1 Department of Pathology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
2 Department of Molecular and Cellular Biology, Baylor College of Medicine, One
Baylor Plaza, Houston, TX 77030, USA
3 Department of Molecular and Human Genetics, Baylor College of Medicine, One
Baylor Plaza, Houston, TX 77030, USA
4 Program in Developmental Biology, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030, USA
* Author for correspondence (e-mail: sgoode{at}bcm.tmc.edu)
Accepted 28 January 2004
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SUMMARY |
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Key words: Epithelial cell cluster, Cluster motility, Cluster polarity, Cell adhesion, Tumor suppressor, Fasciclin 2, Drosophila
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Introduction |
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Drosophila border cells (BCs) provide a simple in vivo model for
deciphering the mechanisms of cell cluster movement
(Montell, 2003). BCs are a
cluster of six to eight somatic follicle cells that differentiate within the
anterior follicular epithelium during mid oogenesis, maintaining some aspects
of epithelial polarity (Niewiadomska et
al., 1999
), while losing others (this work). The BCs then
delaminate from the follicular epithelium, a process that requires the
polarized cluster to coordinately break contact from adjacent epithelial
cells, while simultaneously directing invasion between the germ cells. Once
the BCs exit the epithelium, they take
6 hours to migrate roughly 150
µm between the nurse germ cells to the oocyte
(Spradling, 1993
).
The BC cluster includes two polar cells (PCs) that reside at the center of
the cluster and that do not contact the migration substrate. Preceding BC
differentiation, PCs secrete Unpaired, which determines how many adjacent
epithelial cells will activate the Jak-Stat pathway, and thus become BCs
(Bai et al., 2000;
Beccari et al., 2002
). Stat is
sufficient for expression of the C/EBP transcription factor, Slow Border Cells
(Slbo) (Beccari et al., 2002
;
Montell et al., 1992
;
Silver and Montell, 2001
).
Slbo directs BC differentiation and upregulation of DE-Cadherin, a
cell-adhesion molecule essential for movement
(Liu and Montell, 2001
;
Niewiadomska et al., 1999
).
Two tyrosine kinase receptors that appear to function redundantly, Pvr and
Egfr, guide the BCs to the oocyte (Duchek
and Rorth, 2001
; Duchek et
al., 2001
). In contrast to these genes, Discs large (Dlg)
(Woods and Bryant, 1991
;
Woods and Bryant, 1993
)
prevents BCs from reaching the oocyte prematurely
(Goode and Perrimon,
1997
).
Dlg is a member of a large family of conserved membrane-associated
guanylate kinases (MAGuKs) that lack intrinsic kinase activity
(Anderson, 1996). MAGuKs have
multiple protein-binding domains, including three PDZ domains, an SH3 domain,
and a GuK domain that act as scaffolding modules to assemble specific
combinations of signaling, adhesion and cytoskeletal molecules at cellular
junctions (Sheng, 1996
). In
Drosophila, Dlg scaffolding is crucial for suppressing local tumor
invasion (Goode and Perrimon,
1997
) and metastasis
(Woodhouse et al., 1998
). It
is not known if these requirements reflect Dlg functions in controlling
epithelial polarity, delamination, migration or any combination of these
activities. The mechanisms by which Dlg inhibits migration and tumor invasion
are likely to be conserved across species as two human MAGuKs, DLG and ZO1,
are also implicated in oncogenesis. Human DLG binds to APC, the most commonly
mutated gene in colorectal cancer
(Matsumine et al., 1996
), and
is a target for E6 oncoprotein in human papillomavirus transformation
(Gardiol et al., 2002
). ZO1 is
lost specifically at the transition from in situ to invasive breast cancer
(Hoover et al., 1998
).
How MAGuKs suppress tumorigenesis is not known. In humans, PSD-95 binds a
RasGAP molecule (Kim et al.,
1998), suggesting that MAGuKs may signal through small G proteins.
In Drosophila, Dlg colocalizes and cooperates with two additional
tumor suppressors at epithelial junctions, Lethalgiant-larvae (Lgl) and
Scribbled (Scrib). Loss of either Lgl or Scrib causes a loss of epithelial
polarity and over-proliferation that phenotypically resembles a loss of Dlg
(Bilder et al., 2000
). Lgl
integrates membrane and cytoskeletal organization by binding and repressing
Myosin 2 activity (Peng et al.,
2000
; Strand et al.,
1994
), and regulating vesicular trafficking
(Lehman et al., 1999
). Scrib
is a scaffolding protein containing four PDZ domains and 16 leucin-rich
repeats (Bilder and Perrimon,
2000
). The putative Dlg-Lgl-Scrib complex is believed to mediate
cellular interactions important for epithelial polarity, signaling and
adhesion by clustering selected signaling and adhesion receptors with specific
regulatory, cytoskeletal and trafficking molecules at cellular junctions.
To understand how Dlg scaffolding integrates multiple protein activities to
regulate epithelial polarity and movement, we are analyzing proteins that bind
to distinct Dlg domains. One such protein, Fasciclin 2 (Fas2), is a
transmembrane cell-adhesion molecule (CAM) of the immunoglobulin superfamily.
Fas2 binds Dlg PDZ1+2 domains, and is homologous to vertebrate neural
cell-adhesion molecule (NCAM) (reviewed by
Goodman et al., 1997). The
Fas2 C terminus -SAV-COOH sequence selectively recruits Fas2 to neuromuscular
junctions by binding Dlg PDZ1+2 (Thomas et
al., 1997
; Zito et al.,
1997
). In the absence of the Fas2 C terminus -SAV-COOH, or Dlg,
Fas2 is diffusely localized, resulting in abnormal development of synapse
structure (Thomas et al.,
1997
; Zito et al.,
1997
). The precise spatial and temporal pattern of Fas2 is crucial
for targeted membrane growth, as demonstrated by axon guidance defects
resulting from Fas2 loss or misexpression
(Goodman et al., 1997
).
In the present study, we have examined the role of Fas2, Dlg and Lgl in regulating the motility of an organized cell cluster. We employ a novel method of measuring BC motility that enables us to distinguish the function of Fas2, Dlg and Lgl in regulating the delamination of BCs out of the follicular epithelium from their roles in regulating BC migration. Furthermore, we introduce the use of reproducibly oriented clusters, which enables us to assess the importance of protein localization during delamination and migration. Combined with genetic mosaic analysis, and targeted rescue experiments, these data provide the first model describing the role of neoplastic tumor suppressors in BC movement.
We find that while Dlg and Lgl are constitutively expressed in all follicle cells, Fas2 expression is selectively lost, precisely at the time of BC differentiation, from the anterior follicle epithelium, including the BCs. Fas2 expression is maintained in PCs at the center of the BC cluster. Loss of Fas2 expression in BCs permits a reorganization of Fas2, Dlg and Lgl epithelial polarity, to a motile polarity in PCs, which is crucial for efficient delamination. At the same time, PC Fas2 signals Dlg and Lgl maintenance in BCs, which inhibits the rate of migration. Our data thus demonstrate how dynamic Fas2 expression and polarity regulate epithelial junctions to control cluster motility with temporal precision. Furthermore, our observation that Dlg and Lgl inhibit the rate of movement of a developmentally regulated cell cluster suggests that their loss in metastatic tumors not only facilitates the transition from epithelial to motile polarity, but also directly contributes to tumor motility. The reorganization of molecules important for epithelial polarity to achieve motile cluster polarity has important implications for the coordinate misregulation of epithelial polarity and motility during carcinoma invasion.
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Materials and methods |
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Histochemistry and imaging
Flies were reared on fresh yeast at 25°C. Antibody, phalloidin and
ß-gal activity staining were as previously described
(Goode and Perrimon, 1997).
The following primary antibodies were used: rabbit anti-Amphiphysin (number
9906, 1:500) (Zelhof et al.,
2001
), rat anti-Crb (1:1000; U. Tepass), rabbit anti-Dlg (1:500;
K.-O. Cho), mouse anti-Fas2 (1D4, ppIg 1:1000; DSHB), mouse anti-Fas3 (7G10,
ppIg 1:1000; DSHB), rabbit anti-ß-gal (1:2000; Cappel), mouse
anti-
-Tubulin (1:500; Sigma). Polyclonal anti-Slbo (1:2000) was
produced at Bethyl Laboratories in rabbits with HPLC-purified C-terminal Slbo
peptide 429-VSRVCRSFLNTNEHSL-444, followed by affinity purification. Anti-Lgl
(1:4000) was raised in a sheep against C-terminal Lgl peptide
1146-DNKIGTPKTAPEESQF-1161. Specificity of antibodies was confirmed by ELISA,
western blotting of ovarian proteins and staining wild-type and mutant egg
chambers with immune and preimmune sera. Cy5-, FITC- or rhodamine
red-X-conjugated donkey secondary antibodies (Jackson ImmunoResearch
Laboratories) were used (1:2000). Alexa488- or
Alexa568-phalloidin (1:10; Molecular Probes) were used to visualize
Actin. Images were acquired with a Zeiss LSM 510 confocal microscope or with a
conventional epifluorescence Zeiss Axioplan 2 microscope equipped with a
Hamamatsu ORCA digital camera. Confocal images were processed using Photoshop
software (Adobe).
Cell migration profiles
The traditional method for determining if a mutation perturbs BC migration
is to compare mutant BC migration with migration of the surrounding follicle
epithelium (Lee at al., 1996).
Our purpose for choosing an alternative methodology, by using the oocyte as a
clock to measure the rate of BC migration, is that the genes we examined are
expressed in both BCs and the follicular epithelium, so we could not be
assured the epithelium migrated like wild type. Furthermore, using oocyte
growth as a clock allows us to clearly distinguish the effects of the mutation
on delamination versus migration, and thus to determine the primary cause of
the motility defects. Images of stage 9 phalloidin-stained egg chambers were
captured with a Zeiss Axioplan-2 microscope equipped with a Hamamatsu ORCA
digital camera. Images were measured as described in
Fig. 3B using AxioVision 3.1
software (Carl Zeiss Vision). Morphometric analysis was similar to that
described (Zarnescu and Thomas,
1999
). However, instead of plotting percent BC migration completed
as a function of percent oocyte length, we plotted percent BC migration
completed as a function of percent oocyte area. This gives more reproducible
profiles because oocyte length, but not oocyte area, is expected to oscillate,
owing to expansion and contraction of the surrounding muscle layer. None of
the mutations we analyzed affected linear progression of oocyte growth. Growth
of the oocyte was converted to time by taking wild-type migration as a
standard 6 hours (Spradling,
1993
). To ensure that we measured an unbiased sample, we were
careful to include every s9 egg chamber, even those in which the BCs had just
started to penetrate between the nurse cells, so that all of our analyses are
based on a uniformly distributed set of border cell migration distances (see
Fig. 3E). Tumor development in
dlg and lgl temperature-sensitive allele egg chambers was
not apparent because the flies were shifted to the restrictive temperature
(25°C) for only 8 hours preceding BC analysis (furthermore, the
temperature shift applied to wild-type flies did not affect oocyte growth or
BC migration). SYSTAT 10 software (SSI) was used to complete General Linear
Model analysis of data points from on average 200 egg chambers per genotype.
Statistical significance was taken at a value of P<0.05.
|
Fas2 polarity was measured on PC images by determining the pixel densities of Fas2 staining in the leading and trailing halves of PCs using NIH Image software. Polarity is presented as a ratio of leading Fas2 to trailing Fas2.
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Results |
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Loss of Fas2 expression in BCs permits a shift in Fas2 polarity in PCs
Polarization of Fas2 to the leading half of the PCs, precisely at the time
when Fas2 expression is lost in BCs, suggests that Fas2 loss in BCs might be
crucial for establishing its polarity. To test this hypothesis, we expressed
Fas2 in BCs using the UAS-Gal4 system
(Brand and Perrimon, 1993).
Targeting Fas2 to BCs using Slbo-Gal4 (expressed in BCs but not PCs,
Fig. 2D) causes Fas2 to
accumulate around the circumference of PCs
(Fig. 2B), in a pattern
resembling that found in wild-type clusters preceding BC differentiation. This
experiment demonstrates that loss of Fas2 expression in BCs is crucial for
Fas2 polarization. As a control, we targeted chimeric CD8-Fas2 to BCs.
CD8-Fas2 has the extracellular Ig domain of Fas2 replaced with the
extracellular Ig domain of human CD8. CD8-Fas2 does not significantly affect
Fas2 polarity in PCs (Fig. 2C).
Thus, loss of Fas2 homophilic interactions between BCs and PCs appears to be
crucial for establishing Fas2 polarity.
|
|
We asked if the putative BC receptor is expressed preceding BC differentiation. We reasoned that if the putative receptor is expressed before BC differentiation, then Fas2 should be present in PC membranes even when they contact Fas2null undifferentiated BCs. We generated Fas2null clones and found that loss of Fas2 from presumptive BCs at stage 7 causes loss of Fas2 from the adjacent membrane of the Fas2+ PCs (Fig. 2G), indicating that putative receptor is not expressed before BC differentiation. We also analyzed whether the putative BC receptor is present in other stage 8-9 follicle cells. In mosaics of Fas2null and Fas2+ follicular epithelia, Fas2 is localized only at cell membranes where a Fas2+ cell contacts another Fas2+ cell, and not where Fas2+ cells contact Fas2null cells (Fig. 2F). Thus, there does not appear to be an alternate Fas2 ligand (BC receptor) in other follicle cells.
The above experiments indicate that the putative BC receptor is required to maintain Fas2 in PC membranes. These experiments do not address if the putative BC receptor directs Fas2 polarization to the leading half of the PCs. To test the importance of the putative BC receptor for Fas2 polarization, we targeted CD8-Fas2 to wild-type and Fas2rd1 PCs. CD8-Fas2 is polarized in PCs (Fig. 6J; and data not shown). This suggests that the putative BC receptor does not polarize Fas2, but rather polarization depends on the Fas2 cytoplasmic domain, and presumably interaction with Dlg PDZ domains. In support of this model, dramatic decrease of Dlg or Lgl in dlghf/dlglv55 and lgl4/lglts3 PCs causes loss of Fas2 polarity (see Fig. 6E,G). Thus, Dlg and Lgl, which are polarized to the leading half of PCs (Fig. 2A',A'', Fig. 6A',A''), appear to be crucial for establishing Fas2 polarity.
The simplest synthesis of these results is that developmentally programmed loss of Fas2 expression couples three events crucial for establishing BC polarity: (1) Fas2 homophilic interactions are lost between PCs and BCs, thus permitting formation of Fas2 heterophilic interactions with a putative BC receptor (essential for maintaining Fas2 in PC membranes); (2) Fas2 polarity is directed by Dlg and Lgl polarity in PCs; and (3) accumulation of Fas2 between PCs through homophilic interactions ensures that Fas2 level is kept sufficiently low at the site of contact with BCs to permit its polarization. This novel, precisely timed morphogenetic switch for Fas2 polarization suggests that Fas2 plays a crucial role in regulating cell cluster movement.
Fas2 regulates efficient BC delamination, but inhibits the rate of BC migration
Previous studies suggested that dlg mutant BCs reach the oocyte
prematurely (Goode and Perrimon,
1997). The method used in that study did not allow us to
distinguish if the defect resulted from premature BC delamination or faster BC
migration. To address these alternatives, we used oocyte growth as a clock to
measure the rates of BC delamination and migration
(Fig. 3; Materials and
methods).
We characterized a Fas2 allelic series by measuring Fas2 levels in
the following egg chambers: Fas2null/+ (46±11%
Fas2), Fas2rd1 (6±3% Fas2),
Fas2rd1/Fas2null (3±2% Fas2)
and Fas2null (0% Fas2) (Materials and methods). In 46%
Fas2 clusters, BCs delaminate 15% faster (
1 hour) (P=0.066,
compared with wild type), but migration time is essentially wild type
(Fig. 4). Six percent Fas2 BCs
delaminate faster (not statistically significant) and migrate 35% faster
(P=0.008, compared with wild type)
(Fig. 4). The faster rate of
migration of these clusters is thus quantitatively similar to 50% faster
migration seen when negative regulator of migration Ena/Vasp is removed from
the leading edge of mammalian fibroblasts
(Bear et al., 2000
). Three
percent and 0% Fas2 BCs also have faster migration similar to 6% Fas2 BCs
(P=0.006 and 0.031, respectively, compared with wild type), but
delamination is progressively delayed by 13% (P<0.0005, compared
with wild type) and 35% (P<0.0005, compared with wild type),
respectively (Fig. 4).
|
Fas2 migration defects may result from Fas2 loss in PCs, or from premature loss of Fas2 in early follicle cells (Fig. 1C). To determine if Fas2 is required in PCs, we targeted it to PCs in Fas2rd1 and Fas2null egg chambers using the BA3-Gal4 driver. We observe complete rescue of Fas2 migration defects when Fas2 is targeted to PCs using BA3-Gal4, indicating that PC Fas2 regulates migration of the cluster (rescue of Fas2rd1: P=0.001, compared with Fas2rd1; rescue of Fas2null: P=0.037, compared with Fas2null) (Fig. 4). Moreover, we did not observe rescue of Fas2 migration when BCs were targeted with Fas2 using Slbo-Gal4 (Fig. 4), further demonstrating that Fas2 acts in PCs not BCs to control migration.
To determine if the PDZ-binding domain or extracellular domain of Fas2 is
involved in regulating cluster migration, we targeted Fas23 or chimeric
CD8-Fas2 to Fas2 PCs. The Fas2
3 molecule is missing the last
three amino acids that bind PDZ1 and PDZ2 domains of Dlg
(Thomas et al., 1997
;
Zito et al., 1997
). None of
these two Fas2 derivatives was able to rescue Fas2rd1 or
Fas2null migration defects
(Fig. 4). This indicates that
both the extracellular domain of Fas2 that interacts with a putative BC
receptor and the intracellular domain that binds Dlg are essential for Fas2
function. This assertion is further supported by targeting dominant-negative
Fas2
3 to wild-type PCs, which increases the rate of BC migration by
about 10% (P=0.006, compared with UAS-Fas2
3/+). Likewise,
CD8-Fas2 appears to act in a dominant-negative manner, increasing migration by
10% (UAS-CD8-Fas2/+; BA3-Gal4/+: P=0.025, compared with
UAS-CD8-Fas2/+) (see below and Fig.
4). As PC Fas2 does not contact the migration substrate, we
conclude that Fas2 inhibits rate of migration by signaling to BCs.
Fas2 signals Dlg and Lgl localization in BCs
Fas2 is required for Dlg localization in synapses
(Thomas et al., 1997), and Dlg
is required for Lgl localization in epithelial cells
(Bilder et al., 2000
). We
therefore asked if Fas2 motility defects might result from Dlg and
Lgl mislocalization. In wild-type clusters, polarized Fas2 colocalizes with
Dlg and Lgl in PCs, while Dlg and Lgl colocalize in the cortex of BCs
(Fig. 6A-A''). In both 6%
and 0% Fas2 clusters, Dlg and Lgl levels are lower in the cortex of the BCs,
while cytoplasmic levels of Dlg and Lgl increase
(Fig. 2E',
Fig.
6B',B'',C',C''). This suggests that the
level of Dlg and Lgl did not change, but that the proteins became
redistributed. We confirmed this by quantifying Dlg and Lgl levels in
Fas2+ and Fas2null BCs (Materials and methods).
We find that the levels of Dlg and Lgl do not decrease more than 20%. Further,
these defects are specific, as Crumbs (Crb), a transmembrane protein that
organizes apical polarity, and
-Spectrin (
-Spec), a cortical
molecule that interacts with the cytoskeleton, show no changes in localization
in Fas2null BCs (P.S. and S.G., unpublished).
Mislocalization of Dlg and Lgl in BCs in Fas2 clones might result from loss of Fas2 in PCs, or from premature loss of Fas2 in early follicle cells. To distinguish these alternatives, we analyzed Fas2null mosaic clusters in which only one PC expresses Fas2. Significantly, Dlg and Lgl localize normally in BCs that contact Fas2+ PCs, but mislocalize in BCs that contact 0% Fas2 PCs (Fig. 2E,E'). This suggests that PC Fas2 maintains Dlg and Lgl localization in BCs. However, premature loss of Fas2 in presumptive BCs might also contribute to mislocalization of Dlg and Lgl in BCs, if Fas2null PC clones also include Fas2null BCs. To address this possibility, we analyzed Dlg and Lgl localization in other Fas2+ and Fas2null follicle cells at the time of BC delamination. Dlg and Lgl localization and levels are normal in Fas2null follicle cells (Fig. 2F',F''). PC Fas2 thus ensures Dlg and Lgl localization in BCs, but not in other follicle cells. This interpretation is consistent with the previous observation that the putative BC receptor is expressed in BCs, but not other follicle cells (Fig. 2F,H).
Dlg and Lgl collaborate with Fas2 to regulate delamination and inhibit migration
To determine if Fas2 clusters migrate faster due to
mislocalization of Dlg and Lgl in BCs, we examined migration rates of
dlg and lgl mutant BCs. In dlghf/+ or
lgl4/+ clusters, the BCs delaminate prematurely
(dlghf/+: P=0.001, compared with wild type;
lgl4/+: not statistically significant) and migrate faster
(dlghf/+: P<0.0005 compared with wild type;
lgl4/+: P<0.0005 compared with wild type)
(Fig. 4). This pattern is
similar to motility of 6% Fas2 BCs (Fig.
4), suggesting that mislocalization of Dlg and Lgl in
Fas2 BC clusters causes faster migration.
If decrease in Dlg function in BCs causes faster migration, then targeted
expression of Dlg to dlghf/+ BCs should rescue faster
migration. Targeting Dlg to dlghf/+ BCs completely rescues
faster migration (P<0.0005, compared with
dlghf/+) (Fig.
4). Targeting Dlg to PCs rescues migration only weakly
(Fig. 4). Dlg thus acts in BCs
to inhibit migration. Moreover, targeting wild-type BCs with a truncated Dlg
molecule containing only three PDZ domains, Dlg PDZ1-3, expected to compete
with endogenous Dlg for interaction with PDZ-binding proteins, causes
significant increase in migration rate (UAS-Dlg PDZ1-3/Slbo-Gal4:
P=0.031, compared with Slbo-Gal4/+), and is quantitatively comparable
with 6% Fas2 or dlghf/+ clusters
(Fig. 4). Targeting expression
of Dlg PDZ1-3 to PCs does not significantly affect cluster motility, perhaps
because of the higher level of Dlg in PCs compared with BCs
(Fig. 4). These data support
the hypothesis that Dlg acts in BCs to inhibit movement. To obtain further
evidence that mislocalization of BC Dlg causes faster migration of
Fas2 clusters, we asked if targeting Dlg to BCs could rescue
Fas2 clusters. Targeting Dlg to Fas2rd1 BCs
completely rescues faster migration of Fas2rd1 clusters
(P<0.0005, compared with Fas2rd1;
Slbo-Gal4/+), but no rescue occurs by targeting Dlg to PCs (P=0.910,
compared with Fas2rd1)
(Fig. 4). Moreover, targeting
Fas2 BCs with Fas2, Fas23 or CD8-Fas2 does not rescue faster
migration of BC clusters (Fig.
4). These experiments indicate that faster migration of
Fas2 clusters results from lower levels of Dlg in the BC cortex.
Dlg recruits Lgl to the membrane in epithelial cells and neuroblasts
(Bilder et al., 2000;
Peng et al., 2000
). To
determine if Dlg and Lgl collaborate in BCs, we analyzed Dlg and Lgl
localization in dlghf/+ and lgl4/+
clusters. Whereas levels of cortical Dlg and Lgl are lower in
dlghf/+ clusters (Fig.
6D',D''), only Lgl is lower in
lgl4/+ clusters (Fig.
6F',F''). Dlg thus recruits Lgl to BC plasma membranes
(see also Fig.
6C',C''), but Lgl does not recruit Dlg.
Similar to partial loss of Fas2, partial loss of Dlg and Lgl both increase migration rate, and accelerate delamination (Fig. 4). These data, combined with the rescue and localization experiments, indicate that Fas2, Dlg and Lgl function in a common pathway to control BC movement. If Dlg and Lgl are key collaborators with Fas2 in cluster movement, then further reduction of Dlg and Lgl might cause delayed delamination, similar to that observed in 0% Fas2 clusters. Consistent with this hypothesis, clusters expressing temperature sensitive allele combinations dlghf/dlglv55 and lgl4/lglts3 migrate slightly faster than dlghf/+ and lgl4/+ clusters (dlghf/dlglv55: P=0.012, compared with wild type; lgl4/lglts3: P=0.001, compared with wild type) (Fig. 4). More strikingly, both dlghf/dlglv55 and lgl4/lglts3 clusters have slower delamination (Fig. 4), although only dlghf/dlglv55 cluster delaminate significantly slower (P<0.0005, compared with wild type). There are several possible interpretations for why the delamination of 0% Fas2 clusters is slower than for dlghf/dlglv55 and lgl4/lglts3 clusters (Fig. 4), but the observation that they show the same trend towards slower delamination indicates that Dlg and Lgl collaborate with Fas2 in both delamination and migration. Further evidence for this conclusion comes from the high resolution cellular analysis described below.
Slower delamination of Fas2, dlg, and lgl clusters correlates with defective PC polarity
To determine if there is a common defect in clusters that delaminate
slower, we compared Fas2, Dlg and Lgl localization in clusters with faster
versus slower delamination. 6% Fas2, dlghf/+ and
lgl4/+ BCs delaminate faster, and Fas2, Dlg and Lgl remain
polarized in PCs (Fig.
6B-B'',D-D'',F-F''). 0% Fas2,
dlghf/dlglv55 and
lgl4/lglts3 BCs delaminate slower, and
Fas2, Dlg and Lgl have an apolar pattern in PCs
(Fig.
6C-C'',E-E'',G-G''; polarity is not restored as BC
clusters migrate). Furthermore, loss of Fas2, Dlg and Lgl polarity, owing to
misexpression of Fas2 in BCs in UAS-Fas2/Slbo-Gal4 clusters (see
Fig. 2B for the loss of Fas2
polarity), causes dramatic delay of delamination (P<0.0005,
compared with Slbo-Gal4/+), without affecting migration rate
(Fig. 4). We conclude that when
Fas2, Dlg and Lgl PC polarity is normal, BCs delaminate on time or
prematurely, but when PC polarity is lost, BC delamination is slower. These
data strongly suggest that PC polarity is crucial for organizing the cluster
for efficient delamination.
Ectopic PC Fas2 specifically delays delamination
To further characterize the importance of Fas2 polarity in delamination, we
examined the consequence of disrupting Fas2 polarity by misexpressing Fas2 in
PCs. These clusters have Fas2 around the circumference of the PCs
(Fig. 6H), as in
dlghf/dlglv55 and
lgl4/lglts3 clusters
(Fig. 6E,G). As expected for a
Fas2-binding protein, Dlg becomes localized in the same pattern as Fas2
(Fig. 6H'), together with
Lgl (Fig. 6H''), providing
further evidence that Fas2 can direct Dlg and Lgl localization (see also
Fig. 6C',C''). The
BCs consistently migrate behind the epithelium
(Fig. 5A). We found that,
although migration rate is almost normal, delamination is dramatically delayed
(P<0.0005, compared with UAS-Fas2/+)
(Fig. 4). Consistent with these
data, both the wild-type polarized pattern of Fas2, and timely delamination,
are restored by targeting Fas2 to Fas2rd1 PCs, which
contain polarized Dlg and Lgl (Fig.
6K-K''). However, Fas2 targeted to
Fas2null PCs, which have unpolarized Dlg and Lgl
(Fig. 6C',C''),
fails to rescue polarity (leading-to-trailing Fas2 polarity ratio is
2.1±1.3), and these clusters have slower delamination
(Fig. 4). To determine if the
delamination defects resulting from Fas2 misexpression in PCs result from
higher Fas2 levels, rather than disruption of Fas2 polarity, we targeted
wild-type PCs with Fas23. Polarity of Fas2 is relatively normal in
UAS-Fas2
3/+; BA3-Gal4/+ PCs (Fig.
6I). BCs migrate ahead of the epithelium when Fas2
3 is
targeted to PCs (Fig. 5B), and
this defect results from faster BC migration (P=0.006, compared with
UAS-Fas2
3/+), not premature delamination
(Fig. 4). Targeting CD8-Fas2 to
PCs also does not disrupt Fas2 polarity
(Fig. 6J), but causes faster BC
migration (P=0.025, compared with UAS-CD8-Fas2/+), with no effect on
delamination (P=0.071, compared with UAS-CD8-Fas2/+)
(Fig. 4). These experiments
reveal once again that when Fas2 polarity is maintained, delamination is not
perturbed. Furthermore, as Fas2
3 is not expected to bind to Dlg PDZ
domains, and CD8-Fas2 is expected to interfere with Fas2 interaction with the
putative BC receptor, these data further suggest that Fas2 interactions with
PC Dlg and the putative BC receptor are crucial for inhibiting BC migration.
In conclusion, these results indicate that Fas2 is crucial for timely
delamination through its function in establishing PC polarity, while it
inhibits rate of BC migration through a distinct process that does not depend
on PC polarity.
|
Additional evidence for BC cluster asymmetry comes from localization of
Amphiphysin (Amph), a vesicle trafficking protein important for Dlg and Lgl
localization (Zelhof et al.,
2001). Amph is expressed at higher levels in trailing BCs (not in
contact with PC Fas2) than in leading BCs
(Fig. 7B-C'). Amph
polarity develops around the time of BC differentiation at stage 8 of
oogenesis (Fig. 7A-B'),
supporting hypothesis of its functional significance for cluster motility.
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Discussion |
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A Fas2 morphogenetic switch
Just preceding cluster migration, Fas2 is polarized in PCs with an
orientation that predicts the direction of BC movement
(Fig. 1E'-F). Polarization precedes delamination and migration by 4-6 hours. Furthermore,
Fas2 polarity is not perturbed in clusters that fail to migrate
(slbo1310; data not shown). We conclude that Fas2
polarization is not a consequence of delamination or migration. The timing of
Fas2 polarization is determined by the temporal specificity of developmentally
programmed loss of Fas2 in surrounding BCs at stage 8. As loss of Fas2 in
anterior follicle cells is controlled transcriptionally
(Fig. 1H,I), and includes
stretch cells, not just BCs, it is unlikely that BC specific transcription
factors such as Slbo, Jing or Stat (reviewed by
Montell, 2003) control
developmentally programmed loss of Fas2 expression. Other factors, such as
Eyeless, are expressed in precisely the cells in which Fas2 is lost, while
being lost in PCs (S.G., unpublished). Eyeless thus may be part of a
transcriptional regulatory network that developmentally programs loss of Fas2
expression, as well as programs expression of other genes specifically
involved in morphogenesis of anterior follicle cell motility.
How does developmentally programmed loss of Fas2 expression in BCs permit
Fas2 polarization in PCs? Our data indicate that this is a multistep process.
First, Fas2 homophilic interactions between BCs and PCs are lost, and several
of our experiments indicate that they are replaced by Fas2 heterophilic
interactions with a putative BC receptor. These interactions are essential for
maintaining Fas2 in PC membranes contacting BCs
(Fig. 2F,G). Second, loss of
Fas2 from BCs causes relocation of the majority of PC Fas2 to the interface
between PCs, where it is maintained because of homophilic interactions with
Fas2 from the adjacent PC. In support of this interpretation, misexpression of
Fas2 in PCs appears to oversaturate Fas2 between PCs, causing its
circumferential accumulation at the contact sites with BCs
(Fig. 6H). We conclude that the
accumulation of Fas2 between PCs ensures that Fas2 is kept at sufficiently low
level at the sites of contact with BCs to allow its polarization to the
leading half of PCs. Third, Fas2 polarization is directed by PC Dlg and Lgl,
as loss of function of either protein causes loss of Fas2 polarity
(Fig. 6E,G). However, Fas2 can
also polarize Dlg and Lgl, as loss of Fas2 causes loss of Dlg and Lgl polarity
(Fig. 6C',C''),
while ectopic Fas2 redirects Dlg and Lgl localization
(Fig. 6H',H'').
Thus, Fas2 is in a positive feedback loop with Dlg and Lgl that ensures the
build up of a PC signaling and adhesion complex at the leading half of the
PCs. These data indicate that Fas2 is involved in intercellular interactions
crucial for organizing polarity, an important criterion proposed by Kolega
(Kolega, 1981) for a function
specifically involved in regulation of motility in multicellular clusters.
Significantly, our results indicate that molecules used for polarizing
epithelial cells are reorganized to polarize a motile cell cluster. The timing
of the reorganization of epithelial polarity is crucial for timing
delamination (Fig. 4,
Fig. 6,
Fig. 7D). Fas2 therefore plays
a direct role in mediating intercellular interactions that modulate movement,
a second property proposed by Kolega
(Kolega, 1981) for a function
specifically involved in regulating cluster motility as opposed to single
cells. We conclude that the Fas2 morphogentic switch facilitates development
of motile polarity essential for timely BC delamination. A similar switch
mechanism may be important in other processes that crucially depend on timing
of Fas2 activity, such as axon pathfinding
(Goodman et al., 1997
), and
learning and memory (Cheng et al.,
2001
).
A model for organization of cell cluster motility
Fas2 polarity appears to compartmentalize PCs into distinct functional
domains in order to control functionally distinct intercellular communication
with leading versus trailing BCs (see model,
Fig. 7D). Leading BCs play a
functionally distinct role by pioneering invasion between germ cells while
simultaneously detaching from the epithelium. Trailing BCs are likely to play
a less active role in invasion, but must mediate precisely timed detachment
from the epithelium. Fas2 polarization is thus likely to be crucial for
facilitating coordination of the distinct functional requirements of leading
versus trailing BCs, by establishing distinct sets of intercellular contact
and communication between the PCs and leading versus trailing BCs. In support
of this hypothesis, previous studies have suggested that leading and trailing
BCs are functionally distinct. In BC clusters comprising a mixture of wild
type and slbo, jing, taiman or DE-cadherin mutant cells,
wild-type BCs always lead invasion (Liu
and Montell, 2001;
Niewiadomska et al., 1999
;
Rørth et al., 2000
).
Furthermore, we documented additional structural evidence for cluster
asymmetry. Amph, a vesicle trafficking protein that regulates Dlg and Lgl
localization (Zelhof et al.,
2001
), is expressed at higher level in trailing BCs compared to
leading BCs (Fig. 7B-C').
Amph, Dlg and Lgl, are thus good candidates for proteins that differentially
regulate cortical and cell surface activities needed to mediate distinct
interactions of leading and trailing BCs with adjacent epithelial cells and
germ cells during the delamination process.
As only Dlg and Lgl are mislocalized in Fas2 clusters, but not
Fas3, -Spec or Crb (P.S. and S.G., unpublished), our data suggest that
Fas2 directs localization of specific molecules within distinct regions of
different cells of the cluster to control motility. A putative Fas2-binding BC
receptor may be another molecule whose polarity is controlled by Fas2 (see
previous section). Interaction with this putative receptor appears to
facilitate organization of the global polarity of the cluster, as the
orientation of delamination, mediated by the BCs, directly correlates with
Fas2 polarity in PCs (Fig.
2E,E'). These data thus suggest that Fas2 coordinates
directional mass motion between cells that are potentially capable of motion
in any direction, and that it helps to determine the locomotive-active regions
of these cells, additional criteria proposed by Kolega
(Kolega, 1981
) for a function
specifically involved in regulating cluster motility. Thus, as Fas2 is
required for regulation of several activities that distinguish how single
cells versus clusters move, our data provide the first molecular model for
understanding the organization of epithelial cluster polarity during
delamination and movement (Fig.
7D). One argument against this proposal might be that the PCs
appear to be highly specialized. However, we think this is likely to be of
less significance as PCs express epithelial polarity proteins in a pattern
similar to adjacent follicle epithelial cells
(Fig. 1E-E''',
Fig. 2A-A'',
Fig. 6A-A'').
As we have shown for BC clusters, several vertebrate studies have shown
that transmembrane proteins are differently expressed within different cell
subpopulations in migrating clusters (Hegerfeld et al., 2002;
Nakagawa and Takeichi, 1995;
Toba et al., 2002
).
Furthermore, the structure and functions of Fas2, Dlg and Lgl homologs are
conserved across phylogeny (Abbate et al.,
1999
; Gonzalez-Mariscal et
al., 2000
; Hoover et al.,
1998
; Huang et al.,
2003
; Ito et al.,
1995
; Matsumine et al.,
1996
; Roesler et al.,
1997
; Watson et al.,
2002
). Thus, the involvement of Fas2, Dlg and Lgl in organizing
cell cluster motility also may be conserved. We conclude that although the
precise mechanism of cluster movement may not be conserved in vertebrates, the
information that we glean about how BCs regulate epithelial polarity to
dynamically organize cluster polarity and movement will be generally useful
for understanding how cell cluster motility is organized across phylogeny.
Fas2 intercellular signals inhibit cluster migration
The previous sections discussed the role of Fas2 in delamination; here, we
discuss the role of Fas2 in regulating migration. Loss- and gain-of-function
experiments demonstrate that PC Fas2 acts as a signal to inhibit the rate of
BC migration (Fig. 4). Our work
builds on previous studies demonstrating the importance of PCs in determining
BC fate (Bai et al., 2000;
Beccari et al., 2002
). However,
our work is the first example of an intercellular signal that specifically
organizes cluster movement, rather than determining cell fate. Fas2 clearly
has a signaling function, as PCs do not contact the migration substrate. Thus,
these data demonstrate for the first time the existence of intercellular
communication between cells of a migratory cluster, which is specifically
required to modulate migration (Kolega,
1981
).
Contact inhibition of movement
PC Fas2 signaling inhibits the rate of cluster movement by maintaining Dlg
and Lgl localization in BCs (Fig.
2E,E', Fig.
4). The putative BC receptor that Fas2 interacts with (see
previous section) may control Dlg and Lgl localization in BCs. As Dlg is
localized to the cortex of BCs, Dlg must inhibit the rate of migration through
cortical activities in BCs. One cortical activity controlled by Dlg is the
recruitment of Lgl to the membrane (Fig.
6C'',E''). As lgl clusters have very similar
migration phenotypes to dlg clusters
(Fig. 4), our data indicate
that Lgl and Dlg cooperate to inhibit BC movement. The importance of Dlg and
Lgl in regulating cell movement probably derives from the same scaffolding
activities they use to organize and control membrane, cytoskeletal and
signaling specialization during the polarization of epithelial and neuronal
cells (Bear et al., 2000;
Kim et al., 1998
;
Lehman et al., 1999
;
Peng et al., 2000
;
Strand et al., 1994
). We
propose that Dlg and Lgl scaffolding organizes and integrates transmembrane
signaling and adhesion proteins with signaling, trafficking and cytoskeletal
effectors in the cortex of BCs to mediate contact-inhibition of cluster
movement.
Relevance to tumor cell invasion
BCs resemble dlg invasive tumor cells in that they lose epithelial
polarity by accumulating Dlg and Lgl around their circumference
(Fig. 1F,F', Fig. 2A',A'',
Fig. 6A',A'') (P.S.
and S.G., unpublished), but in contrast to BCs, dlg tumor cells
migrate between germ cells without temporal or spatial control
(Goode and Perrimon, 1997)
(S.G., unpublished). Our data demonstrate that Dlg and Lgl not only control
polarity and delamination of epithelial clusters, but also actively inhibit
movement. Thus, dlg tumor invasion is likely to be caused by a
combination of loss of epithelial polarity and over-activation of motility
pathways. In this context our results appear to be paradoxical in that loss of
epithelial polarity is generally considered to be crucial for facilitating
acquisition of motility, but we see that loss of polarity in normal migrating
clusters delays initiation of movement. Our data resolve this paradox in that
during normal development molecules used for polarizing epithelial cells are
reorganized to polarize a motile cell cluster. It therefore seems likely that
in carcinomas, inappropriate loss of epithelial polarity simultaneously
disrupts acquisition of motile polarity, but this phenomenon is not
appreciated because ultimately the tumor cells migrate. Thus, we postulate
that overactivation of motility pathways, as we see with loss of Dlg and Lgl
in BCs, may be especially crucial for achieving carcinoma invasion. Consistent
with this hypothesis, some dlg mutations that cause loss of
epithelial polarity do not lead to tumor invasion
(Goode and Perrimon, 1997
),
suggesting that acquisition of motility is a separate Dlg function.
Gene expression data for human cancers suggests that mutations that promote
tumor formation, through loss of epithelial polarity and increased
proliferation, may be the same mutations that subsequently cause tumor cell
invasion (Couzin, 2003). Based
on the observation that Dlg is required to maintain polarity, inhibit
proliferation (Woods and Bryant,
1991
) and inhibit movement
(Goode and Perrimon, 1997
)
(this study), we propose that tumor suppressors such as Dlg that regulate
signaling and adhesion at epithelial junctions may unify human gene expression
data by providing an ultrastructural target that controls contact inhibition
of both proliferation and movement. Progressive deterioration of epithelial
junctions may thus provide a common mechanism through which multiple tumor
suppressor pathways impact the cascade from cell proliferation to tumor
invasion, either through mutation or mislocalization of critical junctional
proteins.
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ACKNOWLEDGMENTS |
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