Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
* Author for correspondence (e-mail: gschupbach{at}molbio.princeton.edu)
Accepted 26 August 2003
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SUMMARY |
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Key words: CTP: phosphocholine cytidylyltransferase, Cct1, Drosophila, Oogenesis, Follicle cells
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Introduction |
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Oogenesis begins at the anterior tip of the ovary in the region called the
germarium when a germline stem cell divides to form another stem cell and a
daughter cystoblast [for a review of oogenesis, see Spradling
(Spradling, 1993)]. The
maintenance and division of the germline stem cells require both intracellular
mechanisms and intercellular communication between the germline cells and
three somatic cell types at the anterior of the germarium: terminal filament
cells, cap cells and inner germarial sheath cells (reviewed by
Xie and Spradling, 2001
).
After division of a germline stem cell, the daughter cystoblast undergoes four
rounds of division with incomplete cytokinesis to produce a cyst of 16
interconnected cells. One of the germline cells differentiates into the oocyte
and the other 15 cells become nurse cells. As the germline cyst moves through
the germarium, it becomes surrounded by a layer of somatically derived
follicle cells. During this time, in a process mediated by cadherin- and
ß-catenin-dependent adhesion between the oocyte and follicle cells, the
oocyte becomes positioned at the posterior of the germline cyst
(Godt and Tepass, 1998
;
González-Reyes and St Johnston,
1998
; Peifer et al.,
1993
), thus establishing the anterior-posterior axis of the egg
chamber.
The anteroposterior polarity of the oocyte itself is not established until
mid-oogenesis when Gurken (Grk), a TGF-like ligand, is localized to the
posterior of the oocyte and signals to the overlying follicle cells via
torpedo, the Drosophila EGF receptor, to specify a posterior
follicle cell fate (González-Reyes
et al., 1995
; Roth et al.,
1995
). These follicle cells then signal back to the oocyte,
resulting in a reorganization of the microtubule network within the oocyte and
establishment of its anteroposterior polarity
(Ruohola et al., 1991
;
Theurkauf et al., 1992
). Those
follicle cells at the opposite end of the egg chamber that do not receive a
signal from the oocyte acquire an anterior cell fate. Two signaling pathways
appear to play important roles in anterior patterning. The Janus kinase
(JAK-STAT) pathway is required for the specification of the anterior follicle
cell fate (Xi et al., 2003
),
while the TGFß pathway is required slightly later in oogenesis for
correct patterning of the anterior region of the chorion
(Twombly et al., 1996
).
After signaling to the posterior follicle cells, Grk protein becomes
localized to the future dorsoanterior of the oocyte where it again signals to
the overlying follicle cells via the EGF receptor, this time to establish a
dorsal cell fate (Neuman-Silberberg and
Schüpbach, 1993). Activation of the EGF receptor in the
dorsoanterior follicle cells leads to expression of a number of genes
including broad-complex, rhomboid and argos
(Deng and Bownes, 1997
;
Ruohola-Baker et al., 1993
;
Wasserman and Freeman, 1998
),
which are required for the differentiation of the dorsoanterior follicle cells
in the complex pattern that gives rise to the two dorsal appendages and
operculum.
Although the follicle cells are essential for many processes throughout
oogenesis, it has been difficult to identify genes acting in these cells
through traditional female sterile screens. Many of the genes required in the
follicle cells are also required in other populations of somatic cells early
in development, and as a result, mutations in these genes are often lethal. An
alternative approach to the identification of genes important for patterning
of the egg is to screen for gene expression in particular subsets of follicle
cells. We used enhancer traps to screen for genes that are specifically
expressed in the anterior follicle cells. Through this analysis, we identified
a gene, Drosophila Cct1, that is required in the follicle cells for
anterior patterning, as well as a number of other processes during oogenesis
and ovarian morphogenesis. CTP: phosphocholine cytidylyltransferase (CCT) is
the second of three enzymes in the CDP-choline pathway, through which
phosphatidylcholine is synthesized. CCT catalyzes the conversion of
phosphocholine to CDP-choline, which is a rate-limiting step in the pathway.
Phosphatidylcholine is a major lipid component of all eukaryotic cell
membranes and is the second most abundant phospholipid in Drosophila
cell membranes (Jones et al.,
1992). We have identified two Drosophila homologs of
CCT (Drosophila Cct1 and Cct2) and found that
Cct1 plays unexpected roles in a number of developmental processes.
We describe the effects of mutations in Cct1 on oogenesis and ovarian
morphogenesis.
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Materials and methods |
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Plasmid rescue and cloning of rescue construct
A 0.7kb genomic DNA fragment flanking the P-element BN81 was isolated
by plasmid rescue and sequenced. Blast searches of the Drosophila
genome revealed that BN81 is inserted in the 5'UTR of Cct1,
which is predicted to map to 62A5 based on molecular mapping. Mutations were
generated by excision of the P-element and mapped by PCR analysis. The genomic
rescue construct contains an 11.194 kb BamHI-XhoI fragment
isolated from BDGP P1 clone DS05969 (nucleotides 1371824912)
(Kimmerly et al., 1996
). The
genomic fragment was cloned into pCasper4
(Thummel and Pirotta,
1992
).
After the completion of our experiments, a new gene CG32313 was predicted to map 5' to Cct1 by the BDGP (Fig. 1A). The presence of CG32313, however, should not affect our results because the phenotypes that we described have been observed using the null allele Cct1179, which disrupts only Cct1 (Fig. 1A), and in transheterozygous combinations of this allele with homozygous viable Cct1 alleles.
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In situ hybridizations, X-gal staining, and immunocytochemistry
BDGP EST clone LD34141 (Rubin et al.,
2000) was used to make the Cct1 probes. LD34141 is a
2.49kb full-length cDNA that contains the entire open reading frame and should
detect all Cct1 transcripts. BDGP EST clone GH25855 is a full-length
cDNA that was used to make the Cct2 probes. Digoxigenin-labeled
probes were made using the DIG RNA labeling kit (Boehringer Mannheim). Ovaries
for in situ hybridizations were dissected in PBS and fixed in 4%
paraformaldehyde, 10% DMSO and three volumes of heptane for 20 minutes at room
temperature. Subsequent steps were carried out according to a modified version
of Tautz and Peifle (Tautz and Peifle, 1989), hybridizing at 55°C.
Adult ovaries with GFP-marked clones and ovaries for Arm antibody staining
were dissected in PBS and fixed by incubation in 3.7% formaldehyde for 10
minutes. Monoclonal Arm antibody N2-7A1 was used at 1:5 (gift from E.
Wieschaus) (Peifer et al.,
1994). Adult ovaries used for all other antibody staining were
dissected in PBS and fixed in 4% paraformaldehyde and three volumes of heptane
for 20 minutes at room temperature. Pupal ovaries were dissected in PBS and
fixed in 4% paraformaldehyde for 1 hour at room temperature. Rat anti-Enc
antibody was used at 1:1000 (Van Buskirk
et al., 2000
). Fas3 monoclonal 7G10 was used at 1:4 (Developmental
Studies Hybridoma Bank). Monoclonal Orb antibodies 4H8 and 6H4 were each
diluted 1:60 and mixed together (gift from P. Schedl)
(Lantz et al., 1994
).
-Spectrin monoclonal antibody 3A9 was used at a dilution of 1:40
(Developmental Studies Hybridoma Bank). Rabbit anti-ß-Galactosidase
antibody was used at 1:2000 (Chemicon). Rabbit anti-phosphorylated SMAD (PS1)
was used at 1:650 (gift from T. Tabata)
(Tanimoto et al., 2000
).
Rabbit anti-Vasa was used at 1:2000 (gift from P. Lasko). Myc
expression was induced by heat shocking adult flies at 37°C for 45-60
minutes (Xu and Rubin, 1993
).
Mosaic ovaries were stained with the monoclonal anti-Myc antibody 9E10
(Oncogene) diluted 1:50. All secondary antibodies were obtained from Molecular
Probes and used at 1:1000. Oregon Green and Alexa Fluor 546 phalloidin were
used at 1:500 (Molecular Probes). Hoechst was used at 1 µg/ml (Molecular
Probes).
Ovaries stained for ß-galactosidase activity were fixed in 2.5%
glutaraldehyde for 10-12 minutes and stained as described previously
(Ashburner, 1989).
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Results |
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Mutations in Cct1 and Cct2 were generated by imprecise excision of the P-element insertion BN81. The breakpoints of the deletions were then mapped by PCR revealing two classes of mutation: deletions 5' to the P-element, which are homozygous viable, and deletions 3' to the P-element, which are homozygous lethal (Fig. 1A). The lethal excision Cct1179 is a 5.02 kb deletion that includes the translation start codon, and therefore, is a molecular null allele (Fig. 1A). The lethality of this allele, which disrupts only Cct1, can be rescued with a genomic rescue construct that includes all of Cct1 and part of Cct2 (Fig. 1A). Cct1299 is a large deletion (>12.9 kb) that includes both Cct1 and Cct2 (Fig. 1A). The viability of the Cct199 and Cct1124 excisions, which presumably delete the entire promoter region of Cct1, is most probably due to the presence of alternate promoters in the first intron of the gene, based on the existence of EST clones with transcription start sites in this intron (Fig. 1A). Expression from these promoters appears to be sufficient to rescue the larval lethality of mutations in Cct1, but not sufficient to fully rescue the ovarian phenotypes. All phenotypes described in this paper were seen in ovaries from Cct1BN81 and Cct199 homozygous flies as well as in all transheteroallelic combinations tested.
Cct1 mRNA is expressed in a spatially restricted manner
during oogenesis
The enhancer trap BN81 is expressed in the border cells, the squamous nurse
cell associated follicle cells and the centripetally migrating follicle cells
(Fig. 2A). However, RNA in situ
hybridization using a Cct1 antisense probe revealed that the BN81
insertion only partially reflects the expression of Cct1 in the
ovary. High levels of Cct1 mRNA expression can first be detected in
region 2B of the germarium in the follicle cells that migrate to surround the
flattened germline cyst and separate it from the preceding cyst
(Fig. 2B). Expression of
Cct1 is next observed in a group of anterior follicle cells beginning
at stage 7. During mid-oogenesis, the follicle cells migrate towards the
posterior of the egg chamber so that by stage 10, all but 50 of the
anterior-most follicle cells are surrounding the oocyte. During stage 10B,
Cct1 expression is detected in those remaining 50 follicle cells that
are now stretched over the nurse cells and in a group of follicle cells that
migrate centripetally between the nurse cells and the oocyte
(Fig. 2C). This expression
pattern is the same as that of the BN81 enhancer trap, with the exception of
expression in the border cells, which we cannot detect by in situ
hybridization. Finally, Cct1 is expressed during stages 12-13 in the
two groups of dorsal anterior follicle cells that secrete the dorsal
appendages and in the follicle cells at the posterior of the egg chamber that
secrete the aeropyle (Fig. 2D).
Expression of Cct1 is also detectable in the germline throughout
oogenesis. Although it is difficult to determine expression levels in the
germline cells by in situ hybridization, Cct1 expression in the
oocyte and nurse cells appears to be high early in oogenesis and low during
mid- and late oogenesis compared with the expression of Cct1 in the
follicle cells (Fig. 2B-D).
Cct2 expression could not be detected in the ovary by either Northern
or in situ hybridization analysis (data not shown).
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In order to determine whether Cct1 mutations cause defects in oocyte positioning by affecting shg/arm mediated adhesion, antibodies to each protein were used to look at their expression levels in mutant egg chambers. As in wild-type, Cct1 mutant ovaries with mispositioned oocytes have an accumulation of both DE-cadherin and Arm proteins in the anterior and posterior follicle cells, suggesting that mutations in Cct1 are not causing mispositioning by significantly reducing levels of DE-cadherin or Arm (Fig. 5A-D and data not shown).
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GFP-marked clones were then induced during the larval stages using heatshock-flipase to allow us to examine egg chambers with completely mutant follicle cell epithelia as well as egg chambers in which both the germline and entire follicle cell layer were mutant. When clones were generated in this manner, no mispositioned oocytes were observed when the germline was completely mutant (n=101). By contrast, 1.5% of egg chambers with completely mutant follicle cell layers contained mispositioned oocytes (n=264; Fig. 4I) Two egg chambers with wild-type germline cells and mosaic follicle cell epithelia also contained mispositioned oocytes. Interestingly, when both the germline and follicle cell layer were completely mutant, egg chamber development appeared to arrest during mid-oogenesis, although it was still possible to determine the position of the oocyte. We found that when the germline and follicle cell epithelium were both mutant, 11% of oocytes were mispositioned (n=54; Fig. 4J), which is similar to the frequency of mispositioned oocytes seen in egg chambers from Cct1BN81 homozygous mutant females (see above). These data indicate that Cct1 is expressed in both the germline and follicle cells but that expression in either population of cells is generally sufficient to ensure correct positioning of the oocyte. At a very low frequency, however, partial or complete follicle cell clones can also result in mispositioning, suggesting that the Cct1 acts primarily in the follicle cells.
Mutations in Cct1 affect ovarian morphogenesis
Ovaries from wild-type females consist of ovarioles that are attached near
the oviduct but are otherwise fully separated from each other by epithelial
sheaths that are formed during pupation. These sheaths are composed of muscle
tissue and epithelial cells that secrete a thick basement membrane
(Mahowald and Kambysellis,
1980). In ovaries from females homozygous for mutations in
Cct1, we have observed a novel `branched ovariole' phenotype, in
which multiple ovarioles are attached to one egg chamber
(Fig. 6A). This phenotype is
often seen in ovaries in which some of the ovarioles are separated from each
other as in wild type. In wild-type ovaries, the initial separation of the
ovarioles occurs during metamorphosis, suggesting that the branched ovariole
phenotype may be the result of a requirement for Cct1 during ovarian
morphogenesis. Cct1 mutant ovaries also contain ovarioles that are
curled up within the ovariolar sheath (Fig.
6D) rather than stretched out as in wild-type ovaries
(Fig. 6C). This phenotype has
been previously described for mutations in Drosophila Wnt4 and
components of the Drosophila Wnt4 cell motility pathway and has been
shown to be caused by a delay in the migration of the apical cells during
ovarian morphogenesis that results in a shortened ovariolar sheath
(Cohen et al., 2002
). Given the
similarity between the Drosophila Wnt4 and Cct1 mutant
phenotypes, we reexamined ovaries from flies mutant for Drosophila
Wnt4C1 and found that they also have a branched ovariole
phenotype, which had not been previously described
(Fig. 6B).
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To test whether Cct1 is required in the apical cells for their migration, positively marked apical cell clones mutant for Cct1179 were generated, and their position was examined in ovaries 20 hours APF. Mutant cells appeared to migrate as in wild type, separating the ovarioles and allowing formation of the basal stalks (Fig. 7H). As with the follicle cell clones, this result suggests a non-autonomous effect of Cct1 in the apical cells or a requirement in another population of cells.
Cct1 is required for formation of the operculum
Although females mutant for Cct1 have reduced fecundity, a
sufficient number of eggs are laid to enable us to examine the requirement for
Cct1 in eggshell patterning. One eggshell defect observed is a
reduction in the average length of the operculum. This reduction in length was
quantified by measuring the angle between a line drawn horizontally through
each egg and a line drawn from the anterior-most tip to the posterior-most end
of the operculum. The angles for all the eggs were then averaged. Larger
angles indicate shorter opercula. Eggs from OreR females had an average angle
of 26.58° (±0.82°, n=24)
(Fig. 8A). By contrast, eggs
from females transheterozygous for Cct1 alleles 124 and 299 had an
average angle of 40.22° (±0.60°, n=89)
(Fig. 8B). Thus, a reduction in
the level of Cct1 results in a significant reduction in operculum
length.
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In order to determine whether Cct1 affects operculum length by affecting dpp signaling directly, we examined the expression of an activated form of Mad, using an antibody to the phosphorylated form of the protein (pMAD). In wild-type ovaries, pMAD expression is seen in the centripetal follicle cells during stage 10B. In follicle cell clones mutant for Cct1 (Cct1179 or Cct1299) pMAD expression is still seen in the centripetal follicle cells, indicating that Cct1 most probably does not directly affect the dpp signal to the anterior follicle cells (Fig. 8F). Instead, it probably acts farther downstream and partially independently of dpp to affect operculum formation.
Cct1 expression is regulated by Egfr signaling
In wild-type ovaries, Cct1 is expressed during stages 12 and 13 in
the two groups of dorsoanterior follicle cells that secrete the dorsal
appendages and in the follicle cells at the posterior of the egg chamber that
secrete the aeropyle (Fig. 9A).
Follicle cells respond to grk signaling through the EGF receptor in a
dose-dependent manner, so that a dorsal appendage cell fate is repressed in
the cells at the dorsal midline, which initially receive the highest level of
grk signal, and activated in the more lateral follicle cells, which
initially receive less grk signal (reviewed by
Van Buskirk and Schüpbach,
1999). To determine whether the expression of Cct1 during
stages 12 and 13 is downstream of and regulated by the Egfr pathway,
Cct1 expression was examined in two genetic backgrounds in which the
normal pattern of EGF receptor activation in the dorsoanterior follicle cells
is disrupted. In fs(1)K10 mutants, grk mRNA and protein are
expressed in a ring around the anterior of the oocyte, rather than tightly
localized at the future dorsoanterior
(Roth and Schüpbach,
1994
). This results in activation of the EGF receptor around the
entire anterior circumference of the oocyte. In a fs(1)K10 mutant
background, Cct1 is expressed in a ring of follicle cells around the
anterior of the oocyte, and the gap between the two domains of Cct1
expression increases, consistent with the ectopic activation of the EGF
receptor in the anterior follicle cells
(Fig. 9B). Cct1
expression was also examined in ovaries from flies in which there are four
extra copies of the grk gene
(Neuman-Silberberg and Schüpbach,
1994
). In this background, Cct1 expression is expanded
ventrally, and the width of the gap between the two domains of dorsoanterior
Cct1 expression is increased, again reflecting a
grk-dependent expansion of EGF receptor activation in the follicle
cells (Fig. 9C). These data
confirm that Cct1 expression during late oogenesis is regulated by
the Egfr signaling pathway.
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Discussion |
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We have identified tandem Drosophila homologs of CCT and
shown that Drosophila Cct1 is required at multiple times throughout
oogenesis and ovarian morphogenesis for the correct patterning of the egg.
Thin layer chromatography studies have confirmed that mutations in
Cct1 result in reduced levels of phosphatidylcholine in
Drosophila larvae (Weber et al.,
2003). Although the molecular function of Cct1 suggests a
housekeeping role for the enzyme, experiments carried out in yeast and
mammalian tissue culture have revealed that CCT enzymes may have
additional functions. Studies in yeast, for example, have suggested a role for
CCT in secretion via the Golgi apparatus (reviewed by
Kent and Carman, 1999
). In
addition, phosphatidylcholine is the source of several bioactive lipids,
including diacyglycerol, lysophosphatidylcholine and arachidonic acid,
suggesting a possible role for CCT in signal transduction (reviewed
by Pelech and Vance,
1989
).
Requirement for Cct1 in germline stem cell maintenance
We have found that Cct1 is required during oogenesis for the
maintenance of the germline stem cells. As germline clones do not result in
this phenotype, Cct1 appears to be required in the somatic cells of
the ovary. The somatic cell types associated with germline stem cell
maintenance are the terminal filament, cap, and inner sheath cells. These
three cell types act as a `stem cell niche', which regulates germline stem
cell behavior primarily through the dpp signaling pathway.
dpp is expressed in the cap cells and inner sheath cells and signals
to the germline stem cells to regulate their proliferation
(Xie and Spradling, 2000;
Xie and Spradling, 1998
).
Another signaling molecule that appears to play a more minor role in germline
stem cell maintenance is hedgehog (hh), which is expressed
in the terminal filament and cap cells
(King et al., 2001
). In
addition to these signal transduction factors, the cell adhesion molecules
DE-cadherin and Arm are important for germline stem cell maintenance. Both
DE-cadherin and Arm proteins accumulate at high levels in the junctions
between cap cells and germline stem cells and are required for anchoring the
germline stem cells in the niche (Song et
al., 2002
).
Although we have not been able to detect Cct1 mRNA expression in the terminal filament, cap, or inner sheath cells, the effect of Cct1 mutations on germline stem cell maintenance suggests that it is likely to be required in one or more of these three cell types. It is interesting to note that the dpp pathway is required for germline stem cell maintenance, as Cct1 is expressed in response to dpp signaling later in oogenesis and could also be regulated by the same signaling pathway in the germarium. Another intriguing possibility is that Cct1 is required in the cap cells for their adhesion to the germline stem cells. As discussed below, a function in adhesion could also explain the role of Cct1 in oocyte positioning.
Cct1 function in oocyte positioning
The expression of Cct1 in the follicle cells in region 2B
suggested a direct role for Cct1 in oocyte positioning, similar to
that seen with the adhesion molecules DE-cadherin and Arm (Drosophila
ß-catenin). In wild-type ovaries, both DE-cadherin and Arm proteins are
expressed in all follicle and germline cells but become transiently enriched
in the oocyte and the anterior and posterior follicle cells in the germarium
(Godt and Tepass, 1998;
González-Reyes and St Johnston,
1998
). As the germline cyst moves through the germarium and
becomes rounded, the oocyte contacts the posterior follicle cells and adheres
to them more strongly than to the more lateral follicle cells. Although
mutations in Cct1 may be causing defects in oocyte positioning by
affecting adhesion of the posterior follicle cells to the oocyte, they do not
appear to significantly affect shg- or arm-mediated
adhesion. First, the accumulation of DE-cadherin and Arm proteins in
Cct1 mutant ovaries appears to be similar to wild-type as judged by
immunofluorescence. Second, removal of one copy of shg or
arm does not appear to significantly enhance the mispositioned oocyte
phenotype seen in Cct199 homozygotes (data not shown).
Third, Cct1 mutant follicle cell clones do not behave like
shg mutant clones in that mutant and wild-type cells do not sort out
and form a straight boundary between the two cell populations
(González-Reyes and St Johnston,
1998
).
In addition to an effect on adhesion, it is possible to imagine a number of other mechanisms by which mutations in Cct1 may be causing the mispositioning of the oocyte. One model is that Cct1 is required for the migration of the follicle cells in region 2B to surround the developing germline cyst. If there was a delay in the migration of these follicle cells, they would not be in contact with the oocyte when the germline cyst becomes rounded, and the oocyte would end up randomly positioned within the egg chamber. A defect in the migration of these cells could also explain the packaging defects seen in Cct1 mutants.
Our clonal analysis results indicate that Cct1 is expressed in both the follicle cells and the germline, and that expression in either tissue is sufficient to ensure proper positioning of the oocyte. Because the mispositioned oocyte phenotype is seen in only 11% of egg chambers mutant for the null allele Cct1179, it appears that Cct1 is not absolutely required for oocyte positioning. Instead, Cct1 probably plays a secondary role in the process to ensure that the oocyte is correctly positioned even when there is slight variability in the primary positioning mechanisms. The low frequency of mispositioned oocytes in egg chambers that have a wild-type germline but mosaic (partially mutant) follicle cell epithelium suggests that Cct1 is primarily required in the follicle cells and not the germline for oocyte positioning, but that Cct1 from the germline can generally `rescue' the mispositioned oocyte phenotype when all of the follicle cells are mutant.
Surprisingly, analysis of Cct1 mutant clones also suggested that the gene may be functioning non-autonomously. It is unlikely that Cct1 or phosphatidylcholine itself is being secreted; instead, Cct1 is likely to be required for the production of a secreted molecule. For example, Cct1 could be required for the production of a specific species of phosphatidylcholine that is then converted to a secreted signaling factor.
Requirement for Cct1 during ovarian morphogenesis
We have described a novel phenotype of `branched' ovarioles resulting from
mutations in Cct1, a phenotype that appears to be caused by a delay
in apical cell migration during ovarian morphogenesis. Defects in apical cell
migration have also been described for mutations in Drosophila Wnt4
and the Drosophila Wnt4 cell motility pathway
(Cohen et al., 2002). Mutations
in these genes result in ovarioles in which the ovariolar sheath is shorter
than in wild-type, and the egg chambers are curled up within the sheath rather
than stretched out, a defect that is also seen in Cct1 mutant
ovaries. Drosophila Wnt4 signals through Drosophila Fz2, Dsh
and PKC resulting in an accumulation of focal adhesion kinase in the apical
cells, which is required for their migration. It is unclear how Cct1
is affecting the migration of the apical cells, as our clonal analysis
suggests that Cct1 is either having a non-autonomous effect in the
apical cells, e.g. through the production or secretion of a signaling
molecule, or that Cct1 is required in a different cell population.
Interestingly, we have observed a loss of germline stem cell maintenance,
packaging defects and occasional oocyte mispositioning in Drosophila
Wnt4C1 mutant ovaries (T.G., unpublished; R. Wallace and E.
Wilder, personal communication). In addition, mutations in Cct1 can
suppress the effects of Fz and Dsh overexpression on planar cell polarity
during eye development (U. Weber and M. Mlodzik, personal communication).
These data also suggest a possible link between a requirement for
phosphatidylcholine and Wnt signaling, raising the intriguing possibility that
Cct1 could be required for the secretion of Drosophila
Wnt4.
Mutations in Cct1 affect anterior eggshell patterning
Cct1 is the only gene other than dpp for which a
shortened operculum phenotype has been described. We found that although
dpp signaling is sufficient to drive Cct1 expression in the
follicle cells, it appears to be acting in a redundant or partially redundant
manner with another factor to regulate Cct1 expression. A similar
result was obtained looking at Cct1 expression in
Mad clones in eye imaginal disks
(Weber et al., 2003).
Cct1 was expressed in the absence of dpp signaling in eye
disks, although it was strongly reduced in Mad
clones that were within the antennal disk. It has been suggested that the
expression of Cct1 in Mad clones in eye
disks may be due to redundancy in the dpp and hh signaling
pathways during morphogenetic furrow progression
(Weber et al., 2003
). In the
ovary, it is not clear what other factors are regulating Cct1
expression; however, one possible candidate is the JAK/STAT signaling pathway,
which has recently been shown to be involved in anterior follicle cell
patterning (Xi et al.,
2003
).
Although we do not know the mechanism by which Cct1 is affecting operculum formation, it is likely that additional downstream factors are involved. Cct1 is expressed in the nurse cell associated follicle cells and centripetal follicle cells during mid-oogenesis, and these cells must communicate with the follicle cells over the anterior of the oocyte, which actually secrete the operculum. As Cct1 itself is probably not secreted, it is most probably required for the production of or secretion of another molecule.
In addition to shortened opercula, eggs from Cct1 mutants exhibit
weak ventralization. Although the ventralization phenotype is consistent with
a requirement for Cct1 in the dorsal anterior follicle cells in late
oogenesis, it could also reflect the requirement for Cct1 in the
establishment of anterior cell fates during mid-oogenesis (see above). It has
been shown that dpp signaling is required for the specification of
anterior cell fates and positioning of the dorsal appendages along the
dorsoventral axis (Peri and Roth,
2000). As Cct1 is required for anterior patterning, it is
possible that this requirement also affects proper positioning of the dorsal
appendages. Because it is technically difficult to eliminate Cct1
activity in the dorsoanterior follicle cells without affecting the earlier
activity, we have not been able to determine whether or not this is the
case.
Possible roles for Cct1 in intercellular signaling
Recent studies on the role of Cct1 during Drosophila eye
development have suggested that Cct1 has a specific function in the
regulation of signaling pathways through membrane trafficking
(Weber et al., 2003).
Specifically, Weber et al. argue that during eye development, levels of
phosphatidylcholine affect endocytic pathway components, resulting in an
alteration in the subcellular localization of the EGR and Notch receptors.
Although we have no specific evidence that Cct1 is acting in the same
way during oogenesis, a similar role for Cct1 in signaling in the
ovary could potentially explain the phenotypes that we see. It seems unlikely,
however, that Cct1 would be affecting receptor localization in the
ovary, as many of its effects appear to be non-cell autonomous. Alternatively,
the function of Cct1 in the ovary could be more similar to what has
been suggested by studies carried out in yeast and rat liver cells, which
indicate a role for phosphatidylcholine in secretion through the Golgi
apparatus (reviewed by Kent and Carman,
1999
). In yeast, for example, it has been found that mutations in
sec14, a phospholipid transfer protein that is required for budding
of vesicles from the Golgi, is suppressed by mutations in CDP-choline pathway
enzymes, including yeast Cct1
(Fang et al., 1998
). A model in
which Drosophila Cct1 is involved in the secretion of various ligands
would be consistent with phenotypes that we see during oogenesis. For example,
a role for Cct1 in the secretion of signaling molecules such as
dpp or hh in the germarium could explain the loss of
germline stem cell maintenance, and involvement of Cct1 in secretion
of Drosophila Wnt4 would be consistent with the function of
Cct1 during ovarian morphogenesis and oogenesis.
A second possible function for Cct1 is in the production of a
signaling molecule. As mentioned above, phosphatidylcholine is the source of a
number of bioactive lipids. Some of these molecules, such as diacylglycerol,
function in intracellular signal transduction, whereas others, such as
lysophosphatidic acid, are secreted
(Pelech and Vance, 1989). Work
in zebrafish has shown for the first time that lysolipid phosphates have
essential functions during development. During heart development, for example,
sphingosine-1-phosphate, which is closely related to lysophophatidic acid,
binds to the lysosphingolipid receptor Miles Apart to regulate heart
morphogenesis (Kupperman et al.,
2000
). A role for Cct1 in signaling could explain all of
the phenotypes that we see in the ovary, and conversion of phosphatidylcholine
to a secreted signaling molecule would also explain the non-autonomous
function suggested by our clonal analysis.
Future studies examining the intracellular localization of Cct1 and the identification of downstream factors will be important in determining the molecular basis for the role of this gene during development.
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ACKNOWLEDGMENTS |
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