Dynamics of the basement membrane in invasive epithelial clusters in Drosophila
Caroline Medioni and
Stéphane Noselli*
Institute of Signaling, Developmental Biology and Cancer, UMR 6543 CNRS,
University of Nice Sophia-Antipolis, Parc Valrose, 06108 Nice, cedex 2,
France
*
Author for correspondence (e-mail:
noselli{at}unice.fr)
Accepted 29 April 2005
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SUMMARY
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The basement membrane (BM) represents a barrier to cell migration, which
has to be degraded to promote invasion. However, the role and behaviour of the
BM during the development of pre-invasive cells is only poorly understood.
Drosophila border cells (BCs) provide an attractive genetic model in
which to study the cellular mechanisms underlying the migration of mixed
cohorts of epithelial cells. BCs are made of two different epithelial cell
types appearing sequentially during oogenesis: the polar cells and the outer
BCs. Here, we show that the pre-invasive polar cells undergo an unusual and
asymmetrical apical capping with major basement membrane proteins, including
the two Drosophila Collagen IV
chains, Laminin A and
Perlecan. Capping of polar cells proceeds through a novel, basal-to-apical
transcytosis mechanism that involves the small GTPase Drab5. Apical capping is
transient and is followed by rapid shedding prior to the initiation of BC
migration, suggesting that the apical cap blocks migration. Consistently,
non-migratory polar cells remain capped. We further show that JAK/STAT
signalling and recruitment of outer BCs are required for correct shedding and
migration. The dynamics of the BM represents a marker of migratory BC,
revealing a novel developmentally regulated behaviour of BM coupled to
epithelial cell invasiveness.
Key words: Basement membrane, Border cells, Migration, JAK/STAT, Transcytosis, Collagen IV
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Introduction
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The extracellular matrix (ECM), including the basement membrane (BM), plays
a major role in the establishment and maintenance of epithelial cell
morphogenesis (Sternlicht and Werb,
2001
). Its selective synthesis and degradation is developmentally
regulated to favor cell rearrangements and migration. In cancer cells,
misregulation of ECM break-down strongly promotes cell invasion and metastasis
(Egeblad and Werb, 2002
).
However, the behavior of the ECM in the early stages of migration, such as
during migratory cell selection and delamination from an epithelium, remains
poorly understood.
Drosophila border cells (BCs) represent an attractive genetic
model with which to study the invasiveness of epithelial cell clusters
(Montell, 2003
;
Rorth, 2002
). BCs are made of
two different cell types that appear sequentially during oogenesis, which form
a composite cluster delaminating from the anterior follicular epithelium to
migrate posteriorly into the egg chamber
(Fig. 1A). In early stages, a
pair of polar cells is determined at each pole of the egg chamber. Later, a
ring of six outer BCs, surrounding the anterior polar cells, are specified to
make a mature cluster, which, collectively with polar cells, is called the BC.
We, and others, have recently shown that the formation of the BC cluster
depends on signaling from polar cells to the outer BCs
(Beccari et al., 2002
;
Ghiglione et al., 2002
;
McGregor et al., 2002
;
Silver and Montell, 2001
). In
this process, polar cells express the secreted ligand Unpaired, activating the
JAK/STAT pathway in neighboring cells. Those cells receiving the highest
levels of the Unpaired ligand are committed into the outer BC fate and
participate in the formation of a migratory BC cluster.
Recent work has shown that BCs are guided during their migration towards
the oocyte through the EGFR and the PDGF/VEGF pathways
(Duchek and Rorth, 2001
;
Duchek et al., 2001
;
McDonald et al., 2003
). Other
molecules are also essential for BC migration, including Slbo
(Montell et al., 1992
),
ecdysone (a steroid hormone) (Bai et al.,
2000
) and myosin VI
(Geisbrecht and Montell,
2002
), as is the formation of long cellular actin-containing
extensions (Fulga and Rorth,
2002
). However, the cellular events accompanying the initial
phases of BC formation and delamination are poorly understood, and it is
particularly unclear what are the role and behavior of the BM in this
process.
Here, we show that components of the BM undergo shuttling to the apical
surface in anterior polar cells, through transcytosis involving Drab5 (Rab5 -
FlyBase). This unusual apical capping is both asymmetrical and transient.
Indeed, shortly before invasion starts, JAK/STAT signaling and interaction
with outer BCs is required for shedding of the apical cap in polar cells, thus
coordinating outer BC recruitment and invasiveness of the cluster. Strikingly,
isolated polar cells that are unable to migrate maintain an apical cap.
The apical and transient targeting of BM materials in BCs may represent a
novel marker of migratory cells during development and cancer, as well as a
novel mechanism whereby the transition from pre-migratory to migratory
phenotype is controlled.
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Materials and methods
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Genetics
A description of genetic markers and chromosomes can be found at FlyBase
(http://flybase.bio.indiana.edu).
Protein-trap lines vkgG454 (GFP-Vkg)
(Morin et al., 2001
), and
658, 1700, 1973, 2840, 2867, G6 and G205
(Kelso et al., 2004
), were
used to mark the basement membrane and the apical cap. GAL4-dependent
overexpression in the BC was performed using the slbo-GAL4,
vkgG454/CyO line crossed to UAS-X flies
(with X representing any reporter gene). The UAS lines used in this
study are listed in Table 1. To
overexpress eya, UAS-EYAB1 and UAS-EYAB2 were used (see FlyBase) (Bai
and Montell, 2002), and crossed to Upd-GAL4 (a gift from D. Montell). Clonal
analysis was performed using y w; vkgG454,
FRT40A/CyO crossed to either HS-Flp; HS-c-Myc,
FRT40A/CyO or UB-GFP, FRT40A/CyO; T155-GAL4, UAS-Flp/T155-GAL4,
UAS-Flp. Myc and nls-GFP were used as clonal markers.
Antibodies and western blotting
Polyclonal antibodies against type IV Collagen
1 chain (Cg25C;
1:1000) were from J. Fessler (Blumberg et
al., 1988
) and anti-GFP was a gift from R. Arkowitz (1:10,000).
Monoclonal antibodies against Crumbs (1:50), Armadillo (1:50), Fas2 (1D4;
1:50) and Fas3 (7G10; 1:50) were from the Developmental Studies Hybridoma
Bank; anti-Laminin A (1:3000) was a gift from S. Baumgartner
(Gutzeit et al., 1991
),
anti-ß-galactosidase (1:1000) was from Promega and anti-c-Myc was from
Calbiochem (1:100). Secondary antibodies were anti-rabbit Alexa 488 (1:400) or
Texas Red (1:100), and anti-mouse Texas Red (1:100) or Alexa 488 (1:400), from
Molecular Probes.
Protein extracts were prepared from ovaries, loaded on SDS-PAGE gels and
blotted onto nitrocellulose filters. Type IV Collagen
1 and
2-GFP chains were detected using anti-
1 and anti-GFP antibodies,
respectively.
Confocal microscopy and imaging
From one experiment to another, we found a small variation in the number of
egg chambers showing an apical cap in wild type (85±5%), probably
because of variations in the fixation conditions. To determine the percentage
of egg chambers showing an apical cap (Fig.
4G,H), wild-type and mutant egg chambers were stained and
processed at the same time. Thus, each experiment has its own control, which
is plotted in the histogram. At least three sets of experiments were performed
for each condition.
Images were taken using a Leica TCS-SP1 or a Zeiss LSM 510 Meta confocal
microscope and processed using Photoshop 7.0 (Adobe). Three-dimensional
reconstruction of anterior egg chambers and apical caps was made using
Volocity 2.6 (Improvision).
Statistical analysis
The statistical analysis shown in Fig.
4G was made by comparing the means of the variables in each
experiment. Given the statistically high number of samples, we applied the
Bernoulli Rule. In each experiment, we tested the hypothesis that results are
equal versus the hypothesis that results are different (ShiK44A),
lower (Drab5S43N) or higher (Drab5WT) than the wild
type. We chose a confidence interval (
) and applied Student's
t-test to the data. The result of which indicates that the
probability that Drab5S43N is lower than wild type is 0.99 (99%),
the probability that ShiK44A and wild type are not different is
0.99 (99%), and the probability that Drab5WT is higher than wild
type is 0.85 (85%).
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Results
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Apical capping of anterior polar cells with basement membrane material
In order to analyse the dynamics of the BM in developing BCs, we used a
specific protein-trap line expressing Drosophila type IV Collagen
2 chain [encoded by the viking (vkg) gene] fused to
GFP (Morin et al., 2001
). The
resulting GFP-Vkg fusion protein is expressed under the control of the
endogenous vkg promoter and thus represents a faithful reporter of
Vkg expression. GFP-Vkg strongly accumulates basally in all egg chambers
(Fig. 1B; data not shown),
consistent with Collagen IV being a major component of the BM
(Fessler and Fessler, 1989
).
Most surprisingly, we found that, in stage 8 egg chambers, GFP-Vkg also
accumulates apically into a discrete cap over the anterior polar cells
(Fig. 1B,C), but not over the
posterior ones (Fig. 1D). Only
anterior polar cells will recruit outer BCs and undergo migration
(Fig. 1A). Like GFP-Vkg,
antibodies against the Cg25C/Coll.IV
1 chain
(Fig. 1E-H) (Blumberg et al., 1988
) and
Laminin A (Gutzeit et al.,
1991
) mark the BM and co-localize with GFP-Vkg in the apical cap
(Fig. 1I-K). To determine
whether apical capping represents a general behavior of the BM, a series of
recently developed but uncharacterized protein-trap lines exhibiting BM
localization were analysed (Kelso et al.,
2004
). Sequencing of these protein-trap lines identified a novel
GFP-Vkg fusion (G205; Fig. 1L;
see Fig. S1A in the supplementary material) and several insertions in the
perlecan/CG7981 gene (G6, 658, 1700, 1973, 2840, 2867;
Fig. 1M,N and Fig. S1B in the
supplementary material), which encodes a proteoglycan that is a major
component of BM. These results indicate that most, if not all, of the
components of the BM in stage 8 egg chambers form an apical cap over the
anterior polar cells.

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Fig. 1. Apical capping of anterior polar cells with basement membrane material. (A)
Formation and migration of the BC cluster. (a) A stage 8 egg chamber made of a
monolayer epithelium (follicle cells, light gray) surrounding the germline
(blue, nurse cells; yellow, oocyte). (b) Anterior polar cells (orange) express
the Upd ligand activating the JAK/STAT pathway in follicle cells (arrows). (c)
Cells receiving the highest levels of Upd are committed into the outer BC fate
(beige). (d) A mature cluster delaminates and starts migration. (B) GFP-Vkg
(green) accumulates in the BM. At stage 8, an apical cap forms over anterior
polar cells (arrowhead). (C,D) Close up of the framed regions shown in B.
Polar cells are marked by Fas2 expression (B-D; red). (E-G) Co-localization of
GFP-Vkg and Coll.IV 1 (F,G; red), at the BM and in the apical cap (G).
(H) Antibodies are specific to Coll.IV 1, as they do not recognize
GFP-Vkg. The lower band (asterisk) is non-specific. w, white. (I-K)
Co-localization of GFP-Vkg and Laminin A (red) in the apical cap. (L-N) Apical
cap accumulation of BM protein-trap lines G205 (Vkg), 2840 and 2867 (Perlecan;
see Fig. S1 in the supplementary material). Anterior is to the left. Scale
bars: in B, 10 µM; in C, 5 µM for C,D; in E, 5 µM for E-G; in I, 5
µM for I-K; in L, 5 µM for L-M.
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Cell-type specificity of apical capping
In wild-type egg chambers, the apical cap forms over anterior polar cells
only (Fig. 1B). In order to
demonstrate the cell-type specificity of apical capping, anterior polar cells
were either deleted or made in excess, by manipulating the polar cell fate
repressor eyes absent (eya). Overexpression of eya
using Upd-GAL4 blocks anterior polar cell differentiation (Bai and Montell,
2002) (Fig. 2A-C). As a
consequence, the apical cap is not formed
(Fig. 2A,B; compare with
Fig. 1B,C). Conversely, when
mutant clones for eya are generated in the anterior follicle cells,
ectopic polar cells form and are capped
(Fig. 2D-I). Consistently,
ectopic polar cells made in the posterior region do not show an apical cap
(data not shown), indicating a contribution of anteroposterior patterning in
the regulation of polar cell capping. Altogether, these results demonstrate
that apical capping is a specific feature of the anterior, migrating polar
cells.
Apical capping is asymmetrical and dynamic
Three-dimensional imaging of anterior stage 8 egg chambers shows that the
apical cap is rod-shaped and runs along the apical surface
(Fig. 3A-F; see Movies 1 and 2
in the supplementary material). Interestingly, the apical cap preferentially
associates with one of the two polar cells (see below), thus revealing a
previously unknown intrinsic asymmetry within the pair of anterior polar
cells.
Careful staging of egg chambers indicates that apical capping is highly
dynamic and proceeds through four discrete steps. (1) From stages 1-8 of
oogenesis, GFP-Vkg and other BM component localization is restricted to the
basal surface of epithelial cells (Fig.
3G and data not shown). (2) During stage 8, the formation of a
discrete apical cap above each of the two polar cells is observed, suggesting
that, initially, each polar cell makes its own apical cap
(Fig. 3H, arrowheads;
Fig. 2D,F). Subsequently, when
the apical surface of polar cells constricts, one cap develops (large
arrowhead) while the other remains rudimentary (small arrowhead), thus leading
to asymmetrical capping (Fig.
3I-K). (3) The apical cap is kept in place at stages when polar
cells undergo rounding and detach from the BM
(Fig. 3J). In rare cases, it is
possible to observe an intermediate stage showing two opposite caps, one at
the basal and one at the apical surface
(Fig. 3K). (4) Finally, when
the BC cluster starts to migrate, the apical cap is no longer observed and
only the basal cap remains (Fig.
3L). Thus, apical capping is transient, appearing at stage 8 and
being shed at stage 9. The timing and cell-type specificity of apical capping
indicate that BM cap dynamics are tightly coordinated with the formation of
migratory border cells.

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Fig. 2. Apical capping is specific to anterior polar cells. (A) Overexpression of
the polar cell fate repressor eya. The polar cell marker Fas2 is
shown in red. (B,C) Enlarged views of the framed regions shown in A. Note the
absence of anterior polar cells and an apical cap (compare with
Fig. 1B-D). Egg chambers show
Laminin A (green) and nuclei (blue). (D-I) Mosaic egg chambers containing
eya mutant follicle cells making ectopic polar cells (Fas2, in red).
(D-F) Anterior stage 8 egg chamber with two normal polar cells and their
apical cap (D, arrowheads 1, 2). (E) A ectopic polar cell close by shows a
nascent apical cap (arrowhead 3). (F) Merged image of D and E. (G-I) A group
of five to six ectopic polar cells have assembled two separate and large
apical caps (arrowheads 1, 2). (I) Merged image of G and H. Scale bars: in A,
10 µM; in B, 10 µM for B,C; in D, 5 µM for D-F.
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Fig. 3. The apical cap is asymmetric and dynamic. (A-F) Three-dimensional imaging
and reconstruction of the anterior region of stage 8 egg chambers, showing
GFP-Vkg (green), Fas2 (red) and Hoeschst (blue, in A). (A) The original stack
is a projection of 20 z sections (total depth is 7.6 µm). (B)
Intermediate processing showing BM (green), polar cells (white) and apical cap
(blue). (C) The resulting 3D-reconstructed polar cells with the apical cap.
(D-F) Three other examples of reconstructed polar cells with their apical cap.
Insets in C-F show the apical side of polar cells. The red line is the
boundary between the two polar cells. (G-O) GFP-Vkg (green; G-0) egg chambers
showing Fas2 (G-L), Crumbs (M,O) or Fas3 (N) in red. The process of apical
capping is transient and can be described in four discrete phases (1-4; see
text for details). After shedding (L), the BC delaminate and invade the nurse
cell compartment (M). During migration (phase 5), a BM containing GFP-Vkg is
present in polar cells specifically (M-O), opposite to the apical side (marked
with Crumbs in M and O). The polar cell BM is maintained when BCs reach the
oocyte (O). Anterior is to the left. Scale bars: in A, 5 µM for A-F; in G,
5 µm for G-O.
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Polar cells, but not border cells, maintain a basement membrane during migration
BCs are of epithelial origin and their detachment from the follicular
epithelium requires remodelling of cellular junctions
(Niewiadomska et al., 1999
).
Apical and adherens junction markers are reorganized during the transition
from an epithelial to a migratory phenotype, leading to an extension of the
apical and junctional domains and the adoption of a rosette-like shape
(Fig. 3M)
(Niewiadomska et al., 1999
).
After migration, when BCs reach the oocyte and re-epithelialize, the apical
domain returns to its original organization
(Fig. 3O). Interestingly, we
found that polar cells keep a BM throughout the process, in a position
opposite to the apical side as shown by double staining with the apical marker
Crumbs (Fig. 3M,O). This is in
sharp contrast with outer BCs, whose BM is detached or degraded when they
delaminate from the follicular epithelium. This result indicates that contrary
to outer BCs, polar cells maintain a fully polarized phenotype with a normal
BM during migration.

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Fig. 4. Drab5-dependent basal to apical transcytosis of basement membrane. (A-C)
GFP-Vkg non-expressing cells in a GFP-Vkg/+ background. The Myc
clonal marker is in red; GFP-Vkg non-expressing cells are outlined (dashed
lines). When polar cells only (A), or polar and outer BCs (B,C), do not
express GFP-Vkg, the apical cap forms normally. (D) Wild type; (E,F) polar
cells expressing Drab5S43N (E) do not form an apical cap or (F)
show a `microcap' phenotype. Frequency of caps (G) and microcaps (H) of stage
8 egg chambers in wild-type (n=625) flies, or flies expressing
Drab5S43N (n=298), Drab5WT (n=201) or
ShiK44A (strong; n=183). Data are presented as
mean±s.e.m. (see Materials and methods). Anterior is to the left. Scale
bar: in A, 5 µM for A-F.
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Apical capping is controlled by Drab5-dependent transcytosis
Two simple mechanisms could lead to the formation of an apical cap over
polar cells. In a first scenario, this cap could originate from apical
targeting of neosynthesized BM proteins by polar cells. In a second mechanism,
the apical cap could form by transcytosis of BM material from the basal to the
apical surface. In order to discriminate between these two mechanisms, we
first tested whether inhibition of GFP-Vkg expression in polar cells and/or
BCs could affect apical capping. Clonal analysis was used to make
non-expressing GFP-Vkg cells in a GFP-Vkg background. In such mosaic egg
chambers, polar cells that do not express GFP-Vkg still show an apical cap
(Fig. 4A), indicating that it
is not originating from de novo synthesis in the polar cells. This conclusion
is consistent with the fact that polar cells, unlike other follicle cells, do
not express GFP-Vkg around stage 8, as shown by the absence of GFP-Vkg in the
cytoplasm (Fig. 4 and data not
shown). Apical capping does not depend on GFP-Vkg expression in outer BC
either, as clones of outer BCs that do not express GFP-Vkg do not affect the
assembly of an apical cap (Fig.
4B,C). Thus, BM proteins present in the apical cap have a
non-autonomous origin. We found that the normal BM, which is present in all
stages throughout oogenesis (Fig.
5A), is also made in a non-autonomous manner
(Fig. 5B-G), indicating that it
is not possible to directly test the effect of genetically removing components
of the BM. Indeed, the BM was normal whatever the developmental stage or the
size of the clones; consistently, we found that vkg mutations do not
show any defect in BC migration (data not shown).
The above results suggest that the apical cap originates from outside the
polar cells. Transcytosis is a mechanism allowing proteins from one membrane
sub-domain to be targeted to another domain after internalization
(Tuma and Hubbard, 2003
). To
test whether internalization could be a mechanism by which the apical cap
forms, we blocked Drab5 and Shibire (a Drosophila dynamin homolog)
function in the BCs. These proteins are required in intracellular trafficking
in Drosophila and other organisms
(Seto et al., 2002
), and we
show here that both proteins are essential for BC migration
(Table 1;
Fig. 6). Interestingly, the
expression in BCs of a Drab5 dominant-negative (DN) form
(Drab5S43N) (Wucherpfennig et
al., 2003
) results in the absence of the apical cap in stage 8 egg
chambers, as compared with wild type (Fig.
4D,E,G). Moreover, among clusters still showing a cap in
Drab5S43N egg chambers, 47% show a `micro-cap'
(Fig. 4F,H), which is twice as
frequent as in wild-type egg chambers (20%)
(Fig. 4H). Conversely, the
expression of Drab5WT induces a 5% increase in the number of egg
chambers with an apical cap (Fig.
4G). In this case, micro caps are less frequent (15%) than in wild
type (25%) (Fig. 4H).
The expression of a dominant-negative form of Shibire (ShiS44A)
(Moline et al., 1999
) did not
block the formation of the apical cap (Fig.
4G). Thus, contrary to clathrin-dependent endocytosis, Drab5 and
Shi have distinct roles in the formation of an apical cap over anterior polar
cells.
We also tested the effect of other trafficking molecules and of signaling
pathways known to be important for BC migration (see
Table 1). None of these induced
a defect in apical cap formation, indicating that Drab5 plays a specific role
in this process.
Apical cap shedding requires JAK/STAT signaling and outer BCs
Once BC delaminate and start migration, the apical cap is no longer
observed, indicating that an active process is responsible for removing the BM
apically. It is thus important to determine the mechanism by which apical
shedding and migration are coordinated. One likely possibility is that outer
BCs themselves could control the status of the apical BM in polar cells. In
order to test this possibility, we blocked the formation of outer BCs by
expressing dominant-negative forms of Domeless (Dome) (Dome
Ext and
Dome
Cyt), the receptor of the JAK/STAT pathway. In wild-type egg
chambers, polar cells secrete the Unpaired ligand, which activates the
JAK/STAT pathway in neighboring cells and recruits them as outer BCs
(Fig. 1A) (Beccari et al., 2002
;
Ghiglione et al., 2002
;
McGregor et al., 2002
;
Silver and Montell, 2001
).
Those cells receiving the highest levels of Unpaired are committed into the
outer BC fate and participate in the formation of a migratory BC cluster.
Expression of dominant-negative forms of Dome lacking either the extracellular
or the intracellular receptor domain (Dome
Ext and Dome
Cyt,
respectively), induces most egg chambers to develop without outer BCs
(Ghiglione et al., 2002
).
Consequently, the two polar cells remain at the anterior tip and do not
migrate (Fig. 7B). Strikingly,
the apical cap forms normally in Dome
Cyt stage 9 egg chambers but is
not shed in stage 10, or later, egg chambers
(Fig. 7A,B), suggesting that
outer BCs are required for removing of the apical cap. In order to confirm
this result, and to rule out the possibility that the absence of migration
itself could block shedding, migration of a fully formed BC cluster was
inhibited by expression of either a dominant-negative form of Drac
(DracN17) (Murphy and Montell,
1996
) (Fig. 7C,D)
or a wild-type form of Dome (Ghiglione et
al., 2002
) (Table
1). In both cases, and despite a lack of migration, the apical cap
forms normally and is shed like in wild-type clusters, confirming that outer
BCs are essential for shedding before the cluster starts migration, probably
by interacting back with the polar cells. Note that, in the Drac1 condition,
detachment from the BM appears normal, as indicated by the presence of a basal
cap (Fig. 7D), and the
migratory phenotype originates from actin cytoskeleton defects, as shown
previously (Geisbrecht and Montell,
2004
; Murphy and Montell,
1996
).

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Fig. 5. GFP-Vkg basement membrane is assembled cell non-autonomously. (A) Confocal
image showing the anterior region of an ovariole. Note that BM is present at
all stages of egg chamber development. (B) Scheme of the FLP-FRT-mediated
clonal analysis used to generate mosaic egg chambers containing clones of
cells expressing either no copies (0; blue color code), one copy (gray color
code) or two copies (red color code) of GFP-Vkg. In this scheme, the clonal
marker is a nuclear GFP (nls-GFP, green). (C) A mosaic egg chamber containing
several clones of cells expressing 0, 1 or 2 copies of GFP-Vkg. Note the
uniform basement membrane. In particular, cells that do not express GFP-Vkg
(blue lines) have a normal BM, indicating that GFP-Vkg is not contributed
autonomously to the BM by follicle cells. (D) Another example in which a large
clone of cells expressing two copies of GFP-Vkg (red line) abut cells
expressing no copies of GFP-Vkg (blue line). In this case, as well, there is
no change in the apparent thickness or staining of the BM. (E) The same egg
chamber double labelled with phalloidin-TRITC (red) to outline the cell
boundaries. (F-G') Two egg chambers in which a big clone of cells that
do not express GFP-Vkg (blue line) is adjacent to cells expressing 2 copies of
GFP-Vkg (red lines). In these mosaic egg chambers, the GFP-Vkg BM is uniform.
(C-E) The clonal marker is a nuclear GFP; (F-G') the clonal marker is a
Myc tag (anti-c-Myc shown in red). Scale bars: in A, 10 µM; in C 10 µM
for C-E; in F 10 µM for F-G'.
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The Drosophila genome encodes only two matrix metalloproteinases
(MMPs), which are natural regulators of the extracellular matrix.
Interestingly, Drosophila Dm1-MMP (Mmp1 - FlyBase) and Dm2-MMP (Mmp2
- FlyBase) have been shown to degrade Collagen IV in vitro
(Llano et al., 2002
;
Llano et al., 2000
). However,
we could not see any defect in apical cap dynamics upon expression of Dm1-MMP
(native or activated form) or the Drosophila MMP inhibitor DTIMP
(Timp - FlyBase; Table 1),
which suggests that a different protease or mechanism is responsible for
apical cap degradation. It is also important to mention that neither
Dynamin-dependent endocytosis (ShiK44A) nor lysosomal degradation (Drab7Q67L
and Dor) seem to be involved in cap formation or shedding (see
Table 1).

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Fig. 6. BC migration defects of Drab5S43N and ShiK44A egg
chambers. Expression of Drab5S43N, Drab5WT and
ShiK44A in egg chambers was driven by slbo-GAL4 at 29°C. To
measure the extent of migration, the nurse cell compartment was divided into
four equivalent regions (1 to 4: clusters in region 1 have almost completely
migrated whereas clusters found in region 4 have not migrated at all; see
inset) and the position of the BC relative to this coordinate system
monitored. Drab5S43N and ShiK44A led to similar and
strong migration defects.
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Fig. 7. Apical cap shedding requires JAK/STAT signaling and the recruitment of
outer BCs. GFP-Vkg (green) egg chambers expressing Dome cyt
(A,B) or Drac1N17 (C,D). Follicle cells are marked with phalloidin
(A,C,D; red). In B, polar cells are marked with slbo-lacZ (red). (A)
Blocking outer BC recruitment does not affect the formation of the apical cap
at stage 9. However, shedding does not take place and a cap is still present
in stage 10 Dome cyt egg chambers (B; phenotype schematized
in the upper left panel). Expression of Drac1N17 blocks migration
(D) but has no effect on outer BC recruitment and apical cap formation (C)
(phenotype schematized in the lower left panel). (E) A four-step model for BC
formation and migration: (1) formation of an asymmetrical apical cap (green;
containing Collagen IV 1 and 2 chains, Laminin A, Perlecan) over
the anterior polar cells (orange) through Drab5-dependent transcytosis; (2)
polar cells send a JAK/STAT signal to recruit outer BCs (beige) from the
surrounding follicle cells (light gray); (3) shedding of the apical cap is
dependent on the presence of outer BCs; (4) migration of the mixed BC cluster
starts, with polar cells maintaining a BM. Anterior is to the left. Scale bar:
in A, 5 µM for A-D.
|
|
 |
Discussion
|
---|
The migration of cohorts of cells is an alternative to single-cell
migration, which is used by normal and cancer cells to invade tissues. One
advantage for mixed clusters is to transport tumorigenic (for example,
apoptotic resistant) cells with no migratory abilities to a distant
destination that they could not reach on their own
(Friedl and Wolf, 2003
). In
this case, migration is executed by migratory capable cells within the
cluster. Clusters illustrate how separate functions (tumorigenesis and
migration) can be merged through collaboration between two cell populations.
It is thus important to understand how migrating cell clusters are assembled
and organized. The BC cluster is made of two distinct populations of cells,
i.e. the polar cells and the outer BCs, making it a good model with which to
determine the cellular mechanisms underlying the recruitment and migration of
mixed cohorts of cells. Here, we identify three novel steps in the formation
of BCs (Fig. 7E). First, we
show that a developmentally regulated basal to apical transport of BM material
takes place in the polar cells, the first population of cells to form in the
cluster. The apical cap is the earliest known marker of anterior polar cells.
Second, the asymmetrical positioning of the apical cap suggests that despite
an apparent identity, the two polar cells are different and might play
distinct roles. Third, our data indicate that a two-way interaction takes
place between the two differentiated subpopulations of invasive cells before
they migrate. A first signal, activating the JAK/STAT pathway is sent by the
polar cells to recruit the outer BCs. In a second step, the outer BCs are
essential for shedding the apical cap of polar cells
(Fig. 7E).
Outer BCs are not required for apical cap formation
(Fig. 7A,B). Similarly, outer
BCs form normally in the absence of a cap
(Fig. 4; data not shown),
indicating that apical capping is not a pre-requisite for outer BCs to be
recruited and the cluster to be assembled. Interestingly, we found that
immotile polar cells remained capped (Fig.
7A,B). Thus, a possible role for apical capping is to block the
migration of immature clusters, a finding that could explain the long standing
observation that isolated polar cells cannot migrate on their own. Indeed, the
coordination between apical cap degradation and the recruitment of outer BCs
indicates that degradation of the apical cap could serve as a check point or
quality control ensuring that only finalized clusters can start migration. It
is important to note that degradation of the ECM at the leading edge of
migrating clusters is essential for tumour progression, and examples of cancer
cells showing a reduction or absence of some basement membrane markers,
including Collagen IV, have been reported. In particular, human
3/
4 type IV Collagen is found at the apical surface in normal
colon tissue, but is absent in colorectal neoplastic cells
(Hiki et al., 2002
), making
the differential distribution of type IV collagens potential diagnostic
markers for the invasiveness of cancer cells. The BC model will be central for
future studies aimed at understanding BM dynamics and function in invasive
clusters.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/13/3069/DC1
 |
ACKNOWLEDGMENTS
|
---|
We thank R. Arkowitz, S. Baumgartner, L. Bianchini, N. Brown, L. Cooley, J.
Fessler, M. Gonzalez-Gaitan, P. Léopold, C. Lopez-Otin, D. Montell, X.
Morin, A. Page McCaw, P. Rorth, P. Thérond, the Bloomington Stock
Center and DSHB for providing us with fly stocks, materials and/or for
critical reading; O. Devergne for Fig.
6; P. Spéder for statistical analysis and D. Cerezo for
technical help. Work in S.N.'s Laboratory is supported by grants from CNRS
(ATIPE), ACI, ARC (4673), IFCPAR and EMBO YIP. C.M. is supported by a
fellowship from MNERT and ARC.
 |
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