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First published online 27 April 2005
doi: 10.1242/dev.01839

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Multiple roles of the F-box protein Slimb in Drosophila egg chamber development

Instituto Leloir and IIB, FCEyN-Universidad de Buenos Aires, Patricias Argentinas 435, Buenos Aires, 1405, Argentina

View larger version (97K): [in a new window]   Fig. 1. slmb shows a dynamic expression pattern during egg chamber development. Ovaries from females carrying the slmb00295 enhancer trap were analyzed by X-gal staining or by anti-ß-galactosidase (anti-ß-gal) immunofluorescence. (A) In the ovariole, nurse cells stain positive for X-gal throughout oogenesis (arrows). X-gal signal in FC surrounding the oocyte appears at mid-oogenesis (arrowhead). (B,C) In the germarium, expression of ß-gal (green) is restricted to the germline and is excluded from FC that express Fasciclin 3 (red). (D) Expression of ß-gal cannot be detected in FC at stage 8 (arrowhead) and starts at stage 9 (E, arrowhead). (F) At oogenesis stage 10, strong expression of the enhancer trap occurs in a scattered pattern in the follicular epithelium (arrows). (G) At stage 11, expression becomes stronger in two patches of FC that will form the dorsal appendages (arrows).

View larger version (99K): [in a new window]   Fig. 2. slmb loss of function provokes a wide array of ovarian phenotypes. (A) DAPI staining of a wild-type ovariole (interfollicular stalks are marked by arrows). (B) Some egg chambers from females with slmb germline clones exhibit premature apoptosis (arrow) indicated by the occurrence of fragmented nurse cell nuclei (C, arrowhead). (D) Wild-type follicles always contain 15 nurse cells and one oocyte; in slmb germline clones, egg chambers with decreased (E) or increased (F) number of germ cells are frequently observed, whereas in other follicles germ cell nuclei were heterogeneous in size (G). (H) In females bearing FC clones, egg chambers with two oocytes occur (arrows). (I) Wild-type germarium in which FC are stained for Fas3; the cyst in region IIb shows the typical lens shape (white asterisk) and once in region III the cyst re-shapes into a sphere (yellow asterisk). (J) In slmb somatic clones, the general shape of the germarium is frequently altered and cysts are placed side by side in regions II and III (asterisks). slmb FC clones exhibit a wide array of defects, including abnormal morphology of egg chambers (K; compare with D), oocytes that are mispositioned (L,M, arrows) and lack of interfollicular stalks between adjacent follicles (N,O, arrows; compare with A). (P) slmb/CSN5 genetic interactions; slmb2 germline clones that are heterozygous for CSN5L4032 display enhanced phenotypes such as enlarged germaria (arrow).

View larger version (92K): [in a new window]   Fig. 3. Germ cell mitosis is misregulated in slmb germline clones. Ring canals are visualized by TRITC-Phalloidin staining. (A) In wild-type egg chambers, the oocyte (marked with a broken line) connects to neighboring nurse cells through four ring canals. (B) An oocyte (broken line) from a slmb germline clone exhibits five ring canals, indicating that an extra round of mitosis has occurred. (C) slmb mutant egg chamber with extra germ cells and two oocytes (arrows). (D) Higher magnification image of the oocyte on the right in C, showing that in these follicles, oocytes exhibit four ring canals (arrows). (E) In a slmb mutant ovariole, the egg chamber on the right has only two germ cells connected by a single ring canal (arrow) and exhibit very big nuclei (white arrowhead); in the same ovariole, the egg chamber on the left has the normal complement of germ cells and their nuclei are much smaller (open arrowhead). (F) Western blot analysis of E2f complex components in ovarian extracts with slmb germline clones (GLC), compared with those of wild-type ovaries; whereas E2f1 does not show obvious differences, Dp and E2f2 were clearly reduced in slmb mutant ovaries.

View larger version (85K): [in a new window]   Fig. 4. Delayed FC differentiation and ectopic polar cells occur in slmb somatic clones. (A) In wild-type ovarioles, Fas3 expression (red) refines as oogenesis progresses and from stage 4 onwards becomes restricted to polar cells (arrows). (B) In slmb FC clones, Fas3 remains widespread (egg chambers are outlined). (C) In egg chambers bearing slmb FC clones, large patches of cells expressing Fas3 are still detected at stage 9 (arrow). Mutant clones from the egg chamber shown in C were positively marked with GFP (D, arrow) and the merged confocal image shows that ectopic Fas3 correlates with the position of the clones (E, arrow). Arrowheads in C-E mark a pair of cells that are probably ectopic polar cells. (F-H,J) The PZ80 enhancer trap is specifically expressed in polar cells. In wild-type ovaries at stage 8, the two pairs of polar cells are localized at the anterior and posterior termini of the follicle (F, arrows) at stage 8; at stage 9, anterior polar cells migrate posteriorly between nurse cells (G, arrow); and at stage 10, they have reached the anterior border of the oocyte (H, arrow). (I,K) The slbo-LacZ element is expressed in border cells. In stage 10 wild-type follicles, border cells can be seen at the anterior end of the oocyte (I, arrow). Egg chambers bearing slmb FC clones often exhibit extra polar cells (J, arrowhead) and differentiate ectopic clusters of border cells (K, arrowhead). In these follicles, border cell migration is often delayed and by stage 10, they are located between nurse cells (J,K, arrows).

View larger version (75K): [in a new window]   Fig. 5. slmb mutant clones cause chorion patterning defects. Eggshells were visualized by reflected light or dark-field microscopy. (A) Eggs laid by females with slmb germline clones (right) are often much smaller than wild-type eggs (left). (B) Wild-type dorsal appendage (DA) pattern. (C-F) Egg chambers with slmb somatic clones generate eggshells with variable DA abnormalities, including dorsalized phenotypes (C-E), ramified DA (F), ventralized phenotypes (G,H), and DA shifted to a more posterior position (I), reduced (I,J) or absent (K). In cases where DA are shifted posteriorly (I), the operculum appears enlarged when compared with wild-type eggshells (B); the limits of the opercula are indicated with broken lines.

View larger version (96K): [in a new window]   Fig. 6. Mild overactivation of the Dpp pathway mimics slmb mutant phenotypes. (A) Eggs laid by females bearing the weak Hs-gal4 driver and the strong UAS-dpp element that were subjected to heat shock show a dramatic expansion of the operculum (marked by arrowheads) and lack of DA. (B) DAPI staining of an ovariole dissected from one of these females reveals that many germ cells are surrounded by a single follicular epithelium. (C,D) Upon milder overexpression using the weak UAS-dpp element and the weak Hs-gal4 driver, ventralized eggshells with a single DA occurred. (E,F) Variable chorion phenotypes resulting from overexpression with the same UAS-dpp element driven by e22C-gal4: DA can be ramified (E, arrow), expanded (F) or greatly reduced (G). DA reduction was also observed upon overexpression of Medea with the weak Hs-gal4 driver (H) and in eggs laid by heterozygous sogU2 females (I). Mild overexpression of Dpp with the weak Hs-gal4 or e22C-gal4 drivers (J-M) produced germaria with abnormal morphology (J), egg chambers with extra germ cells (K, arrow), mispositioned oocytes (L, arrow) and ovarioles lacking interfollicular stalks (M, arrowheads).

View larger version (83K): [in a new window]   Fig. 7. Broad-Complex (BR-C) expression and ectopic activation of the Dpp pathway. (A) mRNA in situ hybridization in a stage 10 wild-type egg chamber showing that BR-C is expressed in two patches of dorsoanterior FC. (B-D) In follicles bearing slmb somatic clones, BR-C expression is in some cases expanded (B, arrow) and in others reduced (C, arrow) or split (D, arrows). (E,F) Expression of the dad-lacZ enhancer trap in wild-type ovarioles. (E) In the follicular epithelium, expression is first observed at stage 8 in anterior FC (arrow), being undetectable at stage 6. (F) At stage 9, dad-lacZ can be seen in migrating anterior FC (black arrow) and at stage 10 in stretched cells (arrowhead) and in centripetal cells localized at the anterior border of the oocyte (white arrow). (G) In ovarioles bearing slmb mutant clones, expression of dad-lacZ occurs prematurely at stage 6 (arrow); at stage 10 (H), expression of the enhancer trap often expands posteriorly (arrow). (I) Positive GFP labeling of a slmb mutant clone showing that GFP signal overlaps with ectopic expression of dad-lacZ in the clone. (J,K) In wild-type ovaries, expression of Medea occurs at stage 9 in anterior FC that migrate towards the oocyte (J, arrow) and at stage 10 (K) in all FC, being much stronger in stretched cells (arrowhead) and in centripetal cells localized at the anterior border of the oocyte (arrow). (L) In slmb mutant follicles, strong Medea signal was detected in ectopic patches of columnar FC, at stage 10 (arrow). (M) Positive GFP labeling of the slmb mutant clone overlaps with this patch.

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