ and fuII; Su(fu)LP flies die as late pupae, displaying a strong cos2 phenotype. fuJ denotes the fuJB3 allele. (D) Females were grown at 25°C and dissected 5-8 days after eclosion. *indicates either cos2WI or + (these two different genotypes were not distinguishable in our test). Flies came from the same cross for a given allele. (E) Females were grown at 21°C until late pupal stage, then shifted to 25°C and dissected 8 days after eclosion. Females originated from separate crosses performed in parallel. The total number of ovarioles examined for a given genotype was at least 150, except for fu1 homozygous flies in E (n=39).

View larger version (53K): [in a new window]   Fig. 8. Reduction in Hedgehog signaling levels leads to abnormal prefollicular cell behavior. All ovarioles are oriented with the anterior-most region towards the top or top right-hand corner. hhts2/hhts2 (A,A') and hhAC/hhts2 (B,B') ovarioles dissected from 5-day-old females placed at 29°C immediately after eclosion and double-stained with DAPI (A,B) and anti-Hts antibodies (A',B'). Hts is normally present in spectrosomes and fusomes of germline cells as well as in all somatic cells as of germarial region 2a/2b (Lin et al., 1994). In particular, Hts accumulates at high levels in interfollicular cells. Arrows in A',B' indicate somatic cells aggregated at the periphery of the ovarioles. (C-D') DAPI (C,D) and GFP (C',D', green) staining of hsp-flp/+; Act>CD2>Gal4,UAS-GFP/UAS-cos2 ovarioles. Germarium in D' was additionally stained with anti-Hts antibodies (red). (E-F') DAPI (E,F) and GFP (E',F', green) staining of hsp-flp/+; Act>CD2>Gal4,UAS-GFP/UAS-Cicell ovarioles. All prefollicular cells and their descendants are GFP+ in C' and E', whereas both GFP+ and GFP– prefollicular and follicular cells are present in D' and F'. (C,E) Arrows indicate apposed egg chambers, arrowheads indicate multicyst egg chambers. Scale bars: 10 µm.

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