Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA
* Author for correspondence (e-mail: dmontell{at}jhmi.edu)
Accepted 20 August 2004
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SUMMARY |
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Key words: Par-6, Bazooka, Border cells, Cell migration, Drosophila
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Introduction |
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The migration of the border cells, a small group of follicle cells in the
Drosophila ovary, provides a model system to study these events in
vivo. The border cells originate from the anterior pole of the follicular
epithelium, which surrounds and communicates with an underlying cluster of 16
germline cells to form an egg chamber (Fig.
1A). Migration is initiated when a pair of specialized follicle
cells, known as the polar cells, secrete a cytokine signal that activates the
Janus Kinase/signal transducer and activator of transcription (JAK/STAT)
pathway in the neighboring 6-8 cells, stimulating their motility
(Silver and Montell, 2001).
Migration is completed once the border cells reach the oocyte.
|
Although much has been learned concerning the molecules and mechanisms that
are responsible for generating and maintaining polarity within the follicular
epithelium, little is known of what happens to epithelial polarity when the
border cells change from stationary epithelial cells to invasive cells.
Earlier studies of the organization of the border cell cluster demonstrated
that four to six motile cells are attached to the centrally located pair of
anterior polar cells in a rosette arrangement
(Niewiadomska et al., 1999).
In addition, Crumbs was shown to be localized asymmetrically throughout
migration, although the functional significance of this protein was not
investigated (Niewiadomska et al.,
1999
).
It was not clear from the earlier work whether the asymmetric localization of Crumbs was unique to this protein or whether epithelial polarity was more generally maintained during border cell migration. In this study, we focus on Par-6 and Baz, the Drosophila homolog of Par-3, which are part of an evolutionarily conserved complex that contributes to the polarization of epithelial cells in a variety of organisms. Par-6 and Baz are localized at the apical surfaces of follicle cells and, interestingly, are maintained asymmetrically in the migrating border cells. Loss-of-function mutations in these genes in somatic follicle cell clones result in epithelial defects as previously described. In addition, we show that disruption of par-6 and baz function in the border cells using RNAi results in delayed migration as well as mislocalization of membrane proteins such as E-cadherin and ßps-integrin. Migration delays and mislocalization of E-cadherin and ßps-integrin are also observed when Par-6 and Baz are overexpressed in border cells, using the Gal4/UAS system. Our observations suggest that border cells retain some degree of epithelial polarity during migration, and that Par-6 and Baz are required for the proper distribution of membrane proteins in the border cell cluster and for successful migration.
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Materials and methods |
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To express genes ectopically as well as to express RNAi trangenes, the
Gal4/UAS system was utilized (Brand and
Perrimon, 1993). The Gal4 strains used were Act5c
(Ito et al., 1997
),
e22c (Lawrence et al.,
1995
), c306 (Manseau
et al., 1997
), slbo
(Rorth, 1998
), upd
(Doug Harrison) and tubulin (Lee
and Luo, 1999
). UAS lines used were UAS-Baz
(Kuchinke et al., 1998
),
UAS-Par-6 (J. A. Knoblich, unpublished) and UAS-dome
CYT
(Brown et al., 2001
).
RNAi constructs
The RNAi-par-6 construct contains the 1.5 kilobase coding region
of par-6 from the expressed sequence tag LD08317 cloned into the
Sym-pUAST vector (Giordano et al.,
2002). The RNAi-baz construct was obtained by cloning a
two-kilobase PCR-generated baz fragment (from nucleotides 28506 to
30554 of a genomic clone, GenBank Accession number: AE003504) into the
Sym-pUAST vector. The PCR-amplified genomic fragment was obtained
using the following primer pair: EcoRI-5'
(5'-CGGAATCGGAGGACGACGATCCCAGTCATC) and
EcoRI-3' (3'-CTGAATTCGAGGAGCAAATGCCAC). Each
construct was verified by restriction mapping and DNA sequencing. A control
RNAi-white transgenic fly line was obtained
(Giordano et al., 2002
).
P element-mediated germline transformation
Plasmid DNA to be injected was prepared and purified by using the Qiagen
plasmid kit. DNA was dissolved in injection buffer (0.1 mM phosphate buffer pH
6.8, 5 mM KCl) at a concentration of 500 µg/ml for the transforming plasmid
and 160 µg/ml for the helper plasmid p25.7 (wings-clipped). The RNAi
par-6 construct and RNAi baz construct were injected into
w1118 embryos as described
(Spradling, 1986
). Eighteen
independent insertion lines were obtained for RNAi par-6, and were
combined to create RNAi par-6 lines with two copies of the transgene.
One insertion on the second chromosome was obtained for RNAi baz.
Additional insertions were obtained by mobilizing the original RNAi
baz transgene.
Immunofluorescence and antibody production
Rabbit anti-PAR-6 antibodies were generated against the peptide
HHQQAASNASTIMASDVKDGVLHL, affinity purified, (Proteintech Group) and used at
1:500 dilution. Ovary dissection and fixation, and antibody staining were
performed essentially as described (Bai et
al., 2000; Montell,
1999
). For immunostaining, the following primary antibodies were
used: mouse anti-Armadillo monoclonal [N27A1, 1:75; Developmental Studies
Hybridoma Bank (DSHB)]; rat anti-DE-cadherin monoclonal (DCAD2, 1:10;
(Uemura et al., 1996
); mouse
anti-Fascilin III monoclonal (7G10, 1:50; DSHB); rabbit anti-GFP serum
(1:4000; Molecular Probes); mouse monoclonal anti-ßps-integrin
(CF.6G11, 1:10; DSHB); mouse anti-Singed monoclonal (7C, 1:25; DSHB); mouse
anti-
-tubulin (1:500; Sigma). For rabbit polyclonal DmPAR-6 [1:500
(Petronczki and Knoblich,
2001
)]; rabbit anti-Bazooka N-term (1:250) and rat anti-Bazooka
N-term monoclonal [1:250 (Wodarz et al.,
1999
)] and rabbit anti-nPKC
polyclonal (C20, 1:250; Santa
Cruz Biotechnology), ovaries were first washed with PBS and blocked with 1%
BSA in PBS, then incubated with the primary antibody at 4°C overnight.
Secondary antibodies conjugated to Alexa-488 and Alexa-568 (Molecular Probes)
were used at a dilution of 1:400. F-actin filaments were detected with
rhodamine-phalloidin (Molecular Probes), at a dilution of 1:400. Images were
captured with the Ultraview confocal microscope or with a digital camera on a
Zeiss Axioplan fluorescent microscope. Three-dimensional reconstructions of
confocal image stacks were generated using Volocity software.
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Results |
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To get a more complete view of the distributions of Par-6, Baz and aPKC
during migration, we examined the expression of these proteins in
three-dimensional reconstructions of migrating border cell clusters. Par-6,
Baz and aPKC were found at high levels on one side of the cluster, which was
roughly orthogonal to the direction of migration, whereas E-cadherin could be
detected throughout the lateral surfaces of these cells
(Fig. 1N-P') (see Movie 1
in the supplementary material). Just as in epithelial cells, a small region of
overlap between Par-6, Baz, aPKC and E-cadherin was detected. These findings
are consistent with the previous observation that another apical epithelial
protein, Crumbs, is also found on one side of the border cell cluster during
migration, perpendicular to the direction of migration
(Niewiadomska et al., 1999).
These findings suggest that early in migration the entire cluster rotates so
that the leading edge is roughly orthogonal to the apical domain.
Par-6, Baz and aPKC were not detected at the surfaces of the border cells
in contact with the polar cells. The polar cells occupy a central position in
the border cell cluster during migration, and high levels of Par-6, Baz and
aPKC were observed in a ring-like pattern at the polar cell apices
(Fig. 1N). The asymmetric
distributions of Par-6, Baz, aPKC and Crumbs
(Niewiadomska et al., 1999) in
the migrating border cells indicated that some apical/basal polarity was
maintained.
Requirement for Par-6 and Baz expression in the border cells for proper migration and adhesion within the cluster
The reason for maintaining apical/basal polarity during migration is not
understood. It has been postulated that this would have the advantage that
epithelial polarity does not need to be established de novo when the border
cells reach the oocyte (Niewiadomska et
al., 1999). If this were the only purpose for maintaining polarity
during migration, then the functions of apical proteins would not be required
for migration. Alternatively, or in addition, Par-6 and Baz may play a
specific role in migration, in which case disrupting their expression would
impair migration. To distinguish between these hypotheses, par-6 and
baz homozygous mutant follicle cell clones were generated using the
FLP/FRT system, since null alleles of par-6 and baz are
embryonic lethal.
Follicle cell clones, mutant for the bazxi106 or
par6226 null allele, exhibited epithelial
defects including discontinuity and multilayering of the epithelium, as
previously reported (Abdelilah-Seyfried et
al., 2003
; Huynh et al.,
2001
). Although mutant border cell clusters were rare, we observed
some examples of border cell clusters that failed to migrate to the oocyte
(Fig. 2B,D). In these examples,
the majority of cells in the cluster were homozygous mutant. Clusters with
smaller numbers of mutant cells migrated normally (data not shown), as
previously reported (Abdelilah-Seyfried et
al., 2003
), and mutant cells could be located in any position
within the cluster. The morphology of the mutant clusters was frequently
abnormal (Fig. 2C). In the case
of bazxi106, mosaic clusters were frequently elongated. In
some cases, trailing cells were observed in
par-6
226 and bazxi106
mutant clusters. This suggests that Par-6 and Baz are important for
maintaining adhesion between cells of the migrating cluster.
|
Expression of the RNAi par-6 transgene or the RNAi baz
transgene in the border cells, using slbo-Gal4, inhibited their
migration (Fig. 2J-L). We
observed incomplete border cell migration in 13% (n=685;
P=0.016, Student's t-test) of stage 10 egg chambers from
females carrying two copies of the RNAi par-6 transgene
(Fig. 3). Activation of two
copies of the RNAi par-6 transgene in flies heterozygous for the
par-6226 null allele increased the
severity of this defect to 34% (n=256; P=0.006, Student's
t-test). Similarly, activation of two copies of the RNAi baz
transgene in a bazxi106 heterozygous null background
showed a stronger phenotype (33% migration delay; n=493;
P=0.0001, Student's t-test) than activation of two copies of
the transgene in a wild-type background (14% migration delay; n=685;
P=0.013) (Fig. 3). A
control RNAi white transgene
(Giordano et al., 2002
) did not
cause a migration defect (Fig.
3); nor did it cause a reduction of Par-6 or Baz protein
expression. These data demonstrate that Par-6 and Baz are required in
migratory border cells for normal migration.
|
Depletion of either Par-6 or Baz caused dramatic abnormalities in the organization of border cell clusters and the morphology of the cells, whereas wild-type border cells are typically firmly attached to one another and fairly regular in shape (Fig. 4A). When protrusions are observed in wild type, they are typically seen only in the leading and/or trailing cell of the cluster (Fig. 4B). In cells lacking Par-6 or Baz, border cells frequently appeared to adhere less well to one another and protrusions were observed in many cells and in more of the clusters than in wild type (Fig. 4C,D). Sometimes the protrusions were very long with one end remaining attached to a distant cell (Fig. 4E). These effects were even more obvious and frequent in clusters in which Par-6 or Baz were depleted from both polar cells and border cells than they were in clusters lacking Par-6 or Baz in border cells alone. Taken together these observations suggest that Par proteins are required for adhesion of border cells to each other and to the polar cells.
|
Par-6 and Baz overexpression in the border cells disrupts membrane protein distribution and delays border cell migration
In Drosophila embryos, overexpression of Baz disrupts epithelial
polarity (Petronczki and Knoblich,
2001). To carry out overexpression studies in the ovary, we used
slbo-Gal4 (Rorth et al., 1998). We found that overexpressing Par-6
and Baz in the centripetal cells and posterior cells in the ovary, using
slbo-Gal4, led to an expansion of the domain of expression of aPKC,
an apical marker, into the lateral membrane
(Fig. 5A-D) of these cells.
This suggests that overexpression of Baz and Par-6 disrupts the distinction
between apical and lateral membrane domains. We examined whether
overexpression of Par-6 and Baz in the border cells could alter the
localization of aPKC, a serine threonine kinase that forms a physical complex
with Par-6 and Bazooka. Three-dimensional reconstructions of confocal
micrographs of border cell clusters, under these conditions, showed a
disruption in the localization of aPKC and accumulation of aPKC at ectopic
sites (Fig. 5I-L) (see Movie 2
in the supplementary material) compared to wild type
(Fig. 5E-H) (see Movie 1 in the
supplementary material). In addition, overexpression of Baz resulted in its
accumulation at ectopic sites in border cells, and Par-6 colocalized to these
sites (see Fig. S1 in the supplementary material). However, Par-6
overexpression did not result in the accumulation of Baz at ectopic sites.
|
Overexpressing Par-6 in the border cells caused a delay in migration in 15% (n=119) of stage 10 egg chambers (Fig. 6). Overexpressing Baz in the border cells caused a delay in 32% (n=223) of stage 10 egg chambers (Fig. 6). Overexpression of both Baz and Par-6 enhanced the severity of the migration defect to 83% (n=124) (Fig. 6).
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Discussion |
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The border cells derive from a polarized follicular epithelium, however
little is known of how their epithelial characteristics are modified as they
begin to migrate. In this study, we investigated what happens to the
distributions and functions of epithelial polarity proteins in these migratory
cells. We found that border cells retain an asymmetric distribution of the
apical epithelial proteins Baz, Par-6 and aPKC throughout their migration,
raising the question as to why. One possibility could be that these proteins
contribute to the cells' direction-sensing mechanism. However, neither Par-6
nor Baz localized asymmetrically with respect to the direction of migration,
making this possibility seem less likely. In premigratory border cells, the
apical domain is oriented towards the nurse cells and the direction of
migration. However, once the cells separate from the epithelium, the side of
the cluster with the highest levels of Baz, Par-6 and aPKC was found to be
roughly orthogonal to the direction of migration. These findings are
consistent with previous observations regarding the distribution of Crumbs,
another apical marker (Niewiadomska et
al., 1999), and suggest that early in migration the entire cluster
rotates so that the leading edge is roughly perpendicular to the apical
domain.
A second possibility is that maintaining some aspects of epithelial
polarity during migration eliminates the need to re-establish polarity de novo
when the border cells reach the oocyte
(Niewiadomska et al., 1999).
While possible, this hypothesis is difficult to test and cannot be the only
function for Par-6 and Baz in border cells, since these proteins were also
required during migration.
A third possibility is that cellular asymmetry is retained during border
cell migration in order to achieve the proper asymmetries in the distributions
of other proteins. Consistent with this proposal, the normally asymmetric
accumulations of E-cadherin and ßps-integrin within border
cells were dramatically altered in cells depleted of Baz or Par-6. Previous
studies indicate that loss of E-cadherin from border cells inhibits migration
and that misdistribution of E-cadherin at border cell/nurse cell boundaries
correlates with a migration defect (Bai et
al., 2000; McDonald et al.,
2003
; Niewiadomska et al.,
1999
). The defects in the distributions of E-cadherin and other
membrane-associated proteins in border cells depleted of, or overexpressing,
Par-6 and Baz may collectively lead to the observed migration defect.
We examined a large number of mosaic egg chambers containing clones mutant
for par-6 or baz and observed delays in border cell
migration as well as defects in cohesion within the cluster. It was previously
reported that mosaic clones of baz showed a lack of adhesion within
the border cell cluster but no migratory defects
(Abdelilah-Seyfried et al.,
2003). It is likely that this difference is due to clone size
and/or protein perdurance, since only large clones in which the majority of
the border cells were mutant, showed defects in border cell migration.
Consistent with this, RNAi-mediated reduction of Par-6 and Baz in the border
cells resulted in delayed migration, suggesting that the strongest migration
defects are observed only when all the border cells lack Par-6 or Baz. This is
not unusual. Mutations in slbo, jing, stat92E and shotgun,
which encode E-cadherin, exhibit similar behavior such that clusters
containing some wild-type cells can migrate. These findings seem to indicate
that wild-type cells can `drag' a few mutant cells, but when the number of
migration-defective cells exceeds the number of migration-competent cells,
migration slows or stops.
RNAi-mediated reduction of Par-6 and Baz in the polar cells, in addition to
the outer border cells, exacerbates the defects caused by expression in the
migratory cells alone. This suggests that polar cells contribute to organizing
the cluster. Cohesion of the cluster may be necessary in order for the
migratory cells to receive continuous activation of the JAK/STAT pathway
during migration. Consistent with this, in those clusters that split, those
cells that remain attached to the polar cells migrate further than the cells
that become detached. Polar cells require the migratory cells to reach the
oocyte because they are not motile themselves
(Han et al., 2000), but the
migratory cells also appear to need the polar cells in order to sustain their
motility. This mutual requirement may serve to ensure that the migratory cells
do not run off without the polar cells, since the polar cells are required at
the oocyte surface to form the pore in the micropyle through which a sperm
enters at fertilization.
Migrating border cells possess both epithelial and mesenchymal characteristics
The observations presented here demonstrate that Par-6 and Par-3/Baz are
distributed asymmetrically in migrating border cells, suggesting that not all
epithelial polarity is lost when these epithelial cells become motile. In
spite of this, the morphology of the border cells, particularly at the border
cell/nurse cell interface, can appear fibroblast-like. This interface must
support protrusive behavior and dynamic adhesion, so that the cells can move
along the nurse cells, while they simultaneously remain firmly attached to
each other and to the polar cells. Therefore, migrating border cells possess
both epithelial and mesenchymal characteristics.
We propose that the Par-3/Par-6/aPKC complex functions in these cells, as it does in an epithelium or in asymmetrically dividing neuroblasts, to maintain distinct protein distributions and functional domains in different parts of the cell. In the case of the border cells, three important domains are the interfaces between border cells and nurse cells, between border cells and polar cells and between adjacent border cells. Such distinct domains may be present in other types of cells that maintain contacts with an intact epithelium while they migrate, such as motile keratinocytes at a wound edge or leading endothelial cells during angiogenesis. Tumor cells that metastasize in groups or `nests' may also possess both epithelial and mesenchymal characteristics. Thus the Par-3/Par-6/aPKC complex may contribute to the invasiveness of other cell populations as well.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/21/5243/DC1
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