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First published online 14 June 2006
doi: 10.1242/dev.02423
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1 Department of Biological Sciences, Stanford University, 385 Serra Mall,
Stanford, CA 94305, USA.
2 Zoologisches Institut, Universität Zürich, Winterthurerstr. 190,
CH-8057 Zürich, Switzerland.
* Author for correspondence (e-mail: msimon{at}stanford.edu)
Accepted 3 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Src64, Csk, Oogenesis, Ring canal, Follicle formation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Spradling, 1993;
Robinson and Cooley,
1996).
Significant progress has been made in identifying the molecular mechanisms
that regulate these events. One well-studied process is the regulation of ring
canal morphogenesis by the Src family protein tyrosine kinase (SFK), Src64. In
Src64 mutants, ring canals form normally, but exhibit reduced growth
and stability throughout development
(Dodson et al., 1998). These
defects are correlated with reductions in tyrosyl phosphorylation of ring
canal components such as the protein tyrosine kinase Tec29
(Lu et al., 2004) and the
actin-bundling protein Kelch (Kelso et
al., 2002). Additionally, Src64 mutants exhibit reduced
rates of actin polymerization at the ring canal inner rim
(Kelso et al., 2002),
suggesting that the defects observed in Src64 mutants are due to
direct effects on actin cytoskeletal dynamics.
By contrast to our growing understanding of the downstream consequences of
Src64 function at the ring canal, the mechanisms that regulate Src64 activity
during oogenesis are unknown. Src64 is a member of a conserved group of SH3-
and SH2-domain-containing protein tyrosine kinases (PTKs) that have been
extensively studied in vertebrate systems, where nine SFK members
phosphorylate dozens of target proteins. Activation of vertebrate SFKs occurs
by two primary mechanisms. First, engagement of the SH3 or SH2 domain by an
appropriate binding partner promotes the `open' or active conformation of
SFKs. Alternatively, dephosphorylation of a key tyrosine residue in the
C-terminal tail of an individual SFK (Y547 in Src64)
(Kussick and Cooper, 1992)
disrupts its autoinhibited form, allowing activation to occur. The reverse
reaction, phosphorylation of the same C-terminal tyrosine residue, promotes
the `closed', inactive conformation and is performed by C-terminal Src kinase
(Csk) family members. The balance of negative regulation, largely by Csk, and
positive regulation by a variety of signals determines SFK localization and
activity levels, and subsequently the levels of tyrosyl phosphorylation on key
target proteins (Brown and Cooper,
1996).
To understand Src64 regulation during oogenesis, we generated complete loss-of-function alleles of Src64 (Src64KO) or Csk and examined their effects on morphological events in the germ cells. As expected, Src64KO ovaries exhibited small ring canals as well as catastrophic ring canal attachment defects, demonstrating the increased phenotypic severity of ovaries lacking all Src64 function compared with previously characterized hypomorphic mutations. By contrast, loss of Csk did not dramatically affect ring canal growth, suggesting that other methods of Src64 regulation are crucial during this process. Importantly, we found that regulation of Src64 by Csk was involved in the packaging of germline cysts by follicle cells, demonstrating that Src64 is differentially regulated during distinct events. Together, these results define key roles for regulation of Src64 by Csk during oogenesis and uncover novel functions for Src64/Csk in morphological events.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and the pEndsOut2 vector (kindly provided by J. Sekelsky).
In Src64KO, the white gene replaces 5550 bp of
the Src64 locus from -802 bp to +971 bp relative to the ATG and stop
(Genome release 4.3 coordinates: 3L bp 4,574,517-4,580,068).
w;Src64
17
(Dodson et al., 1998), and
B1-93F (Ruohola et al., 1991)
were described. Rescue experiments were performed using
w,P[OS64-WT]/+;Src64mut/Src64mut where the
wild-type Src64 transgene is expressed under oskar promoter
control (Ephrussi and Lehmann,
1992; Smith et al.,
1992). w;P[UASp-Src64Y547F] lines express
constitutively activated Src64 under UAS control. To express
[Src64Y547F], the transgenic line was crossed to
w1118;P[w[+mC]=GAL4::VP16-nos.UTR]MVD. For clonal
analysis, w;Src64mut,FRT2A/GFP,FRT2A larvae were heat
shocked on days 3,4,5 after egg lay, and clonal phenotypes in adult ovaries
assessed.
w;Cskj1D8 and w;CskS017909 were
from Bloomington and Szeged stock centers, respectively.
w;CskS13 is an imprecise
excision of the P-element in w;CskS017909. Csk coding
region mutations were generated by EMS mutagenesis and affect Csk protein as
follows: w;CskQ156Stop, is a presumed null;
w;CskD589N is a mutation in the catalytic aspartic acid,
and w;CskE481K disrupts the kinase active site. Clonal
phenotypes of Csk mutants were assessed using the FLP/DFS technique
or by marking clones with GFP.
Egg lay and hatch rate analysis were as described
(Dodson et al., 1998).
Generation of antibodies and immunoblotting
Affinity purified rabbit anti-Src64CT antibodies are directed against a
peptide containing the terminal 13-amino acids of Src64. Rabbit anti-pY434
activation-specific Src64 antibodies were generated against the peptide
RVIADDEpYCPKQG. Irrelevant antibodies were subtracted by passing whole serum
over an unphosphorylated Y434-BSA column. Anti-pY434 antibodies were affinity
purified on a BSA-pY434 column (Miermont
et al., 2000). Immunoblot analysis was as described
(Lu et al., 2004) using
anti-Src64CT (1/1000) or anti-BicD antibodies (DSHB).
Immunocytochemistry and imaging
Ovary dissections and fixation were described
(Guarnieri et al., 1998).
Seven-day-old virgin females were placed with males on yeasted vials for 2
days before ovary isolation. Phalloidin (Molecular Probes) or propidium iodide
staining was described (Guarnieri et al.,
1998), as was immunofluorescence analysis with antibodies against
Src64CT (1/8000), Hts (1/1000) (Xue and
Cooley, 1993), Vasa (1/2000)
(Hay et al., 1990), Orb (1/10,
DSHB), Bazooka (1/500) (Wodarz et al.,
1999), DE-Cadherin (1/25) (Oda
et al., 1994), Armadillo (1/100, DSHB), Fas3 (1/1000, DSHB), Cut
(1/10, DSHB), pY434 (1/100), ß-Gal (1/1000, Promega). Images were
collected using a Bio-Rad MRC1024 confocal laser scanning microscope and
Lasersharp software. Image processing was done using Image J and Adobe
Photoshop software.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) to
delete the Src64 coding region. In crosses between heterozygous
flies, Src64KO flies eclosed at the expected rate,
indicating that zygotic Src64 function is dispensable for viability.
As expected, Src64 protein expression was undetectable by immunofluorescence
or immunoblotting in Src64KO ovaries
(Fig. 1A,B). By contrast, the
previously described allele
Src6417
(Dodson et al., 1998) had low
levels of Src64 protein that were detectable only by immunoblotting
(Fig. 1B).
Src64KO females displayed severe fertility defects,
including dramatically reduced egg lay and hatch rates
(Fig. 1C,D). Egg lay rates in
Src64KO mutants were far more severe than those observed
in Src6417 hypomorphic mutants,
suggesting that the small amount of Src64 protein found in
Src6417 mutants was sufficient
to provide substantial Src64 function (Fig.
1C,D).
We next examined the effects of complete loss of Src64 function on ring
canal morphology. Homozygous Src64KO ring canals were
smaller than both wild-type and
Src6417 ring canals
(Fig. 2A,B,
Table 1), and had undetectable
levels of tyrosine phosphorylation (data not shown). Additionally,
Src64KO mutants exhibited severe defects in ring canal
attachment to the cortical membrane relative to
Src6417
(Fig. 2C).
Src64KO/Src6417
exhibited intermediate effects (Fig.
2C). Src64 ring canal defects were rescued substantially
by germline-specific expression of wild-type Src64
(Fig. 1B,
Fig. 2,
Table 1), consistent with our
previous reports (Dodson et al.,
1998; Guarnieri et al.,
1998). These results provide supporting evidence that Src64
function is crucial during ring canal morphogenesis and demonstrate that flies
lacking all Src64 function exhibit more severe defects than flies retaining
low expression levels.
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; Guarnieri et
al., 1998; Kelso et al.,
2002; Lu et al.,
2004; Roulier et al.,
1998). Whereas Tec29 and Kelch are regulated by localization,
Src64 localizes uniformly to all germ-cell membranes, including the ring
canals (Fig. 1A)
(Dodson et al., 1998),
suggesting that Src64 function is regulated predominantly at the level of
activity rather than localization. Although proteins that activate
Drosophila SFKs have not been identified, Drosophila Csk
(Csk) has been shown to phosphorylate the regulatory C-terminal tyrosine of
Src64 in vitro (Pedraza et al.,
2004), demonstrating that this negative regulatory mechanism is
conserved between flies and vertebrates. If negative regulation of Src64 activity by Csk is important for regulation of ring canal growth, then three predictions can be made: (1) Csk mutants should exhibit ring canal growth defects; (2) constitutive activation of Src64 in the germ cells should mimic the effects of loss of Csk; and (3) reducing Csk levels in Src64 mutants that retain a small amount of protein should lead to increased Src64 activity and suppression of defects.
To examine potential roles for Csk during ring canal morphogenesis, we
generated Csk mutant germline clones
(Chou and Perrimon, 1996).
Homozygous mutation of previously described Csk alleles
(Stewart et al., 2003;
Read et al., 2004) and their
derivatives did not affect germ cell development
(Table 2,
Table 4), but these alleles are
not predicted to affect all Csk transcripts. We therefore examined
three coding region mutations in Csk that eliminate Csk kinase
activity and affect all Csk transcripts (see Materials and methods).
Surprisingly, loss of Csk had no significant effect on average ring
canal size relative to controls (Fig.
3A, Table 2).
Similarly, normal-sized ring canals were observed in germ cells either
expressing wild-type ([Src64WT]) or highly activated
([Src64Y547F])
(Kussick and Cooper, 1992)
Src64 transgenes in addition to endogenous Src64
(Fig. 3B,
Table 1). These results
indicate that increasing Src64 activation in germ cells does not affect ring
canal growth and that Csk is not the major regulator of Src64 during this
process. However, we found that removal of one or two copies of Csk
in Src6417 mutants suppressed
Src6417 ring canal size defects
(Table 2). These results
suggest that Csk can regulate Src64 to control ring canal size when Src64
levels are limiting, but mechanisms that lead to Src64 activation normally are
more crucial during this process.
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). By
contrast to Src64LOF, wild-type egg chambers always
contained 16 germ cells (Table
3). The total germ cell number in defective
Src64LOF egg chambers was usually a multiple of 16, with
the expected 15:1 ratio of nurse cells to oocytes
(Fig. 4A,B). In cases where
adjacent egg chambers were abnormal, the sum of the germ cells within both egg
chambers often totaled a multiple of 16 and exhibited a 15:1 ratio of nurse
cells to oocytes (Fig. 4C,D).
The percentage of Src64LOF egg chambers with aberrant cell
numbers increased with age and temperature, and defective egg chambers were
most prevalent when females were raised under optimal egg laying conditions.
Germline expression of a wild-type Src64 transgene in
Src64LOF backgrounds fully rescued the defects
(Table 3), and egg chambers
containing Src64LOF germline clones also exhibited
aberrant nurse cell numbers (Fig.
4F), demonstrating that Src64 is required in the
germline.
|
). If germ cells undergo an extra cell division, we expect
oocytes to have five ring canals instead of four
(Hawkins et al., 1996). Most
Src64KO egg chambers exhibited severe ring canal
attachment defects, including floating ring canals and cortical membrane
collapse, making it difficult to accurately score ring canal number in
defective egg chambers (Fig.
4G). Therefore, oocyte ring canal number was scored in
Src6417 egg chambers that
exhibited aberrant cell numbers. Every oocyte (n=52) in
Src6417 egg chambers with
aberrant cell numbers had exactly four ring canals, indicating that extra cell
division with incomplete cytokinesis could not explain the phenotype. Egg
chambers with too many cells might also arise from extra cell divisions
characterized by completed cytokinesis, leading to extra cells but fewer ring
canals. Wild-type egg chambers have equal numbers (15 each) of nurse cell
nuclei and ring canals. Similarly, equal numbers of nurse cell nuclei and ring
canals were found in Src6417
mutant egg chambers with aberrant germ cell numbers (n=10). Taken
together, these results indicate that Src64 mutant egg chambers with
aberrant cell numbers do not arise due to changes in germ cell proliferation
patterns.
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;
Schupbach et al., 1978;
Wieschaus and Szabad, 1979;
Margolis and Spradling, 1995).
This process ensures the genotypic equivalence of all cells in an individual
cyst. We then looked for the presence of both GFP-labeled and unlabeled cells
in Src64LOF egg chambers with aberrant cell numbers, a
phenotype that would suggest the packaging of cells derived from more than one
cyst into a single egg chamber. Using this method, we found
Src64LOF egg chambers containing labeled and unlabeled
cells (Fig. 4H), demonstrating
that the aberrant egg chambers arose due to defective packaging.
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Src64 is regulated by Csk during packaging
To assess potential roles for Csk in regulating Src64 during packaging, we
examined the phenotypes of Csk mutant ovaries. Whereas homozygous
CskS13 ovaries are
indistinguishable from wild type, Csk germline mutants that lack kinase
activity had effects on packaging that mimic expression of the highly
activated [Src64Y547F] (Fig.
5, Tables 3,
4). Csk egg chambers
with aberrant cell numbers resulted from defective packaging, as oocytes
(n=24) always had four ring canals, demonstrating that germ cell
proliferation was normal, and GFP-labeled and unlabeled cells could be seen
packaged together (Fig.
5C).
To test whether Csk regulates Src64 during packaging, we removed one copy
of Csk in Src6417
mutants, predicting that the activity of the small amount of Src64 present in
Src6417 mutants would increase
upon removal of its negative regulator, leading to suppression of
Src6417 phenotypes. Consistent
with this prediction, the high penetrance of packaging defects exhibited in
Src6417 mutants was suppressed
upon removal of one copy of Csk in transheterozygous Src64 mutants
(Src6417,Csk/Src64KO,+),
and dramatically suppressed in
Src6417 homozygotes
(Src6417,Csk/Src6417,+)
(Table 4). By contrast, the
penetrance of packaging defects in Src64KO homozygotes was
unaffected by removal of one copy of Csk
(Table 4). These results
support a model in which Src64 activity is regulated by Csk during packaging,
and imply that packaging defects observed in Csk germline clones are a
consequence of unregulated Src64 activation.
|
). Additionally, Orb and Bicaudal D (BicD), which mark
all germ cells at early stages and become concentrated in the oocyte by stage
1 (Lantz et al., 1994;
Suter and Steward, 1991), were
expressed and localized normally in mispackaged Src64 egg chambers
(Fig. 6B), suggesting that
oocytes are properly specified.
Although oocyte specification occurred normally in most mispackaged egg
chambers, some Src64 egg chambers exhibited 16 polyploid nuclei and
lacked oocytes. In most cases, oocyte absence was correlated with cortical
membrane collapse, and sometimes leaking of Orb protein could be observed
(Fig. 6B). Egg chambers
containing 16 polyploid cells in which the cortical membrane appeared intact
might arise due to defects in transport of oocyte-specific materials early in
development (Djagaeva et al.,
2005); however, these egg chambers are extremely rare and do not
represent a major Src64LOF phenotype. Our results suggest
that oocytes were generally specified normally in Src64KO
mutants, but defects in ring canal attachment led to the release of
oocyte-specific components and subsequent conversion to a nurse cell fate.
The integrity of individual germline cysts must be maintained by adhesion
between the germ cells for proper packaging to occur. In cases where germ
cell-germ cell adhesion is compromised, follicle cells can invade
inappropriately, separating nurse cells
(Abdelilah-Seyfried et al.,
2003; Goode and Perrimon,
1997; Peifer et al.,
1993). If Src64 packaging defects are due to defects in
germ cell-germ cell adhesion, we predict that germ cell shape and localization
of key adhesion molecules will be altered in cysts lacking Src64
(Peifer et al., 1993).
However, Src6417 nurse cells
exhibited normal morphology, including appropriate size, straight membranes
and localization of germ cell-germ cell adhesion molecules such as Bazooka
(Baz) and Armadillo (Arm) (Peifer et al.,
1993; Oda et al.,
1997; Goode and Perrimon,
1997; Godt and Tepass,
1998; Huynh et al.,
2001; Cox et al.,
2001) (Fig. 7D,E).
Src64KO germ cells were indistinguishable from wild-type
or Src6417 germ cells in terms
of size and localization of adhesion molecules in the germarium. However, in
many cases nurse cell morphology was subsequently affected by detachment of
ring canals from the cortical membrane as early as stage 1 (data not shown).
These changes in nurse cell morphology are unlikely to be the primary cause of
Src64LOF packaging defects, because
Src6417 mutants exhibited
dramatic packaging defects without changes in germ cell morphology.
Src64 mutants exhibit normal follicle cell proliferation, differentiation and polarity
Defects in follicle cell proliferation, differentiation or morphology
frequently lead to dramatic packaging defects
(Horne-Badovinac and Bilder,
2005). Follicle cells with diminished proliferation rates fail to
produce sufficient cell numbers to surround each germline cyst, resulting in
gaps in the somatic epithelium and packaging defects
(Jackson and Blochlinger,
1997; Zhang and Kalderon,
2000; Oh and Steward,
2001; Besse et al.,
2002). Conversely, maintenance of follicle cells in an extended
proliferative state prevents the differentiation of specialized subpopulations
of follicle cells, including the stalk cells, which are crucial for proper
packaging (Ruohola et al.,
1991; Bilder et al.,
2000; Grammont and Irvine,
2001; Lopez-Schier and St.
Johnston, 2001). In the absence of stalk cells, adjacent egg
chambers can collapse into one another, forming fused egg chambers
(Torres et al., 2003).
Finally, the maintenance of follicle cell morphology, and particularly the
apical-basal organization of the nascent epithelium, is essential for the cell
migration events that control packaging
(Goode et al., 1996;
Goode and Perrimon, 1997;
Abdelilah-Seyfried et al.,
2003).
|
;
Deng et al., 2001;
Lopez-Schier and St. Johnston,
2001) (Fig.
7A).
Furthermore, polar and stalk cell differentiation occurred normally in
Src64 mutants. Stalk formation depends on a Delta-Notch signaling
relay system that specifies anterior polar cells, which then induce stalk cell
formation (Torres et al.,
2003). Egg chambers lacking Notch in the follicle cells
or Delta in the germline fail to produce polar and stalk cells and
exhibit dramatic packaging defects
(Ruohola et al., 1991;
Xu et al., 1992;
Lopez-Schier and St. Johnston,
2001; Torres et al.,
2003). To assess whether the germline-specific requirement for
Src64 in packaging is mediated through control of Delta/Notch
signaling and/or stalk cell specification, we examined Src64 mutants
for downstream markers of Delta/Notch signaling. These markers include Cut,
which is expressed in polar and stalk cells
(de Celis et al., 1996;
de Celis and Bray, 1997;
Sun and Deng, 2005), Fas3,
which localizes to polar cells by stage 3
(Ruohola et al., 1991), or the
ß-galactosidase-expressing enhancer trap line B1-93F, which marks stalk
cells (Ruohola et al., 1991).
Expression of each marker in wild-type, Src64KO or
[Src64Y547F] ovarioles was indistinguishable
(Fig. 7A-C). Additionally,
Src64 egg chambers were always separated by stalks, even when egg
chambers were mispackaged (Fig.
7), indicating that specification of the polar and stalk cells was
normal in Src64LOF and Src64GOF
mutants.
We next examined whether the polarity of the follicle cells was altered in
Src64 germaria. Localization of proteins that mark apical (Baz),
basolateral (Arm) and basal (ßPS integrin) domains
(Tepass et al., 2001) were
identical in wild-type and Src64KO mutants
(Fig. 7D,E and data not shown).
Similar results were seen in [Src64Y547F]-expressing
germaria (Fig. 7D,E),
demonstrating that the polarity of the follicle cells is normal when Src64
levels are altered.
Src64 mutants exhibit defects in the germarium during packaging
To further define the timing of the initial Src64 defect, we
analyzed Src64 germaria for potential packaging defects. The
encapsulation of cysts in wild-type germaria can be visualized by labeling
follicle cells and their projections with anti-Fas3 antibodies and germline
cysts with anti-Vasa antibodies. In wild-type germaria, 16 germ cells are
always surrounded by a single layer of epithelial follicle cells, and follicle
cell projections extend to the anterior of flattened germline cysts
(Fig. 7F). However, in
Src6417, Src64KO, or
[Src64Y547F] germaria, cysts with aberrant cell numbers
were frequently observed (Fig.
7G-I), and, in rare cases, individual germ cells were separated
from the rest of the cyst (Fig.
7G). The follicle cells in Src64LOF germaria
extended projections and migrated to fully surround germline cysts, supporting
the idea that Src64 mutation does not affect follicle cell function.
Germline cysts in Src64KO germaria often failed to flatten
in region 2B, resulting in the simultaneous packaging of side-by-side cysts
(Fig. 7H), a defect that
probably contributes to faulty packaging. The penetrance of
Src6417 packaging defects
within germaria paralleled that observed in vitellaria (Tables
2, 5), suggesting that later
defects are a consequence of aberrations in initial encapsulation.
Src64KO and [Src64Y547F] mutants
exhibited a higher penetrance of packaging defects in germaria versus
vitellaria (Tables 2,
5), a discrepancy that is
probably due to the difficulty of scoring mispackaging in older egg chambers
with collapsed cortical membranes. Together, these data indicate that
Src64 mutant packaging defects occur before the specification of
polar and stalk cells and are distinct from these events. Instead,
Src64 mutant packaging defects occur while follicle cells are
invading the germarium to surround germline cysts, suggesting that defective
communication or adhesion between cells may occur in
Src64LOF mutants.
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|
).
Antibodies that specifically recognize this phosphorylated tyrosine (Y416 in
chicken c-Src) have been used as markers for SFK activation
(Miermont et al., 2000). We
generated and purified antibodies against the analogous phosphorylated
tyrosine, pY434 in Src64, and tested their specificity. The antibody
specifically recognized pY434, as S2 tissue culture cells expressing
[Src64WT] or [Src64Y547F] transgenes
stained with anti-pY434 antibodies, whereas cells expressing
[Src64Y434F/Y547F], a version of Src64 that is open but
lacking the autophosphorylation site, did not (data not shown).
Ring canals in wild-type egg chambers stained robustly with anti-pY434
throughout oogenesis (Fig. 8A),
consistent with known roles for Src64 at ring canals
(Dodson et al., 1998).
Anti-pY434 ring canal staining was eliminated by competition with the pY434
peptide antigen, whereas incubation with the unphosphorylated peptide (Y434)
or an unrelated pY peptide had minimal effects (data not shown). Finally, no
anti-pY434 signal was observed in either
Src6417 or
Src64KO mutants (Fig.
8B, Fig. 9A and
data not shown), demonstrating that Src64 expression is required for the
pattern observed. Germline expression of [Src64WT] rescued
anti-pY434 ring canal staining in Src64LOF mutants, but
egg chambers expressing a catalytically inactive version of Src64
([Src64KD]) exhibited no staining
(Fig. 8B). These experiments
suggest that anti-pY434 antibodies can be used to mark sites of Src64
activation in vivo.
We next assessed whether reducing Csk function in wild-type or
Src64LOF backgrounds affected anti-pY434 staining patterns
or levels. We were unable to detect increased anti-pY434 staining on ring
canals in Csk mutant ovaries with wild-type Src64 levels. By
contrast, removing one copy of Csk in
Src6417 mutants
(Src6417,Csk1T6/Src6417,+)
enabled us to detect weak anti-pY434 staining on some ring canals
(Fig. 8C). No anti-pY434 ring
canal staining was observed when one copy of Csk was removed in
Src64KO mutants (data not shown). These results are
consistent with our observation that reducing Csk levels suppressed
Src64 ring canal growth defects when Src64 protein levels were
limiting (Table 2) and support
a model in which Csk directly regulates Src64.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Actin polymerization is a crucial component of ring canal growth regulation
(Hudson and Cooley, 2002), and
mutation of genes that control actin dynamics causes dramatic ring canal
defects (Hudson and Cooley,
2002; Somogyi and Rorth,
2004). Src6417 ring
canals are smaller than wild type (Dodson
et al., 1998) and exhibit diminished actin polymerization
(Kelso et al., 2002). Recent
work has shown that Src64-mediated phosphorylation of the actin-bundling
protein Kelch is crucial for regulating actin polymerization during ring canal
growth. Whereas the Src6417
ring canal defects are strikingly similar to those observed in germ cells
expressing only [Kelch YA], which cannot be tyrosyl phosphorylated
by Src64 (Kelso et al., 2002),
we found that Src64KO ring canal growth defects are more
severe than those in Src6417
(Fig. 2,
Table 1) or, by inference,
[KelchYA] mutants. This result suggests that
Src64 may control additional signals during this process. Cortactin
or members of the WASP/SCAR protein family promote actin polymerization
through Arp2/3 complex activation (Weaver
et al., 2003) and are required for ring canal growth regulation
(Somogyi and Rorth, 2004;
Zallen et al., 2002). Both
types of protein are known vertebrate SFK substrates
(Brunton et al., 2004),
suggesting the possibility that several Src64-dependent routes may drive the
actin polymerization required for ring canal growth.
|
). The ring
canal-specific pattern of activated Src64 staining contrasts with the
localization of Src64 protein to all germ cell membranes
(Dodson et al., 1998), (Figs
1,
8,
9), suggesting that Src64
activators are present specifically at ring canals. SFKs can be activated
either through SH3-SH2 domain binding to ligand or PTP-mediated
dephosphorylation of the C-terminal regulatory tyrosine
(Brown and Cooper, 1996). Csk
opposes PTP action by phosphorylating the SFK C-terminal tyrosine, thus
promoting the inactive state. If the primary mechanism that determines Src64
activation at the ring canal is PTP-mediated dephosphorylation, we would
expect that loss of Csk should have dramatic effects on ring canal growth.
However, we found no significant effects on ring canal growth in germ cells
lacking Csk (Table 2)
or that express a version of Src64 that cannot be regulated by Csk
([Src64Y547F], Table
1). Our results suggest that a minimum threshold of Src64 activity
is required for regulation of ring canal growth and, once this threshold is
reached, the Src64-mediated response is saturated. Consistent with this idea,
reduction of Csk function can suppress Src64 mutant defects and
partially restore Src64 activation under limiting Src64 conditions
(Table 2,
Fig. 8C). Taken together, these
results suggest that Src64 is predominantly regulated by SH3-SH2 domain
engagement at the ring canal and that Csk plays a minor role in this
process.
In addition to Src64 ring canal defects, deviation from wild-type
Src64 activity levels leads to the formation of egg chambers containing
aberrant germ cell numbers surrounded by a normal follicular epithelium
(Fig. 4)
(Djagaeva et al., 2005). Egg
chambers containing incorrect germ cell numbers can arise due to germ cell or
follicle cell proliferation defects, failure to properly differentiate the
stalk cells that separate adjacent egg chambers, or as a result of defective
packaging of germline cysts by follicle cells within the germarium
(de Cuevas et al., 1997;
Horne-Badovinac and Bilder,
2005). In this work, we show that both
Src64LOF and Src64GOF mutants exhibit
normal proliferation patterns in both follicle cells and germ cells, and that
follicle cell polarity and differentiation are unaffected by Src64
mutation. Instead, defects in the initial separation of germline cysts by
invading follicle cells are responsible for Src64 mutant packaging
defects.
Two previously identified genes, egghead (egh) and
brainiac (brn) are required in the germline to regulate the
migration of follicle cell precursors during packaging
(Goode et al., 1996). When
germ cells lack egh or brn, follicle cell precursors
frequently fail to extend projections, leading to the packaging of multiple
germline cysts into one compound egg chamber. Mutations in egh or
brn also affect follicle cell polarity and later migration events.
Similarly, genes such as Delta, toucan or BicD are involved
in germline-derived signals that affect follicle cell differentiation or
morphogenesis (Grammont et al.,
1997; Lopez-Schier and St.
Johnston, 2001; Oh and
Steward, 2001). These results suggest that instructive cues
generated by the germ cells direct follicle cell morphogenesis during
packaging.
Although Src64 is required in the germ cells, Src64
mutant phenotypes are inconsistent with a similar role for Src64 in regulating
follicle cell morphogenesis. No defects in follicle cell proliferation,
process extension, migration, differentiation or polarity are observed in
Src64 mutants. Importantly, Src64 is activated at contact points
between germ cells and follicle cells while packaging occurs
(Fig. 9). This finding implies
that contact between follicle cells and germ cells leads to changes in the
germ cell surface over which follicle cells migrate, indicating that germ
cells actively respond to follicle cell-derived signals. Roles for SFKs in
dynamic regulation of endothelial cell surfaces that act as substrata for
attachment and migration of leukocytes or metastatic tumor cells have been
previously proposed (Eliceiri et al.,
1999; Weis et al.,
2004). In endothelial cells lacking SFK activity, leukocyte
attachment and migration is defective, and metastatic colon cancer cells fail
to penetrate the endothelial barrier. These results demonstrate crucial roles
for SFKs in establishing an appropriate substratum for cell migration.
We propose that Src64 functions in an analogous manner during packaging. In this model, Src64 is activated by contact between follicle cell projections and germ cells. The precise Src64 activity levels are determined by the balance between contact-dependent activators and Csk. Src64-dependent activation of downstream pathways may then establish the germ cell surface as an appropriate substratum for follicle cell attachment and migration. Defects in adhesion or the underlying cytoskeleton resulting from inappropriate Src64 activation levels would lead to defective adhesion by invading follicle cells, resulting in packaging defects.
E-cadherin and Arm/ß-catenin are important regulators of adhesion
between germ cells within an individual cyst as well as adhesion between germ
cell and follicle cell surfaces. Germline mutation of arm or
shotgun (shg), which encodes E-cadherin, leads to ring canal
attachment defects, failure of germline cysts to flatten across the germarium,
packaging defects and oocyte mislocalization
(Peifer et al., 1993;
Oda et al., 1994;
Godt and Tepass, 1998;
Gonzalez-Reyes and St. Johnston,
1998). These phenotypes overlap with Src64 mutant
defects, suggesting that Src64 might function within germ cells to regulate
E-cadherin complexes. Vertebrate SFKs can dynamically alter the adhesive
strength of E-cadherin-mediated complexes through catenin phosphorylation
(Brunton et al., 2004;
Reynolds and Roczniak-Ferguson,
2004; Lilien and Balsamo,
2005), supporting the idea that Src64 may function similarly
during oogenesis. Although direct regulation of E-cadherin-mediated adhesion
by Src64 is an attractive model, we did not observe changes in the levels of
E-cadherin or Arm at germ cell or follicle cell membranes in Src64
mutants (Fig. 7E and data not
shown), shg is dispensable for Src64 activation (A.M.O. and M.A.S.,
unpublished), and the most prominent phenotype observed in shg or
arm mutants is oocyte mislocalization
(Peifer et al., 1993;
Godt and Tepass, 1998;
Gonzalez-Reyes and St. Johnston,
1998), a phenotype that occurs in less than 1% of Src64
mutant egg chambers (Djagaeva et al.,
2005) (data not shown). It is possible that Src64 selectively
regulates E-cadherin complexes that mediate ring canal attachment and the germ
cell-follicle cell interactions that occur during packaging without affecting
oocyte localization. Alternatively, Src64 may target a different adhesion
complex, the disruption of which indirectly affects E-cadherin-dependent
events. Further analysis of the relationships between Src64 and E-cadherin
complex members is required to distinguish between these possibilities.
The incomplete penetrance of packaging defects in Src64 mutants suggests that follicle cells can package germline cysts properly even when an ideal substratum is lacking, that Src64 plays a modifying role in this process, or that additional unidentified mechanisms function redundantly with Src64-controlled events. Future identification of upstream activators and downstream consequences of Src64 activation will contribute significantly to the understanding of its role in regulating the germ cell surface during packaging.
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ACKNOWLEDGMENTS |
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REFERENCES |
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