Multiple roles of the F-box protein Slimb in Drosophila egg chamber development
Mariana Muzzopappa and
Pablo Wappner*
Instituto Leloir and IIB, FCEyN-Universidad de Buenos Aires, Patricias
Argentinas 435, Buenos Aires, 1405, Argentina
*
Author for correspondence (e-mail:
pwappner{at}leloir.org.ar)
Accepted 29 March 2005
 |
SUMMARY
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Substrate-specific degradation of proteins by the ubiquitin-proteasome
pathway is a precise mechanism that controls the abundance of key cell
regulators. SCF complexes are a family of E3 ubiquitin ligases that target
specific proteins for destruction at the 26S-proteasome. These complexes are
composed of three constant polypeptides Skp1, Cullin1/3 and Roc1/Rbx1
and a fourth variable adapter, the F-box protein. Slimb (Slmb) is a
Drosophila F-Box protein that fulfills several roles in development
and cell physiology. We analyzed its participation in egg chamber development
and found that slmb is required in both the follicle cells and the
germline at different stages of oogenesis. We observed that in slmb
somatic clones, morphogenesis of the germarium and encapsulation of the cyst
were altered, giving rise to egg chambers with extra germline cells and two
oocytes. Furthermore, in slmb somatic clones, we observed ectopic
Fasciclin 3 expression, suggesting a delay in follicle cell differentiation,
which correlated with the occurrence of ectopic polar cells, lack of
interfollicular stalks and mislocalization of the oocyte. Later in oogenesis,
Slmb was required in somatic cells to specify the position, size and
morphology of dorsal appendages. Mild overactivation of the Dpp pathway caused
similar phenotypes that could be antagonized by simultaneous overexpression of
Slmb, suggesting that Slmb might normally downregulate the Dpp pathway in
follicle cells. Indeed, ectopic expression of a dad-LacZ enhancer
trap revealed that the Dpp pathway was upregulated in slmb somatic
clones and, consistent with this, ectopic accumulation of the co-Smad protein,
Medea, was recorded. By analyzing slmb germline clones, we found that
loss of Slmb provoked a reduction in E2f2 and Dp levels, which correlated with
misregulation of mitotic cycles during cyst formation, abnormal nurse cell
endoreplication and impairment of dumping of the nurse cell content into the
oocyte.
Key words: Drosophila, Slmb, Oogenesis, Egg chamber, Eggshell
 |
Introduction
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Destruction of intracellular proteins at the 26S proteasome is a finely
regulated process that determines the half-life of most key cell regulators
(Hershko and Ciechanover,
1998
). The proteasome is a 25 MDa multi-subunit protease complex
that recognizes and degrades proteins that have been tagged with a
poly-ubiquitin chain (Voges et al.,
1999
). The poly-ubiquitin chain is synthesized on the substrate
that must be degraded by the sequential activities of three enzymes: an
ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an
E3 ubiquitin ligase. It is now widely accepted that specificity of the
ubiquitination process relies on the selective recognition of substrates by
specific E3 enzymes (Pickart,
2001
).
SCF complexes are a family of E3 ubiquitin ligases that have been reported
to target different signaling molecules and cell cycle regulators
(Deshaies, 1999
;
Zheng et al., 2002
). They are
composed of three relatively constant polypeptides Skp1, Cullin1/3 and
a Ring finger domain protein, Roc1/Rbx1
(Kamura et al., 1999
;
Ohta et al., 1999
) and
one variable component the F-box protein that is specific for
a particular substrate or small group of substrate proteins
(Patton et al., 1998
;
Winston et al., 1999
). The
Drosophila F-box protein Slimb (Slmb) fulfills several functions in
development and cell physiology: it participates in E2f destruction at the
beginning of the S phase of the cell cycle
(Heriche et al., 2003
); it is
necessary for normal circadian rythmicity by targeting the clock protein
Period (Grima et al., 2002
;
Ko et al., 2002
); it plays a
role in limiting centrosome duplication
(Wojcik et al., 2000
) by
destroying the inhibitor of the Anaphase Promoting Complex Emi1
(Margottin-Goguet et al.,
2003
); it represses the immune response through downregulation of
the transcription factor Relish (Khush et
al., 2002
); and it participates in imaginal wing, limb and eye
development through the modulation of Wingless, Hedgehog and Dpp/TGF-ß
pathways (Jiang and Struhl,
1998
; Miletich and
Limbourg-Bouchon, 2000
; Ou et
al., 2002
; Theodosiou et al.,
1998
). In order to assess new functions of the Slmb-containing SCF
complex in Drosophila development, we have begun to study the role of
Slmb during oogenesis.
The Drosophila ovary is made up of 16-20 chains of egg chambers of
progressive age, called ovarioles. Egg chambers are formed at the anterior end
of each ovariole in a structure called germarium that harbors both germline
and somatic stem cells (Margolis and
Spradling, 1995
; Wieschaus and
Szabad, 1979
). The germarium can be divided into three regions. In
region I, at the anterior part, a germline stem cell divides asymmetrically
producing one daughter stem cell and one cystoblast. The cystoblast undergoes
four rounds of mitosis with incomplete cytokinesis, giving rise to a cyst
formed by 16 germ cells interconnected by ring canals
(de Cuevas et al., 1997
;
Spradling, 1993
). At germarium
region IIa, the two germ cells with four ring canals enter meiosis and become
pro-oocytes; afterwards, one of them is selected to become the oocyte while
the other 15 cells develop as nurse cells. In region IIb, the cyst is
contacted posteriorly and surrounded by somatically originated follicle cells
(FC), thus acquiring a lens shape. Later on, in region III, the cyst re-shapes
into a sphere, giving rise to an egg chamber that buds off from the germarium
covered by a monolayer of FC (van Eeden
and St Johnston, 1999
). At this stage, the oocyte localizes to the
posterior of the cyst, attaching through specific interactions to posterior FC
of the egg chamber (Godt and Tepass,
1998
; Gonzalez-Reyes and St
Johnston, 1998
).
As the egg chamber leaves the germarium, three types of FC have
differentiated: stalk cells, polar cells and cuboidal FC
(Dobens and Raftery, 2000
;
Torres et al., 2003
). Stalk
cells are a subtype of five to eight FC that separate adjacent egg chambers;
polar cells are two pairs of FC located at the anterior and posterior termini
of the follicle that induce a terminal cell fate on their neighboring FC
(Beccari et al., 2002
;
Xi et al., 2003
). The third
subtype, the cuboidal FC, form an epithelium that surrounds the cyst. At
mid-oogenesis, cuboidal FC are patterned by the combined activities of
Epidermal Growth Factor Receptor (EGFR) and Decapentaplegic (Dpp) pathways
that determine the size, shape and position of specific chorion structures
such as the operculum and dorsal appendages
(Dobens and Raftery, 2000
;
Peri and Roth, 2000
).
In this paper, we report that Slmb plays several different roles in
oogenesis: it is required in FC for normal morphogenesis of the germarium, for
cyst encapsulation, for timely differentiation of FC into different
subpopulations and for chorion patterning. We present evidences that Slmb
downregulates the Dpp pathway in FC, suggesting that most of the above
phenotypes might be caused by overactivation of this pathway. In addition, we
show that slmb loss of function in the germline provokes
misregulation of cystocyte divisions, nurse cell endoreplication defects and
incomplete dumping. These germline phenotypes correlate with a sharp decrease
in the levels of the E2f subunits E2f2 and Dp, suggesting that Slmb
participates in the regulation of the network of cell cycle modulators.
 |
Materials and methods
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Fly strains
The following slmb alleles were used:
slmb00295 (provided by T. Xu), slmb1
and slmb2 (provided by J. Jiang). The following markers
were incorporated or recombined into slmb2 P{neoFRT82B}/TM3B
Sb genotype: slbo01310
(Montell et al., 1992
), PZ80
and dad1883 (obtained from A. Spradling). For
overexpression studies, the following lines were used: K25sevHs-gal4
(strong); Hs-gal4 (weak); CY2-gal4
(Queenan et al., 1997
);
T155-gal4, e22C-gal4 (Duffy et
al., 1998
); UAS-dpp (strong)
(Ruberte et al., 1995
);
UAS-dpp (weak) (Staehling-Hampton
and Hoffmann, 1994
); UAS-medea
(Marquez et al., 2001
);
UAS-activated tkv (provided by S. Cohen) and UAS-slmb
(Grima et al., 2002
).
sogU2, dadj1E4, CSN5L4032,
dCul112764 and dCul110494 lines were
obtained from the Bloomington Drosophila Stock Center. Canton-S was
used as wild type.
Induction of constructs and mosaic clones
For Gal4/UAS induction, flies were grown at 18°C until eclosion and
then transferred to 25°C. Heat shocks were performed for 20 minutes at
37°C on three consecutive days. Eggs from these flies were collected or
females were dissected 2-6 days after the last heat shock. Mutant clones were
generated by FLP-mediated mitotic recombination
(Xu and Rubin, 1993
). Briefly,
females y w; slmb00295, 1 or 2 P{neoFRT82B}/TM3B Sb; were
mated with males y w P{Hs-FLP}; P{neoFRT82B}
P{w[+mC]=ovoD1}3R/TM3 to generate germline clones
(Chou and Perrimon, 1996
); with
males w; P{en2.4-GAL4}e22c P{UAS-FLP1.D}JD1/CyO; P{neoFRT}82B
ry506 to generate follicle cell clones; or with males w
P{GawB}elav[C155], P{UAS-eGFP} P{Hs-FLP}; P{neoFRT}82B
P{tubP-GAL80}LL3/TM6B to generate positively marked GFP somatic clones
(Lee et al., 2000
).
Antibodies and cDNAs
For immunostaining, the following primary antibodies were used: rabbit
anti-ß-galactosidase (1/1200); anti-GFPmAB 3E6 (1/500) and rabbit
anti-GFP (1/200; Molecular Probes); 7G10 anti-Fasciclin 3 (1/100,
Developmental Studies Hybridoma Bank); and rabbit anti-Medea (1/500; a gift
from Laurel Raftery). For western blot, the following antibodies were used:
guinea pig anti-E2f1 (1/2000; a gift from Terry Orr-Weaver); anti-E2f2 mei8
(1/2) and anti-Dp yun-6 (1/3) (Frolov et
al., 2001
); mouse monoclonal anti-CycE (1/10; a gift from Helena
Richardson); and mouse anti-hsp70 (1/5000, Sigma). The secondary antibodies
Cy3-conjugated donkey anti-mouse, Cy2-conjugated donkey anti-rabbit, donkey
anti-mouse-HRP and goat anti-rabbit-HRP were from Jackson ImmunoResearch;
anti-guinea pig-HRP was from Sigma. The Broad-Complex core domain
cDNA (Deng and Bownes, 1997
)
was kindly provided by Mary Bownes.
 |
Results
|
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Slimb expression during oogenesis
As a first step in the analysis of Slmb function in oogenesis, we wanted to
assess the cell types and stages in which the gene is expressed. We failed to
detect the transcript by in situ hybridization, suggesting that slmb
is expressed at very low levels. Although enhancer traps do not always
reproduce all aspects of endogenous transcription, they might provide a useful
and often more sensitive method for studying gene expression when transcripts
cannot be detected directly. We used the slmb00295
enhancer trap line (Jiang and Struhl,
1998
) that revealed a widespread and dynamic expression profile of
slmb during oogenesis. High levels of expression were observed in
nurse cells all throughout oogenesis (Fig.
1A). From the germarium until stage 8 of oogenesis, expression was
excluded from FC (Fig. 1B-D)
and at stage 9, low ß-galactosidase levels could be detected
(Fig. 1E). From stage 10
onwards, strong expression was recorded in patches of FC surrounding the
oocyte (Fig. 1F), being still
detected by the end of oogenesis (not shown). Remarkably, at stage 11 very
strong expression was observed in two dorsal patches of FC that give rise to
dorsal appendages (Fig. 1G)
(Deng and Bownes, 1997
;
Sapir et al., 1998
;
Wasserman and Freeman, 1998
).
As the expression pattern suggested a function for slmb in both the
germline and FC, we wanted to determine whether slmb loss of function
provokes defects in oogenesis.

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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).
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Slimb is required in both germline and follicle cells for egg chamber development
We tested three different slmb alleles: slmb1,
previously described as a hypomorphic allele; and slmb2
and slmb00295, reported to be null alleles
(Jiang and Struhl, 1998
;
Theodosiou et al., 1998
). As
in all cases homozygous or heteroallelic combinations were lethal at different
developmental stages (Jiang and Struhl,
1998
), we decided to examine the effect of slmb loss of
function in homozygous germline clones generated by the
FLP/OvoD method
(Chou et al., 1993
;
Chou and Perrimon, 1996
) or in
somatic clones induced with the FC driver e22C
(Duffy et al., 1998
) (see
Materials and methods). slmb00295 clones were recovered at
low rates, probably owing to poor cell viability
(Jiang and Struhl, 1998
);
slmb1 and slmb2 alleles exhibited
similar highly reproducible egg chamber defects and were used in most of the
experiments throughout this study. DAPI staining of egg chambers bearing
germline clones revealed that 17.4% of the follicles (n=483)
exhibited different kinds of abnormalities. To rule out the possibility that
some of the observed defects arise from an effect of the
OvoD itself, we focused our phenotypic analysis on
ovarioles with vitellogenic cysts, a situation never found in ovaries from
females carrying one copy of the OvoD transgene
(n=100 ovarioles). Germline phenotypes included follicles that
degenerated, exhibiting features of apoptosis (3.7%,
Fig. 2A,B), such as germ cell
nuclear fragmentation (Fig.
2C); consistent with this, TUNEL-positive staining at early stages
of oogenesis was recorded (data not shown). Another frequently observed
phenotype consisted of follicles displaying either increased (9.9%) or reduced
(1.7%) numbers of germ cells (Fig.
2D-F), as well as germ cell nuclei that were heterogeneous in size
(2.9%, Fig. 2G). These results
suggest that mitotic divisions, as well as the switch from mitosis to
endoreplication, might be misregulated in these cysts (see below). Remarkably,
in egg chambers with a reduced number of germ cells, the nuclei were bigger
than normal (see below Fig.
3E), suggesting that these cells failed to divide and, instead,
entered endoreplication prematurely. In another set of experiments, we
analyzed the effect of slmb loss of function in FC clones. Although
slmb00295 enhancer trap analysis indicated that expression
in FC apparently starts at stage 9 (Fig.
1E), we frequently observed two oocytes and extra nurse cells
within a single follicular epithelium (Fig.
2H). At vitellogenic stages, we never found supernumerary
follicles exhibiting one single oocyte, suggesting that the phenotype arise
from encapsulation defects rather than from an extra round of mitosis in the
cyst. Consistent with encapsulation defects, we observed that morphology of
mutant germaria was altered: in wild-type germaria region IIb, the cyst has
acquired a lens shape, spanning the whole width of the germarium
(Fig. 2I); instead,
slmb mutant ovarioles often exhibited germaria in which two or more
cysts were placed side by side in regions IIb and III
(Fig. 2J). In addition, egg
chambers with somatic clones often displayed an aberrant shape
(Fig. 2K), suggesting that
normal formation of the follicular epithelium was impaired. Moreover, we
observed that oocytes were frequently mispositioned within the follicle,
adopting an anterior (Fig. 2L)
or lateral (Fig. 2M) location,
indicating that axial polarity of the egg chamber was altered. Finally,
another category of phenotypes consisted of ovarioles lacking interfollicular
stalks between adjacent follicles but separated, instead, by a layer of
epithelial cells (Fig. 2N,O).
Previous work has shown that posterior localization of the oocyte depends on
the presence of a stalk that links the egg chamber with the neighboring older
follicle (Torres et al.,
2003
). We observed that in some of the follicles with mislocalized
oocytes, posterior interfollicular stalks were missing (9/15), while in others
posterior stalks were present (6/15), suggesting that alterations in axial
polarity in the latter egg chambers involved a different mechanism.
As Slmb is a substrate recognition subunit of an SCF complex
(Bocca et al., 2001
;
Feldman et al., 1997
;
Skowyra et al., 1997
;
Yaron et al., 1998
), mutations
affecting other components of the complex or molecules required for its
ubiquitin ligase activity should render overlapping biochemical effects and
similar phenotypes in oogenesis. We induced cullin1 (lin19
FlyBase) (dCul110494)
(Bocca et al., 2001
;
Filippov et al., 2000
;
Heriche et al., 2003
;
Khush et al., 2002
) general
clones by the Hs-FLP/FRT method that rendered ovarioles with cyst
encapsulation defects, lack of interfollicular stalks and nurse cell nuclei of
heterogeneous size (data not shown). Moreover, mutations affecting the
subunits CSN4 and CSN5 of the COP9 signalosome, a highly conserved complex
that regulates the activity of SCF complexes
(Bech-Otschir et al., 2002
;
Schwechheimer and Deng, 2001
;
Seeger et al., 2001
), have
recently been reported to cause phenotypes that are also very similar to those
provoked by slmb mutations
(Doronkin et al., 2002
;
Doronkin et al., 2003
). To
study if Slmb might be a component of an SCF complex in the ovary, we looked
for genetic interactions in egg chambers with slmb2
germline clones that were at the same time heterozygous for
dCul112764 or CSN5L4032 alleles.
Strong interactions were detected, as the proportion of egg chambers with
extra germ cells increased from 9.9% (n=483) in ovaries with
slmb2 homozygous clones to 32.1% in those with
dCul112764/+; slmb2/slmb2
clones (n=156) and to 25.4% in ovaries with
CSN5L4032/+; slmb2/slmb2
clones (n=130). Moreover, the phenotypes were enhanced in these
genotypic combinations, as egg chambers with more than 32 germ cells and
enlarged germaria (Fig. 2P)
were observed in addition to the phenotypes described above. These results
suggest that Slmb functions as a component of an SCF complex in oogenesis.

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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).
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Control of germ cell divisions is impaired in slimb germline clones
Phenotypes described in Fig.
2E,F suggested that slmb loss of function in the germline
might cause an impairment in the regulation of mitotic divisions that
originate the cyst (de Cuevas et al.,
1997
). Among follicles with extra germ cells (n=38), two
types of egg chambers could be recognized: those with one single oocyte
(16/38) and those with two oocytes (22/38). In order to determine if extra gem
cells arise from an additional round of mitotic divisions, we analyzed the
pattern of ring canals in these cysts. In wild-type cysts, four rounds of
mitotic divisions take place, giving rise to oocytes with four ring canals
clearly visible by phalloidin staining
(Fig. 3A)
(Hawkins et al., 1996
). By
contrast, in slmb germline clones, all oocytes from follicles with
extra nurse cells and one single oocyte exhibited five ring canals instead of
four (16/16; Fig. 3B),
indicating that an extra round of mitosis occurred. However, all the observed
egg chambers bearing two oocytes exhibited four ring canals per oocyte (22/22;
Fig. 3C,D), suggesting that in
these cases mitotic divisions were normal. We believe that the latter
phenotype did not reflect slmb loss of function in the germline but
instead was due to the occurrence of slmb FC clones that provoked
defects in cyst encapsulation as described above. Interestingly, egg chambers
with fewer than 16 germ cells (n=8) exhibited a number of ring canals
that was always consistent with the predicted rounds of cell divisions (i.e.
two cells=one ring canal; four cells=no more than two ring canals per cell,
etc.) (Fig. 3E). From these
experiments, we hypothesized that Slmb is required in the germline for the
regulation of mitotic cycles during cyst formation.
It has been reported that Drosophila Dp (Dp), which forms
heterodimers with E2f transcription factors (E2f1 and E2f2), is required for
essential processes during oogenesis. Dp mutant germline clones often
execute an extra round of mitosis, giving rise to follicles with extra nurse
cells (Myster et al., 2000
;
Royzman et al., 2002
;
Taylor-Harding et al., 2004
).
To gain insights about the mechanism underlying misregulation of the cell
cycle in slmb germline clones, we performed western blot analysis of
Dp, E2f1 and E2f2. As can be seen in Fig.
3F, E2f1 protein levels were not affected in slmb mutant
ovaries but, by contrast, a major decrease in E2f2 and Dp levels occurred. As
a Slmb direct target is expected to be increased in slmb mutant
clones, we believe that the reduction of Dp and E2f2 probably occurs through
an indirect mechanism.

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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.
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Slimb somatic clones cause defects in differentiation of follicle cells and generate ectopic polar cells
In the germarium, a lineage of intercyst FC differentiates into two
subpopulations that stop dividing and originate stalk and polar cells
(Bai and Montell, 2002
;
Margolis and Spradling, 1995
;
Tworoger et al., 1999
). The
lack of interfollicular stalk cells observed in slmb loss-of-function
ovarioles suggested that differentiation of FC into different subpopulations
might be impaired. In wild-type germaria, undifferentiated FC express high
levels of Fasciclin 3 (Fas3), and by oogenesis stage 4, expression becomes
restricted to polar cells (Fig.
4A). In slmb somatic clones refinement of Fas3 expression
was often delayed (Fig. 4B),
being still detected in large patches of cells beyond oogenesis stage 9
(Fig. 4C). In order to examine
cell autonomy of this effect, clones positively marked with GFP were generated
by using the MARCM system (Lee et al.,
2000
). As can be seen in Fig.
4C-E, Fas3 labeling largely overlaps with cell patches expressing
GFP, indicating that the effect is cell autonomous. In some mosaic egg
chambers, Fas3 expression refined into a pattern that seemed to correspond to
ectopic polar cells and, interestingly, these cells were mutant for
slmb, as indicated by GFP positive staining
(Fig. 4C-E, arrowheads). As
Fas3 is not a specific polar cell marker
(Lopez-Schier and St Johnston,
2001
; Zhang and Kalderon,
2000
), we used the PZ80 enhancer trap that is specifically
expressed in these cells (Fig.
4F-H) (Karpen and Spradling,
1992
; Spradling,
1993
). In wild-type follicles, anterior polar cells induce
differentiation of five to eight FC into a border cell fate. The whole border
cell cluster, including polar cells, expresses the slow border cells
gene (slbo), as revealed by expression of the slbo-lacZ
enhancer trap (Fig. 4I) (Grammont, 2002; Liu and Montell,
1999
; Montell et al.,
1992
; Rorth et al.,
2000
). At oogenesis stage 9, polar cells surrounded by the whole
cluster start migrating posteriorly between nurse cells
(Fig. 4G), and by stage 10 they
have reached the anterior border of the oocyte
(Fig. 4H). In slmb
mutant egg chambers we clearly observed the occurrence of ectopic polar cells
(Fig. 4J) and consistent with
this, ectopic clusters of border cells differentiated
(Fig. 4K). Interestingly,
migration of border cell clusters was often impaired in these follicles
(Fig. 4J,K).
Slimb is required for chorion patterning
Eggs laid by females in which slmb mutant clones were induced with
the Hs-FLP driver exhibited diverse chorion patterning defects. Some
of them, like a smaller size or abnormal shape of the egg
(Fig. 5A; data not shown)
probably derive from dumping defects or from alterations in the general
morphology of the egg chamber (Fig.
2K). Interestingly, we also observed a wide array of eggshell
phenotypes in which the size, shape and/or position of dorsal appendages (DA)
was altered. In wild-type eggs, the dorsoanterior region of the chorion
displays a pair of filaments (DA) separated by a gap
(Fig. 5B). Some eggshells
derived from slmb mosaic ovaries had a dorsalized phenotype
(Sapir et al., 1998
;
Wasserman and Freeman, 1998
)
characterized by laterally expanded DA
(Fig. 5C) or in more severe
cases, by DA surrounding all the anterior circumference of the egg
(Fig. 5D). In other cases,
ectopic DA formed (Fig. 5E) or
DA deranged, giving rise to ramified structures
(Fig. 5F). Another category of
phenotypes was that of eggshells apparently ventralized
(Schupbach, 1987
;
Wasserman and Freeman, 1998
),
with DA partially or totally fused (Fig.
5G,H). Finally, in the last category, eggshells exhibited a
slightly enlarged operculum [with DA shifted to a more posterior site
(Fig. 5I)] or a greatly reduced
amount or absence of DA material (Fig.
5J,K).

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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).
|
|
In order to distinguish which chorion phenotypes arise from slmb
loss of function in the germline and which ones from clones in the follicular
epithelium, we induced clones through the OvoD method or
the FC driver e22C. The latter method caused the same DA phenotypes described
above, indicating that abnormalities reflected slmb loss of function
in the follicular epithelium. By contrast, eggs of smaller size, which derived
from dumping defects (data not shown), were not observed upon induction of
somatic cell clones but rather, were laid by females with germline clones (8%;
n=902), indicating that this phenotype was due to Slmb loss of
function in the germline.

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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.
|
|
Mild overexpression of Dpp in follicle cells mimics slimb loss-of-function phenotypes
It has been reported that high levels of Dpp at the anterior-most region of
the chorion prevent DA formation, leading to differentiation of the operculum
(Peri and Roth, 2000
;
Twombly et al., 1996
). As
eggshells derived from follicles with slmb somatic clones exhibited
phenotypes resembling those that occur upon overactivation of the Dpp pathway
(Fig. 5I), we decided to
perform further ectopic Dpp expression experiments and look for novel eggshell
phenotypes and for abnormalities in egg chamber development. We used various
Gal4 drivers and UAS constructs for inducing the Dpp pathway at different
levels. At 25°C all crosses were lethal at pre-imaginal stages and only at
18°C some of the tested combinations rendered viable adults that allowed
ovary and chorion analysis. As depicted in
Table 1, in the strongest
viable combinations overexpressing Dpp, opercula expanded dramatically and DA
were absent (Fig. 6A) as was
previously reported (Peri and Roth,
2000
; Twombly et al.,
1996
). In these cases, we observed germaria with tumorous arrays
of germline stem cells (not shown) (Xie
and Spradling, 1998
) as well as aberrant follicles with large
numbers of germ cells engulfed in the same follicular epithelium
(Fig. 6B). Interestingly, upon
milder induction of the Dpp pathway, we observed variable DA phenotypes, very
similar to those caused by slmb loss of function in somatic cells
(Table 1). These included
eggshells exhibiting one thin dorsal filament
(Fig. 6C), fused or ramified DA
(Fig. 6D,E), DA spanning the
whole anterior circumference of the egg
(Fig. 6F) and reduced DA
(Fig. 6G). Similar phenotypes
were observed (1) upon overexpression of an activated form of the type I
receptor Thick veins (not shown); (2) by overexpressing the co-Smad protein
Medea (Fig. 6H); and, (3) in
low proportion, in eggs laid by females heterozygous for short
gastrulation (sog) or for daughters against dpp
(dad) genes (Fig. 6I;
Table 1), which encode negative
regulators of the pathway (Araujo and Bier,
2000
; Francois et al.,
1994
; Tsuneizumi et al.,
1997
). When we analyzed ovarioles from females subjected to the
same mild overexpression conditions of Dpp, we observed a wide array of
phenotypes similar to those observed in slmb mutant somatic clones.
These included abnormal germaria, two complete cysts encapsulated within a
single follicular epithelium, mislocalized oocytes and lack of interfollicular
stalks between adjacent follicles (Fig.
6J-M). In order to determine whether loss of Slmb might cause
increased activation of the Dpp pathway leading to the above phenotypes, we
initially tested whether overexpression of Slmb was able to antagonize the
effect of ectopic Dpp on eggshell morphogenesis. Indeed, Slmb overexpression
rendered full reversion of the `expanded operculum' phenotype (n=376;
Fig. 6A) of eggs laid by
females overexpressing Dpp under control of a CY2-gal4 driver
(Table 1); no rescue was
observed when an UAS-EGFP transgene was overexpressed instead. Thus,
based on similarities of slmb loss-of-function phenotypes with those
caused by Dpp mild overexpression and, considering that the strong ectopic Dpp
eggshell phenotype could be rescued by simultaneous overexpression of Slmb, we
hypothesized that Slmb negatively regulates Dpp pathway in ovarian somatic
cells.

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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).
|
|
Ectopic activation of the Dpp pathway occurs in slmb loss-of-function follicles
In stage 10 wild-type egg chambers, the intersection of Dpp and EGFR
pathways induces the expression of the Broad-Complex (BR-C)
gene in two patches of dorsoanterior FC
(Fig. 7A), promoting DA
specification and positioning (Deng and
Bownes, 1997
; Peri and Roth,
2000
). We found that in egg chambers bearing slmb FC
clones the BR-C pattern was altered, being in some cases expanded
(Fig. 7B), whereas in others it
was reduced (Fig. 7C) or
partitioned (Fig. 7D). As we
did not detect alterations in the EGFR pathway (data not shown), we analyzed a
possible effect of slmb mutations on Dpp signaling, by using the
dad1883 enhancer trap (dad-LacZ)
(Casanueva and Ferguson, 2004
).
In wild-type ovarioles, expression of dad-lacZ is first detected at
stage 8 in terminal anterior FC that migrate towards the oocyte
(Fig. 7E). By oogenesis stage
10, dad-LacZ expression is detected in centripetal and border cells
located at the anterior border of the oocyte, as well as in stretched cells
which surround the nurse cells (Fig.
7F). Analysis of dad-lacZ in ovaries bearing
slmb mutant clones revealed that expression started prematurely at
stage 6 (Fig. 7G) and later on,
at stages 9-10, the pattern was often expanded posteriorly
(Fig. 7H,I). These results
suggest that loss of slmb might cause ectopic activation of the Dpp
pathway. The ubiquitin/proteasome system regulates the stability of various
Smad proteins through the activity of different E3 ubiquitin ligases
(Casanueva and Ferguson, 2004
;
Fukasawa et al., 2004
;
Fukuchi et al., 2001
;
Li et al., 2004
;
Liang et al., 2004
;
Wan et al., 2004
). Because in
mammalian cell culture proteasomal degradation of Smad4, a common signal
transducer in the TGFß signaling pathway, is regulated by the Slmb
ortholog, ßTrcp1 (Wan et al.,
2004
), we decided to explore whether a similar mechanism operates
in the Drosophila ovary. We initially analyzed the expression of the
Drosophila Smad4 homologue Medea
(Sutherland et al., 2003
) in
wild-type follicles. At stage 8-9, expression was restricted to migrating
anterior cells (Fig. 7J) and at
stage 10 expression could be observed in stretched cells as well as in all FC
surrounding the oocyte, being remarkably stronger in the centripetally
migrating FC, located at the anterior border of the oocyte
(Fig. 7K). Interestingly, in
egg chambers bearing slmb clones, ectopic patches expressing high
levels of Medea were observed (Fig.
7L,M). Taken together, these results suggest that at least some of
the slmb mutant phenotypes might have been caused by increased levels
of Medea protein, which led to overactivation of the Dpp pathway.

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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.
|
|
 |
Discussion
|
---|
Slimb function in the germline
We have shown that the F-box protein Slmb is required for oogenesis in both
the germline and FC. In the germline, we found that Slmb plays a role in the
control of mitotic cycles during cyst formation, in regulation of nurse cell
endoreplication and in nurse cell dumping. Recent reports have demonstrated
that Slmb can control cell cycle progression in different experimental
settings. It has been reported that, following DNA replication, Slmb is
required in larval wing discs for proteolysis of a specific cell cycle
modulator: the transcription factor E2f1
(Heriche et al., 2003
).
Remarkably, the E2f complex was implicated in cell cycle control of ovarian
germ cells, in nurse cell transition from polyteny to polyploidy and in
dumping of the nurse cell content into the oocyte
(Myster et al., 2000
;
Royzman et al., 2002
). In this
study, we found that two subunits of the E2f complex, Dp and E2f2, were
downregulated in ovaries bearing slmb germline clones, while E2f1 did
not change. Differences in Cyclin E levels, another cell cycle regulator
involved in cyst formation (Doronkin et
al., 2003
; Lilly and
Spradling, 1996
), could not be detected in these clones (data not
shown). We want to stress the good correlation that exists between the
phenotypes observed in slmb germline clones (this study) and in
Dp germline clones (Myster et
al., 2000
), as in both cases an additional round of cystocyte
mitotic divisions occurs. In order to understand the molecular mechanism
causing Dp and E2f2 reduction in slmb germline clones, a detailed
analysis of the alterations of the network regulating the cell cycle is
required.
Slimb function in follicle cells
Although expression levels in somatic cells in the germarium are too low to
be detected through an enhancer trap or by in situ hybridization,
loss-of-function experiments suggest that slmb is needed in these
cells for normal morphogenesis of the egg chamber and for encapsulation of the
cyst. In addition, our results suggest that Slmb is required for timely
differentiation of FC that is reflected by the refinement of Fas3 expression;
this is accompanied by the occurrence of ectopic polar cells, lack of
interfollicular stalks and disruption of normal egg chamber polarity. Later in
oogenesis, Slmb is expressed at high levels in FC surrounding the oocyte and
participates in chorion patterning, contributing to define the shape and
position of DA.
It has been reported that slmb mutant clones induce ectopic
activation of the Hedgehog (Hh) pathway in limb discs
(Jiang and Struhl, 1998
;
Theodosiou et al., 1998
).
Notably, some of the phenotypes observed upon slmb somatic clone
induction were similar to those originated by overactivation of the Hh pathway
in FC. These include a delay in FC differentiation, development of ectopic
polar cells and mislocalization of the oocyte
(Forbes et al., 1996a
;
Zhang and Kalderon, 2000
).
Nevertheless, excessive activation of the Hh pathway also causes FC
over-proliferation that results in excess of undifferentiated somatic cells
that form very long interfollicular stalks between egg chambers
(Forbes et al., 1996a
;
Forbes et al., 1996b
;
Zhang and Kalderon, 2000
). By
contrast, we observed that slmb loss of function in FC caused a lack
rather than an excess of interfollicular cells. Finally, dominant genetic
interactions were not detected between slmb and negative regulators
of Hh pathway and the ptc-LacZ enhancer trap, which was reported to
be activated in FC by the Hh pathway
(Forbes et al., 1996b
), was
not induced ectopically in slmb mutant clones (data not shown). These
results indicate that, despite some similarities between slmb
loss-of-function and hh gain-of-function phenotypes, Slmb is unlikely
to be a negative regulator of Hh pathway during oogenesis.
In limb discs, Slmb was also reported to be a negative regulator of the Dpp
pathway, although the molecular mechanism involved is still unclear
(Miletich and Limbourg-Bouchon,
2000
; Theodosiou et al.,
1998
). We have shown that mild overexpression of Dpp caused a wide
spectrum of phenotypes that were largely coincident with those caused by
slmb loss of function in FC. Supporting the idea that loss of
slmb might cause hyperactivation of the Dpp pathway, the strongest
chorion phenotypes originated by overexpression of Dpp were completely
antagonized by simultaneous overexpression of Slmb in FC. Moreover, expansion
of dad-lacZ expression occurred in slmb mutant follicles,
further suggesting that ectopic induction of the Dpp pathway indeed occurs as
a consequence of slmb loss of function. Consistent with this, we
found that a downstream component of the Dpp pathway, the co-Smad protein
Medea, was upregulated in slmb mutant egg chambers. Because in
mammalian cell culture it was demonstrated that Smad4 is a direct target of
the mammalian Slmb ortholog, ßTrcp1
(Wan et al., 2004
), we believe
that Medea could be a direct target of Slmb. Further molecular work is
required to assess whether this is indeed the case or if alternatively, the
effect of Slmb on Medea is indirect.
 |
ACKNOWLEDGMENTS
|
---|
We thank Maki Asano, Konrad Basler, Mary Bownes, Stephen Cohen, Nick Dyson,
Jing Jiang, Bernardette Limbourg-Bouchon, Stuart Newfeld, Terry Orr-Weaver,
Laurel Raftery, Helena Richardson, Francois Rouyer, Danny Segal, Benny Shilo,
Allan Spradling, David Stein, Tian Xu, the Bloomington Drosophila
Stock Center and the DSHB for stocks and/or antibodies. We thank Helena Araujo
and Amir Sapir for technical advice; and Hernán López-Schier,
Fernanda Ceriani, Lázaro Centanín and members of P.W.'s
laboratory for discussion. Wellcome Trust Grant 070161/Z/03/Z, Universidad de
Buenos Aires X411 and ANPCyT 01-10839. M.M. is a fellow and P.W. is a career
investigator of CONICET.
 |
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