Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA
Author for correspondence (e-mail:
dmontell{at}jhmi.edu)
Accepted 23 April 2003
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SUMMARY |
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Key words: PDGF, VEGF, PVF, Ecdysone, Border cells, Cell migration, Drosophila
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INTRODUCTION |
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Regulation of border cell migration by a second signaling pathway might
contribute to the timing of migration. The Drosophila steroid hormone
ecdysone provides a signal for the border cells to begin migrating at stage 9
(Bai et al., 2000) and controls
the movements of other follicle cells at this stage (J.A.M. and D.J.M.,
unpublished). The role of this pathway in border cell migration was suggested
by identification of loss-of-function mutations in the ecdysone receptor
co-activator taiman (tai), which cause severe border cell
migration defects (Bai et al.,
2000
). The ecdysone pathway, via the ecdysone receptor,
tai and as-yet-unidentified transcriptional target genes, regulates
the distribution of Drosophila E-cadherin (DE-cadherin) in border
cells (Bai et al., 2000
).
DE-cadherin protein is expressed at the cortex of all follicle cells and nurse
cells, and is upregulated in the border cells
(Bai et al., 2000
;
Niewiadomska et al., 1999
).
During migration, DE-cadherin appears to localize in a punctate pattern at the
interface between border cells and nurse cells, whereas a higher level and
more uniform distribution are observed between cells within the cluster
(Bai et al., 2000
;
Niewiadomska et al., 1999
).
DE-cadherin is important because loss of DE-cadherin in either the border
cells themselves or the nurse cells disrupts migration
(Niewiadomska et al., 1999
;
Oda et al., 1997
). Loss of
tai in the border cells results in an abnormal accumulation of
DE-cadherin at the interface between border cells and nurse cells
(Bai et al., 2000
). Thus,
disruption of either the normal expression or distribution of DE-cadherin is
associated with defective migration. An important unresolved question is how
DE-cadherin is dynamically regulated at the interface of border cells and
nurse cells.
To identify genes that play a role in border cell migration, we undertook a
screen for mutations that exhibit dominant genetic interactions with a
mutation in tai. 199 deficiencies were screened, 16 of which showed
some degree of inhibition of border cell migration when heterozygous with
tai. In one case, we identified a P-element insertion that showed
dominant interactions with tai and also exhibited border cell
migration defects when homozygous mutant. This P-element causes a null
mutation of Pvf1 (Duchek et al.,
2001). In order to test whether PVF1 or other growth factors are
capable of guiding the border cells, we ectopically expressed PVF1, PVF2 or
GRK in random cells to see whether the border cells could be attracted to a
new source of growth factor. PVF1 was capable of attracting the border cells
to a new target but GRK and PVF2 were not. Our results indicate that PVF1 acts
as a guidance cue but additional factors likely contribute to the robust
ability of these cells to find their target.
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MATERIALS AND METHODS |
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We used the FLP/FRT system (Xu and
Rubin, 1993) to generate mosaic mutant follicle cell clones of
tai61G1, FRT40A and
Pvrc2195, FRT40A. Mosaic mutant clones marked
for loss of GFP were induced as described
(Bai et al., 2000
;
Silver and Montell, 2001
)
using females of the genotype hs-FLP; ubiquitin-nuclear-GFP,
FRT40A. Positively marked clones were induced using the MARCM
system (Lee and Luo, 1999
) as
described (Silver and Montell,
2001
).
Screen for dominant genetic interactions with tai
The tai61G1 allele was recombined with a viable
enhancer trap expressed in the border cells, PZ8685 (D.J.M.,
unpublished). The tai61G1, PZ8685/CyO stock was crossed to
the deficiency kit lines from the Bloomington stock center
(http://fly.bio.indiana.edu/).
We dissected ovaries from three to five female progeny lacking the balancer
chromosomes and performed ß-galactosidase activity staining. Egg chambers
double heterozygous for tai and the deficiency were examined for
delays in border cell migration compared with tai61G1,
PZ8685/CyO. A deficiency was considered phenotypic if ≥8% of the egg
chambers scored showed delay of border cell migration. The average of at least
two experiments is reported in Table
1. Two deficiencies were identified that exhibited partial
haploinsufficiency with respect to border cell migration
(Table 1).
|
To make clones of cells ectopically expressing a gene under the control of
GAL4/UAS, we used the `FLP-out' GAL4 system (AyGAL4)
(Ito et al., 1997). To induce
clones, adult females were heat-shocked at 37°C for 1 hour and incubated
for 2 days at 25°C before having their ovaries dissected. Clones were
detected by expression of UAS-lacZ using an anti-ß-galactosidase
antibody. For UAS-Pvf1 and UAS-s-grk, we confirmed by
antibody staining that the respective proteins were expressed.
Production of antisera, ß-galactosidase activity and
immunofluorescence
The last 311 amino acids of PVR were cloned into the pGST-Parallel 1
expression vector (Sheffield et al.,
1999). The PVR C-terminal fragment was expressed as a fusion
protein in Escherichia coli [strain BL21(DE3)] and affinity purified.
Purified protein was used to immunize rabbits and rats (Covance). Animals were
boosted a total of five times, resulting in optimal signal in egg chambers at
1:1000 to 1:2000 dilution.
Ovary dissection and fixation, ß-galactosidase activity staining and
antibody staining were performed essentially as described
(Bai et al., 2000;
Montell, 1999a
). The following
primary antibodies were used: mouse anti-Armadillo monoclonal (N27A1; 1:75)
(Developmental Studies Hybridoma Bank); rabbit anti-ß-galactosidase serum
(1:3000; Cappel); rat anti-DE-cadherin monoclonal (DCAD2; 1:10)
(Uemura et al., 1996
); rabbit
anti-GFP serum (1:4000; Molecular Probes); mouse anti-PVF1 serum (1:200)
(Duchek et al., 2001
); rat
anti-PVR serum (1:1500); mouse anti-Singed monoclonal (7C; 1:25)
(Cant et al., 1994
); and rabbit
anti-TAI serum (1:1000) (Bai et al.,
2000
). Secondary antibodies conjugated to alexa fluor 488 and
alexa fluor 568 (Molecular Probes) were used at a dilution of 1:400. To
visualize actin, rhodamine-phalloidin (Molecular Probes), at a dilution of
1:400, was added during secondary antibody incubation. Images were captured
with the Ultraview confocal microscope or with a digital camera on a Zeiss
Axioplan fluorescent microscope.
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RESULTS |
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In order to maximize the number of genes that could be tested for interaction with tai, we screened a collection of deficiencies, which in total removes ≥75% of the genome, for loci that showed border cell migration defects when doubly heterozygous with a null allele of tai (tai61G1) (see Materials and Methods; Fig. 1D). Of 199 deficiencies tested, 16 deficiencies, defining 14 separate interacting regions, exhibited a border cell migration defect in heterozygous combination with tai (Figs 1, 2, Table 1). The proportion of egg chambers with border cell migration defects varied between 8% and 47%, depending on the deficiency (Fig. 1B,C, Table 1). In order to confirm that the deficiencies specifically interacted with tai, we re-screened the interacting deficiencies with another tai allele, taik05809, which is a P-element insertion allele (Table 1). Most of the deficiencies interacted with both tai alleles with a few exhibiting different strengths of interactions (Table 1). In several instances, we were able to define smaller interacting regions by testing overlapping deficiencies (Table 1).
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tai and Pvf1 affect
DE-cadherin distribution in the border cells
To investigate the basis for the genetic interaction between Pvf1
and tai, we first tested whether ecdysone signaling affected
expression of either PVF1 or its receptor PVR. TAI is expressed in all
follicle cells (Fig. 4A)
(Bai et al., 2000), as is PVR
(Fig. 5A,B)
(Duchek et al., 2001
). Because
TAI encodes a transcriptional regulator, we examined the expression of PVR
protein in follicle cells that were mutant for tai. No change in the
levels of PVR expression was observed either in the columnar follicle cells
surrounding the oocyte (Fig.
4C,D) or in the border cells
(Fig. 4F). PVF1 was also
expressed normally in ecdysoneless mutant egg chambers (data not
shown), which are defective in the synthesis of ecdysone. We then examined the
expression of TAI protein in Pvf1 mutant egg chambers
(Fig. 4A,B). Pvf1
mutant egg chambers displayed normal levels of TAI protein in border cells and
other follicle cells (Fig. 4B).
We conclude that ecdysone signaling does not affect PVF or PVR protein
expression in follicle cells, nor does the Pvf1 pathway regulate the
expression of TAI in follicle cells.
|
|
Loss of the PVF1 receptor disrupts border cell migration
In order to better understand the role of Pvf1 in regulating
border cell migration, we next examined the contribution of the PVF1 receptor,
PVR (Cho et al., 2002;
Duchek et al., 2001
;
Heino et al., 2001
). A
dominant-negative version of PVR causes an incompletely penetrant border cell
migration defect (Duchek et al.,
2001
). It is not clear whether the mild phenotype actually
represents the effect of a null mutation in the receptor or reflects a partial
loss-of-function effect of the dominant negative mutation. Moreover, the
Drosophila genome encodes two additional ligands related to PVF1,
called PVF2 and PVF3 (or VEGF27Cb and VEGF27Ca), which are expressed in the
ovary (Duchek et al., 2001
) and
could possibly contribute to border cell migration. However, there is only a
single receptor, therefore we tested to what extent a Pvr mutation
disrupted border cell migration (Fig.
5). An allele of Pvr (Pvrc2195) was
recently reported (Cho et al.,
2002
). We generated homozygous mutant clones in follicle cells
using this allele. We found that PVR was undetectable in mutant cells
(Fig. 5C,D) and so
Pvrc2195 is a strong loss-of-function, and possibly null,
allele. Border cell clusters in which all cells were homozygous for the
mutation displayed delays in border cell migration, but the phenotype was
incompletely penetrant (Fig.
5E,G) and many border cell clusters completed their migration to
the oocyte (Fig. 5F). This
phenotype is indistinguishable from the null phenotype for Pvf1
(Fig. 5G).
Ectopic expression of PVF1 is sufficient to misguide the border
cells
PVF1 is expressed in the oocyte and its receptor is expressed in all
follicle cells (Fig. 5A,B), and
so it has been proposed to guide the border cells
(Duchek et al., 2001). However,
this has not been directly tested. We used the `FLP-out' GAL4 system
(Ito et al., 1997
) to express
PVF1 in random groups of follicle cells in order to test whether PVF1 was
sufficient to guide the border cells to a new source of ligand
(Fig. 6;
Table 2). We scored border cell
migration in egg chambers that expressed PVF1 in the anterior follicle cells
that surround the nurse cells (squamous follicle cells) but not in border
cells (Fig. 6). Antibody
staining confirmed that PVF1 was actually expressed in the follicle cell
clones (Fig. 6A-C). The levels
of ectopic PVF1 expressed in clones of anterior follicle cells appeared to
exceed the concentration of endogenous PVF1 in the nurse cells.
|
|
Although it appeared that the levels of ectopic PVF1 were higher than the
local concentration of endogenous PVF1, it seemed possible that endogenous
PVF1 was somehow more effective and therefore that the ectopic protein was not
very active because of the presence of endogenous protein. Therefore, we
induced clones of cells expressing ectopic PVF1 in a
Pvf1EP1624 mutant background, which lacks endogenous PVF1
protein (Duchek et al., 2001).
Similar results were obtained whether or not endogenous PVF1 was present
(Table 2), making it unlikely
that endogenous PVF1 interfered with the ability of ectopic PVF1 to guide the
border cells to new targets in the egg chamber. We also considered the
possibility that PVF1 expressed in follicle cells might be sequestered by
endogenous PVR, which is expressed in all follicle cells, such that little
PVF1 ligand could be secreted by these follicle cells. However reducing by
half the dose of PVR had little or no effect on the ability of ectopic PVF1 to
redirect the migration (Table
2).
Most egg chambers expressing ectopic PVF1 exhibited normal border cell
migration, so we tested whether other ligands could misguide the border cells,
either alone or in combination with PVF1. PVF2 is a protein homologous to PVF1
that is capable of redirecting hemocyte migration in the Drosophila
embryo, even though PVF1 cannot (Cho et
al., 2002). We therefore tested whether PVF2 could misguide the
border cells when ectopically expressed in follicle cells. PVF2 alone did not
cause any border cell migration delays and did not guide the border cells to
new targets (Table 2). Specific
misexpression of PVF2 in the border cells using slbo-GAL4 did not
affect their migration either (data not shown), therefore the border cells
appeared to be unresponsive to PVF2 alone. The results of co-expressing PVF1
and PVF2 in follicle cell clones are shown in
Table 2. There was a small
increase in the proportion of egg chambers that fell into class IV, indicating
that PVF2 might be more effective in combination with PVF1 than alone.
The most dramatic effects on border cell migration were observed when two copies of the UAS-Pvf1 transgene were included in the experiment, thus presumably doubling the concentration of ectopic PVF1. The proportion of class IV egg chambers jumped to 13% and that of egg chambers showing normal migration was reduced to 57% (Table 2).
EGFR and PVR might function redundantly to guide the border cells to the
oocyte, because expression of dominant-negative EGFR and PVR together disrupt
border cell migration more potently than either dominant-negative alone
(Duchek et al., 2001). If these
factors function redundantly to guide the border cells then either factor
should be sufficient on its own to redirect the migration. We tested whether
expression of a secreted form of GRK (s-GRK)
(Van Buskirk and Schupbach,
1999
) was sufficient to attract the border cells. In all cases, we
observed normal border cell migration following expression of s-GRK in
follicle cell clones (class I; Table
2). To test whether expression of PVF1 and s-GRK together was a
more effective guidance cue than either alone, we coexpressed them in follicle
cell clones. We obtained similar results to ectopically expressing PVF1 alone
(Table 2), indicating that
ectopic s-GRK in anterior follicle cells did not affect border cell migration.
Ectopic expression of GRK in border cells, in contrast to PVF2, was able to
disrupt border cell migration to a small degree (data not shown)
(Duchek and Rørth,
2001
).
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DISCUSSION |
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Relationship between TAI and PVF1
The genetic interaction between Pvf1 and tai indicates
that the regulation of border cell migration timing and guidance might be
linked. What is the nature of the interaction between tai and
Pvf1 during border cell migration? Ecdysone signaling did not
regulate PVF1 or PVR expression nor did Pvf1 regulate TAI expression,
but the ecdysone and Pvf1 pathways both affected the distribution of
DE-cadherin and Arm. We favor a model whereby tai and Pvf1
interact because they both regulate adhesion complex localization or turnover.
The tai and Pvf1 genes could act independently to regulate
cadherin-dynamics. Alternatively, tai and Pvf1 might
function in a common pathway. TAI and PVR both function autonomously in the
border cells, although they are unlikely to bind directly to each other
because TAI localizes to the nucleus and PVR is a receptor tyrosine kinase
localized to the membrane. One possibility is that PVR activates (or
represses) the function of a protein whose expression is dependent on TAI, and
that this protein in turn regulates cadherin dynamics in the border cells.
Tyrosine phosphorylation of ß-catenin, the Arm homolog, causes
destabilization of adhesion complexes in other cell types
(Lilien et al., 2002), so
perhaps PVR activity destabilizes E-cadherin/Armadillo complexes specifically
in the border cells. Identification of additional genes identified in this
screen, in particular those that affect adhesion turnover in border cells,
should help clarify the biochemical relationship between TAI and PVF1.
PVF1 functions as a concentration dependent guidance cue
evidence for additional guidance cues?
The results reported here demonstrate that ectopic expression of PVF1 is
sufficient to redirect border cells even though, in Pvf1 null
mutants, border cell clusters migrate normally in the majority of egg
chambers. When PVF1 was ectopically expressed in random follicle cells, the
border cells were attracted to these sources of PVF1. The border cells were
attracted more efficiently to sources of PVF1 signal close to the anterior
pole, indicating that they respond better to high concentrations of the
ligand. The finding that doubling the dose of ectopically expressed PVF1
dramatically increased the frequency with which the cells responded to the
ectopic signal confirmed the idea of a concentration dependent effect.
The concentration of ectopic PVF1 at the anterior end of the egg chamber appeared to exceed the concentration of endogenous PVF1 at that position, even when only a single UAS-Pvf1 transgene was included in the experiment. Consistent with that idea, elimination of endogenous PVF did not significantly alter the response of the border cells to ectopic ligand. The border cells still migrated normally in many cases, apparently ignoring ectopically expressed PVF1. The most likely explanation for this is that there are additional germ-line-derived attractive cues that instruct the cells to migrate correctly in the absence of endogenous PVF1 and in the presence of ectopic PVF1.
PVF2 does not seem to be a good candidate for a redundant guidance cue
because loss of function of the PVR receptor produced a phenotype that was
indistinguishable from loss of PVF1 alone. Moreover UAS-Pvf2 was not
able to redirect the border cells. This finding is surprising because PVF2 is
thought to bind and activate the same receptor as PVF1. It is especially
surprising because only PVF2 (expressed from the same UASPVF2 transgene) and
not PVF1 is effective at misguiding hemocytes in the embryo
(Cho et al., 2002). Together,
these findings suggest a striking, and as-yet inexplicable, specificity of
ligand action that will be interesting to study further.
We also found that GRK, the major EGFR ligand in the ovary, was not an
effective guidance cue for the border cells, either when expressed alone or in
combination with PVF1. The inability of GRK to affect border cells was
striking because even class II and III phenotypes were absent, even though
these were not uncommon following PVF misexpression. This is consistent with
the observation that migration of the border cells to the oocyte is completely
normal in grk mutant egg chambers and in mosaic egg chambers in which
border cells lack EGF receptor function
(Duchek and Rørth,
2001). GRK does, however, have a role in the dorsal migration of
the border cells after they reach the oocyte
(Duchek and Rørth,
2001
). Currently, the evidence supporting a role for GRK in
migration of the border cells to the oocyte is the combined effect of
dominant-negative PVR and dominant-negative EGFR
(Duchek et al., 2001
). Taken
together with the results supplied here, the evidence in favor of a role for
EGFR is somewhat better than the evidence in favor of a role for GRK, possibly
suggesting the involvement of other EGFR ligands.
In addition to ligands for PVR and EGFR, this study might imply the
existence of other, as-yet-unidentified cues, that participate in the
long-range guidance of the border cells. We propose that PVF1, and possibly
additional unknown ligands, guide the border cells to the oocyte. Similarly,
in the Drosophila central nervous system, multiple short-range and
long-range cues are required to guide motor axons properly to their
appropriate muscle targets (Winberg et
al., 1998). Perhaps even a simple migration, such as that of the
border cells, uses multiple cues, each of which might only have a small
contribution. Screens such as the one reported here might help us to identify
the full set of border cell migration cues as well as additional genes that
function in adhesion complex turnover.
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ACKNOWLEDGMENTS |
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Footnotes |
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