Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Anatomy, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
* Author for correspondence (e-mail: n.brown{at}gurdon.cam.ac.uk)
Accepted 1 September 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Integrin, Migration, Drosophila, Extracellular matrix, Cell adhesion
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of integrin function during development has revealed that they
contribute to the formation of most tissues
(De Arcangelis and Georges-Labouesse,
2000; Bokel and Brown,
2002
). Their function in mediating adhesion between tissue layers
via the intervening ECM is well documented in C. elegans, Drosophila,
mice and humans. However, other potential functions of integrins during
development appear to be more variable between experiments and model
organisms, as demonstrated by the following examples. Integrins have been
shown to contribute to the cell fusion that gives rise to muscles in mouse,
both in vitro and in vivo (Schwander et
al., 2003
). Yet, in Drosophila, mutations in the only
integrin known to be expressed in muscles did not impair fusion
(Brown, 1994
). Integrins
function in mammalian cells in culture in establishing cell polarity and
providing anchorage-dependent growth
(Juliano, 1996
;
Zegers et al., 2003
), but
similar functions have not been apparent from genetic analysis of integrin
function in Drosophila or C. elegans. In these cases of
conflicting results, it is important to re-evaluate the genetic experiments to
be sure that the mutant condition does result in complete loss of function.
The goal of eliminating integrin function can be confounded in several ways:
the mutations may not be amorphic (null); maternally deposited protein or mRNA
may ameliorate the zygotic mutant phenotype; and related genes may be
providing redundant, compensatory function. In Drosophila, the first
two potential problems have been resolved in the case of the widely expressed
ßPS subunit, as well characterized null alleles are available
(Bunch et al., 1992
) and one
can remove both maternal and zygotic contributions by making germline clones
of the null allele (Roote and Zusman,
1995
; Martin-Bermudo et al.,
1999
). However, the potential for redundancy has not yet been
addressed.
The completion of genome sequences allowed a confident tabulation of the
number of integrin genes in different organisms: C. elegans, one
ß and two subunits; Drosophila, two ß and five
subunits; mouse and human, eight ß and 18
subunits
(Brown, 2000
;
Hynes, 2002
). It is possible
for a gene to be missed if it happens to be embedded in highly repetitive DNA,
e.g. heterochromatin, but the integrin gene number in Drosophila
melanogaster can be compared with those from the recent genome sequences
from Drosophila pseudoobscura and the mosquito Anopheles
gambiae (D.D. and N.H.B., unpublished). Each insect species contains only
two integrin ß subunits, called ßPS and ß
. If both ß
subunits could be eliminated, this should result in a complete absence of
integrin function, as the
subunits are not expected to have any
function in the absence of a ß partner. This is because only
ß heterodimers are transported from the endoplasmic reticulum to
the cell surface (Kishimoto et al.,
1987
; Leptin et al.,
1989
).
The majority of studies of Drosophila integrin function have
focused on integrins containing the ßPS subunit, which is the orthologue
of C. elegans ßpat-3 and vertebrate ß1 (reviewed by
Brown et al., 2000;
Brower, 2003
). The ßPS
subunit is widely expressed, and mutations in this subunit cause a wide range
of morphogenetic defects during development. Yet the ßPS subunit mutant
phenotype does not include defects in processes that are integrin dependent in
other systems, such as muscle fusion, or establishment of polarity or
proliferation. Is this a difference between the organisms, or is it due to a
failure to eliminate completely integrin function in Drosophila?
Furthermore, some processes dependent on the function of ßPS integrins
are not completely defective, suggesting partial redundancy with another ECM
receptor. The best example of this is during the migration of the primordial
midgut cells. These cells arise from two regions of the blastoderm embryo, at
the anterior and posterior. They delaminate from the epithelium and migrate
towards each other along a substrate provided by the visceral mesoderm
(Reuter et al., 1993
;
Tepass and Hartenstein,
1994b
). In the absence of zygotically expressed ßPS, this
process occurs normally, but if the maternal contribution is also eliminated
then there is a severe delay in the migration
(Roote and Zusman, 1995
;
Martin-Bermudo et al., 1999
).
However, the primordial midgut cells still do manage to complete the
migration, suggesting that another receptor is able to partially substitute
for the ßPS containing integrins. Is this predicted receptor the other
integrin?
The goal of this work was to discover the contribution to development made
by the only other ß subunit in Drosophila, ß. It is
less conserved in its sequence than other ß subunits, being
33%
identical to ßPS and each of the previously known vertebrate ß
subunits (Yee and Hynes,
1993
), compared with 47% identity between ßPS and vertebrate
ß1. Furthermore, it has diverged faster within dipterans: ßPS is 62%
identical between Drosophila and Anopheles, while ß
is only 39% identical. In the embryo, ß
is most strongly expressed in
the endodermal cells of the developing midgut, and this midgut-specific
expression is maintained in the larva and pupa
(Yee and Hynes, 1993
). This
tissue-specific expression of the ß
subunit suggested that it was
unlikely to provide redundant functions for the ßPS subunit outside the
midgut epithelium. However, the maternal contribution of the ßPS subunit
is at levels below detection with our antibody staining methods (D.D. and
N.H.B., unpublished), yet its function has been clearly revealed by the
enhancement of the mutant phenotype when it is removed
(Wieschaus and Noell, 1986
).
Thus, it is possible that low levels of ß
are normally expressed in
other tissues, or that ß
becomes abnormally expressed in other
tissues in response to the absence of ßPS, and in either case provides
compensatory integrin function.
To elucidate the contribution made by the ß subunit, we generated
null mutations in the gene encoding ß
. Despite its conservation in
other insect species, we found that ß
is not essential for viability
or fertility. We examined the ability of ß
to compensate for the loss
of ßPS and found that it does so, but only in the tissue in which it is
highly expressed the midgut.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of mutant embryos and clones
The ßPS integrin mutant allele mysXG43, described
by Bunch (Bunch, 1992), was
used. For talin mutant embryos, the null allele rhea79B
was used (Brown et al., 2002
).
Germline clones were generated using the FLP/FRT system
(Chou et al., 1993
).
mysXG43 FRT101/ovoD1 FRT101; hsFLP38/+ or
mysXG43 FRT101/ovoD1 FRT101;
ß
1; MKRS hsFLP99/+ larvae were heat shocked for
2-3 hours at 37°C. Females with germline mys clones were
out-crossed to FM7gfp or FM7gfp;
ß
1 males to discriminate between zygotically
rescued female embryos and hemizygous germline mutants. Talin germline clones
were generated by crossing rhea79B FRT2A/TM3 virgin
females to y w hsFLP/Y; ovoD FRT2A/TM3 and heat shocking
their progeny (Brown et al.,
2002
).
To generate clones in the follicular epithelium, adult flies of the
genotype mysXG43 FRT101/GFP FRT101; hsFLP38
ß2/ß
1 were heat
shocked at 37°C for 1 hour, 24 hours after hatching. Ovaries were allowed
to develop for 24-48 hours, then were dissected, fixed in 4% paraformaldehyde,
and stained with anti-DE-cadherin antibodies. To generate clones in the
imaginal discs, the heat shock was performed on first instar larvae.
Cuticle preparations
Cuticles from mysXG43 and mysXG43;
ß1 germline clone embryos were prepared after 24
hours of development by placing a small drop of 1:1 Hoyer's medium:lactic acid
onto dechorionated and fixed embryos. Embryos were cleared after a 24 hour
incubation at 65°C and imaged with a Leica DMR microscope with a MacroFire
digital camera and PictureFrame image grabbing software (Optronics).
Immunofluorescence microscopy
Embryos were fixed in 4% paraformaldehyde and antibody stained using
standard methods. For phalloidin staining, embryos were fixed in 8%
paraformaldehyde and devitellenized in 80% ethanol rather than methanol. All
antisera dilutions and incubations were made in PBS + 0.1% Triton+ 0.5% BSA.
Antibodies were used at the following dilutions: rabbit anti-lamininA
(Gutzeit et al., 1991) at
1:500, rat anti-DE-cadherin (Uemura et
al., 1996
) at 1:200, rabbit anti-Vasa at 1:5000 (R. Lehmann),
rabbit anti-
PS3 at 1:100 (Grotewiel
et al., 1998
), rat anti-
PS2 at 1:5
(Bogaert et al., 1987
), rabbit
anti-talin at 1:1000 (Brown et al.,
2002
), rat anti-Cheerio (Sokol
and Cooley, 1999
) at 1:500 and mouse anti-Fasciclin 3 at 1:5
(Brower et al., 1980
).
Fluorescently labelled secondary antibodies were used at a 1:200 dilution and
rhodamine-labelled phalloidin was used at 1:1000 (all from Molecular Probes).
Images were obtained by confocal microscopy on a BioRad Radiance.
In situ hybridization
In situ hybridization of whole-mount embryos was performed as described by
Tautz and Pfeifle (Tautz and Pfeifle,
1989) with digoxigenin-labelled RNA probes corresponding to the
antisense strand of ß
and the sense stand as a negative control.
Labelled RNA probes were made using the DIG RNA labelling kit (Roche) and
hydrolyzed by alkali treatment (Na2CO3-NaHCO3
buffer pH 10.5). Probes were detected with anti-digoxigenin Fab fragments
(Boehringer) at a 1:5000 dilution.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Flies homozygous for ß1 or
ß
2 were viable and fertile, and can be kept as a
stock, ruling out any rescue by a maternal component or a grandchildless
phenotype. Their adult morphology appeared normal
(Fig. 1C). Thus, the generation
of null mutations in the ß
locus has demonstrated that this integrin
subunit is not essential for development or viability.
ß function in the developing midgut is revealed in the absence of ßPS
The ß subunit is most highly expressed in the developing midgut
(Yee and Hynes, 1993
), but we
were unable to detect any defects in midgut development in the absence of
ß
(data not shown). This is consistent with earlier findings that
removing ß
and adjacent genes with overlapping deficiencies did not
cause a defect in midgut development
(Reuter et al., 1993
). To test
whether the function of ß
is masked by the presence of ßPS, we
examined midgut development in embryos lacking both integrins.
We first examined whether the absence of ß would enhance the
midgut defects caused by the absence of zygotic expression of ßPS, and
found that it does. The first defects are detectable at the step when the
three initial midgut constrictions would normally lengthen and fold the gut
into a highly convoluted tube (stage 15-16)
(Wright, 1960
;
Roote and Zusman, 1995
).
Instead of becoming convoluted like the wild type
(Fig. 2A), in the absence of
ßPS the constrictions failed to lengthen, resulting in a poorly
convoluted midgut (Fig. 2C).
Removal of ß
both maternally and zygotically, as well as of zygotic
ßPS, significantly enhanced the strength of the phenotype so that the
midgut lost the constrictions and became a simple yolk-filled sac
(Fig. 2E). For practical
reasons, in our experiments embryos were examined that lacked both the
maternal and zygotic contribution of ß
; it seems likely that
ß
function is provided zygotically, but we have not tested whether
there is a functional maternal contribution of ß
.
|
We examined the mutant embryos prior to the appearance of these
morphological defects to see if there was an underlying molecular defect in
the generation of epithelial polarity in the midgut cells. This epithelium
forms anew following the migration of the mesenchymal primordial midgut cells.
In contrast to ectodermally derived epithelia such as epidermis, the embryonic
midgut epithelium does not contain zonula adherens or other junctional
complexes (Tepass and Hartenstein,
1994a). Nevertheless, these cells do adopt a columnar morphology
and are polarized by virtue of the asymmetric localization of DE-cadherin
apically and by laminin deposition basally
(Fig. 3; D.D. and N.H.B.,
unpublished).
|
In the absence of ß and ßPS primordial midgut cell migration completely fails
As mentioned in the introduction, ßPS-containing integrins are known
to play an important role in midgut cell migration, but they are not
absolutely essential (Roote and Zusman,
1995; Martin-Bermudo et al.,
1999
). At a time when the midgut cells in wild-type embryos were
actively migrating, those in embryos lacking both maternal and zygotically
contributed ßPS appeared less motile, and the substrate for migration,
the visceral mesoderm, was less well organized
(Fig. 4B). However, the
primordial midgut cells eventually recover and complete their migration to
meet at the centre of the embryo (Fig.
4F). These findings suggest that there must be another receptor
that is capable of promoting migration in these cells, and we show that this
receptor is ß
.
|
Several conclusions can be drawn from this finding. The first is that
migration of the primordial midgut is absolutely dependent on integrin
function. The second is that ßPS is capable of mediating this migration
on its own, as migration was normal in the absence of ß (data not
shown). Third, ß
is not capable of mediating normal migration on its
own, as there was a substantial delay when ßPS is removed, even when the
normal ß
gene was still present. One possible explanation for this
delay is that at the start of migration, ß
is expressed at levels too
low to mediate migration. We tested this by precocious expression of
ß
with the Gal4 line 48Y. It has been shown previously that
normal migration was restored when this line was used to express UAS::ßPS
in embryos mutant for ßPS
(Martin-Bermudo et al., 1999
).
In place of a UAS::ß
line, we used an EP insertion line that inserts
a GAL4-dependent UAS promoter upstream of the ß
transcription unit,
the EP line EP(2)2235. Combining EP(2)2235 with 48Y
successfully expressed ß
mRNA at an earlier stage
(Fig. 5B), but this failed to
rescue the migration delay (Fig.
5D). This suggested that ßPS has a specific ability to
mediate the early phase of migration, not shared with ß
.
|
|
|
|
As above, we compared embryos lacking both ß and ßPS to those
just lacking ßPS. We examined the muscle detachment phenotype
(Fig. 9A,B), germ band
retraction and dorsal closure defects (Fig.
9C), and the gross morphology of the central nervous system by
staining for actin (data not shown). We did not find that removal of
ß
enhanced ßPS mutant phenotypes in tissues other than the
midgut. We also did not detect any new phenotypes during embryogenesis.
Notably, integrins are not essential for myoblast fusion as they are in
mammalian cells (Schwander et al.,
2003
), nor the initial attachment of muscles
(Fig. 9B). Thus, in the embryo
we have only detected a compensatory function of ß
in the midgut, the
tissue where it is most highly expressed.
|
Previous studies have shown that cells lacking the PS integrins can
proliferate to make large clones of cells in the Drosophila wing or
eye (Zusman et al., 1990;
Brower et al., 1995
). We
tested whether the removal ß
would reduce the ability of the ßPS
mutant cells to proliferate, but it did not (e.g.
Fig. 10A,B). Thus, cell
proliferation in imaginal disc or follicle cell epithelia is not dependent on
integrin function. Follicle cells lacking ßPS are defective in the
organization of stress fibre-like actin bundles on the basal surface of the
follicle cells (Bateman et al.,
2001
), and this was not enhanced by the additional removal of
ß
(data not shown). We also observed that follicular epithelium cells
lacking ßPS (regardless of whether ß
was also removed) had a
tendency to become multilayered, especially if they were positioned over the
posterior end of the oocyte (Fig.
10C). This defect did not appear to be an inability of cells to
polarize, as cells lacking both integrins were still correctly polarized when
in contact with the oocyte, as assayed by DE-cadherin
(Fig. 10C') and DPatj
(data not shown). The cells that form the abnormal layer not in contact with
the oocyte lacked normal distribution of these markers, suggesting that
detachment is followed by loss of polarity. Thus, combining these results with
those from the embryonic endoderm, we conclude that integrin mediated cell-ECM
adhesion at the basal surface is not a primary cue for establishing apical
polarity in Drosophila, but contributes to its maintenance.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One of the key questions to emerge from this work is what changes 2
hours after the time when primordial midgut migration is normally initiated,
so that ß
can now mediate migration? It seems likely that the
developmental change that permits ß
integrin-dependent migration is
the synthesis of an essential protein or proteins. It is not just the timing
of ß
synthesis itself that is regulated, as expression of ß
earlier than normal did not alleviate the delay in migration, even though
expression of ßPS with the same approach did successfully restore
migration at the normal time in embryos lacking endogenous ßPS
(Martin-Bermudo et al., 1999
).
Therefore, at least one additional protein is required for ß
-mediated
migration. This protein could have one of several possible functions: an
integrin subunit heterodimeric partner; an extracellular matrix
ligand; an intracellular protein required to link integrins to the
cytoskeleton, vital for cell movement; and/or a regulator of any of these
proteins. We know that the
PS3 subunit is present in the midgut at the
early stages of migration because eliminating it along with the
PS1
subunit results in delayed migration
(Martin-Bermudo et al., 1999
),
so we can rule out the possibility that
PS3 is a limiting factor for
ß
-mediated migration. Curiously, eliminating the
PS1 and
PS3 subunits zygotically did not block migration completely
(Martin-Bermudo et al., 1999
),
as we might expect if
PS3 is the major
subunit partner for
ß
. Either there is a substantial maternal contribution of the
PS3 subunit or perhaps the
PS4 or
PS5 subunits also
function in the midgut.
Evidence to suggest that integrins containing the two ß subunits
mediate migration by interacting with different cytoplasmic linker proteins
came from our analysis of the role of talin in midgut migration. Talin binds
directly to integrins, with more than one binding site, and the detailed
nature of one crucial interaction has been characterized at high resolution
(Garcia-Alvarez et al., 2003).
We show that the defect in midgut migration caused by the loss of talin
resembles the defect caused by the loss of ßPS rather than both ß
subunits. This is consistent with the divergence of the ß
cytoplasmic
tail, particularly a lack of conservation of a tryptophan that makes a key
contact with talin in ß3. Therefore, it seems likely that ß
makes interactions with an alternate cytoplasmic linker, and it may be that
the synthesis of this protein is what permits ß
-dependent migration.
One candidate protein, filamin 1, appears to be ruled out by the observation
that its localization is not ß
dependent.
The functions we have observed for ß are seen only in the absence
of ßPS, so it is still unclear what ß
does under normal,
wild-type conditions. Although a relatively divergent integrin, other insect
genomes that have been sequenced also contain an orthologue of the ß
gene (D.D. and N.H.B., unpublished), suggesting that it does have a function
worthy of retaining through evolution. One possibility is that ß
contributes to the architecture of the midgut in way that is not obvious by
appearance but that makes an important contribution to the physiology of this
organ. If the flies lacking ß
integrin are unable to digest their
food as well as their wild-type counterparts, this would probably make these
flies less competitive in the wild.
Morphogenesis in the absence of integrins
Creating the tools to eliminate all integrin heterodimers allowed us to
address whether some integrin functions that are well-established in
vertebrates and cell culture are important in Drosophila. For
example, cell cycle progression in cultured mammalian cells is absolutely
dependent on their adhesion to an extracellular matrix and, consequently,
cells in suspension will arrest their cell cycle
(Clarke et al., 1970;
Juliano, 1996
). Integrins are
the major adhesion molecules that control this phenomenon called
`anchorage-dependent growth' (Juliano,
1996
). However, the developmental relevance of this phenomenon is
not completely clear because in an intact organism most cell types, other than
those of the circulatory system, will never be in suspension. Genetic
experiments in mouse are beginning to address this issue and have demonstrated
that integrins are important for proliferation during the development of a
number of tissues including skin, bone and embryonic ectodermal ridge cells
(De Arcangelis et al., 1999
;
Raghavan et al., 2000
;
Aszodi et al., 2003
). We have
tested whether integrins are required for cells to divide in
Drosophila, and, surprisingly, we find that they are not. By
comparing the size of clones of cells generated by mitotic recombination, we
found that double integrin mutant epithelial cells are able to proliferate at
approximately the same rate as their siblings that just lack ß
. Thus,
it appears that integrins have adapted an additional function in regulating
cell proliferation during the evolution of the vertebrate lineage. Perhaps
this additional level of cell cycle regulation arose with the massive increase
in the number of cell divisions that mammals undergo throughout their
development. It is clearly advantageous for cells to arrest proliferation in
the absence of adhesion to prevent the growth of metastatic tumours, so
perhaps another, non-integrin adhesion molecule is permissive for growth in
Drosophila epithelia.
There is a substantial amount of data to suggest that integrins play a role
in epithelial architecture and polarity
(Sheppard, 2003;
Zegers et al., 2003
), but the
data are conflicting regarding the precise roles. Experiments using 3D
epithelial cysts demonstrated a role for integrins in orienting the direction
of polarity, but not for establishing distinct apical and lateral membrane
domains (Wang et al., 1990
;
Ojakian and Schwimmer, 1994
).
However, experiments in vivo suggest that integrins maybe required for the
initial establishment of polarity. For example, epiblasts derived from
laminin-/- or ß1 integrin-/- ES cells fail to form
a polarized epithelium or a proamiotic cavity
(Aumailley et al., 2000
;
Murray and Edgar, 2000
;
Li et al., 2002
). Furthermore,
laminin mutants in C. elegans show numerous polarity defects, such as
non-basal integrin adhesions and ectopic adherens junctions
(Huang et al., 2003
). We took
advantage of our double ß integrin mutants to address the role of
integrins in Drosophila epithelia and found that they are important
for the maintenance, but not the establishment of polarity in secondary
epithelia. In embryos lacking ß
and zygotic ßPS, the integrity
of the endoderm is severely compromised, although its polarity is initially
established. Furthermore, when we eliminated integrin heterodimers in clones
of cells, we observed the initial distribution of apicolateral markers in the
follicular epithelium to be normal. However, epithelial integrity was
compromised because cells eventually rounded up and formed multiple layers, a
common phenotype seen in mutants that disrupt polarity or epithelial integrity
(Bilder et al., 2000
;
Tanentzapf et al., 2000
).
Thus, cells cannot retain their polarity without basal adhesion, but integrins
are not necessary to set up the establishment of apical and lateral domains in
Drosophila.
Although the roles of ß during Drosophila morphogenesis
are found to be minor, the important findings of this study are twofold.
First, the presence of another integrin ß subunit in Drosophila
had required cautious interpretation of previous genetic analyses of PS
integrin mutants. Therefore, it was important to establish whether redundancy
by ß
masked any important roles for the PS integrins in
Drosophila. However, this study has shown that removal of ß
does not enhance ßPS mutant phenotypes in the muscle, CNS or embryonic
epidermis. It can now be said with confidence that in tissues other that the
midgut, ßPS-containing integrins are the primary players in cell-matrix
adhesion in Drosophila. Hence, for all practical purposes, germline
depletion of ßPS essentially eliminates integrin function in all
embryonic tissues other than the midgut. Second, we have now established that
Drosophila integrins are important for some but not all of the
functions that they mediate in vertebrates. Although important for muscle cell
fusion, establishment of epithelial polarity and cell proliferation in
mammals, integrins are not required for these processes in
Drosophila.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aszodi, A., Hunziker, E. B., Brakebusch, C. and Fassler, R.
(2003). Beta1 integrins regulate chondrocyte rotation, G1
progression, and cytokinesis. Genes Dev.
17,2465
-2479.
Aumailley, M., Pesch, M., Tunggal, L., Gaill, F. and Fassler,
R. (2000). Altered synthesis of laminin 1 and absence of
basement membrane component deposition in (beta)1 integrin-deficient embryoid
bodies. J. Cell Sci.
113,259
-268.
Bateman, J., Reddy, R. S., Saito, H. and van Vactor, D. (2001). The receptor tyrosine phosphatase Dlar and integrins organize actin filaments in the Drosophila follicular epithelium. Curr. Biol. 11,1317 -1327.[CrossRef][Medline]
Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W.,
Tsang, G., Evans-Holm, M., Hiesinger, P. R., Schulze, K. L., Rubin, G. M. et
al. (2004). The BDGP gene disruption project: single
transposon insertions associated with 40% of Drosophila genes.
Genetics 167,761
-781.
Bilder, D., Li, M. and Perrimon, N. (2000).
Cooperative regulation of cell polarity and growth by Drosophila tumor
suppressors. Science
289,113
-116.
Bloor, J. W. and Brown, N. H. (1998). Genetic
analysis of the Drosophila alphaPS2 integrin subunit reveals discrete
adhesive, morphogenetic and sarcomeric functions.
Genetics 148,1127
-1142.
Bogaert, T., Brown, N. and Wilcox, M. (1987). The Drosophila PS2 antigen is an invertebrate integrin that, like the fibronectin receptor, becomes localized to muscle attachments. Cell 51,929 -940.[Medline]
Bokel, C. and Brown, N. H. (2002). Integrins in development: moving on, responding to, and sticking to the extracellular matrix. Dev. Cell 3,311 -321.[Medline]
Brower, D. L. (2003). Platelets with wings: the maturation of Drosophila integrin biology. Curr. Opin. Cell Biol. 15,607 -613.[CrossRef][Medline]
Brower, D. L., Smith, R. J. and Wilcox, M. (1980). A monoclonal antibody specific for diploid epithelial cells in Drosophila. Nature 285,403 -405.[Medline]
Brower, D. L., Bunch, T. A., Mukai, L., Adamson, T. E., Wehrli,
M., Lam, S., Friedlander, E., Roote, C. E. and Zusman, S.
(1995). Nonequivalent requirements for PS1 and PS2 integrin at
cell attachments in Drosophila: genetic analysis of the alpha PS1 integrin
subunit. Development
121,1311
-1320.
Brown, N. H. (1994). Null mutations in the
alpha PS2 and beta PS integrin subunit genes have distinct phenotypes.
Development 120,1221
-1231.
Brown, N. H. (2000). Cell-cell adhesion via the ECM: integrin genetics in fly and worm. Matrix Biol. 19,191 -201.[CrossRef][Medline]
Brown, N. H., Gregory, S. L. and Martin-Bermudo, M. D. (2000). Integrins as mediators of morphogenesis in Drosophila. Dev. Biol. 223,1 -16.[CrossRef][Medline]
Brown, N. H., Gregory, S. L., Rickoll, W. L., Fessler, L. I., Prout, M., White, R. A. and Fristrom, J. W. (2002). Talin is essential for integrin function in Drosophila. Dev. Cell 3,569 -579.[Medline]
Bunch, T. A., Salatino, R., Engelsgjerd, M. C., Mukai, L., West,
R. F. and Brower, D. L. (1992). Characterization of mutant
alleles of myospheroid, the gene encoding the beta subunit of the Drosophila
PS integrins. Genetics
132,519
-528.
Chou, T. B., Noll, E. and Perrimon, N. (1993).
Autosomal P[ovoD1] dominant female-sterile insertions in Drosophila and their
use in generating germ-line chimeras. Development
119,1359
-1369.
Clarke, G. D., Stoker, M. G., Ludlow, A. and Thornton, M. (1970). Requirement of serum for DNA synthesis in BHK 21 cells: effects of density, suspension and virus transformation. Nature 227,798 -801.[Medline]
De Arcangelis, A. and Georges-Labouesse, E. (2000). Integrin and ECM functions: roles in vertebrate development. Trends Genet. 16,389 -395.[CrossRef][Medline]
De Arcangelis, A., Mark, M., Kreidberg, J., Sorokin, L. and
Georges-Labouesse, E. (1999). Synergistic activities of
alpha3 and alpha6 integrins are required during apical ectodermal ridge
formation and organogenesis in the mouse. Development
126,3957
-3968.
Garcia-Alvarez, B., de Pereda, J. M., Calderwood, D. A., Ulmer, T. S., Critchley, D., Campbell, I. D., Ginsberg, M. H. and Liddington, R. C. (2003). Structural determinants of integrin recognition by talin. Mol. Cell 11,49 -58.[Medline]
Grotewiel, M. S., Beck, C. D., Wu, K. H., Zhu, X. R. and Davis, R. L. (1998). Integrin-mediated short-term memory in Drosophila. Nature 391,455 -460.[CrossRef][Medline]
Gutzeit, H. O., Eberhardt, W. and Gratwohl, E. (1991). Laminin and basement membrane-associated microfilaments in wild-type and mutant Drosophila ovarian follicles. J. Cell Sci. 100,781 -788.[Abstract]
Huang, C. C., Hall, D. H., Hedgecock, E. M., Kao, G., Karantza,
V., Vogel, B. E., Hutter, H., Chisholm, A. D., Yurchenco, P. D. and Wadsworth,
W. G. (2003). Laminin alpha subunits and their role in C.
elegans development. Development
130,3343
-3358.
Hynes, R. O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell 110,673 -687.[Medline]
Juliano, R. (1996). Cooperation between soluble factors and integrin-mediated cell anchorage in the control of cell growth and differentiation. Bioessays 18,911 -917.[Medline]
Kishimoto, T. K., Hollander, N., Roberts, T. M., Anderson, D. C. and Springer, T. A. (1987). Heterogeneous mutations in the beta subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency. Cell 50,193 -202.[Medline]
Leptin, M., Bogaert, T., Lehmann, R. and Wilcox, M. (1989). The function of PS integrins during Drosophila embryogenesis. Cell 56,401 -408.[Medline]
Li, S., Harrison, D., Carbonetto, S., Fassler, R., Smyth, N.,
Edgar, D. and Yurchenco, P. D. (2002). Matrix assembly,
regulation, and survival functions of laminin and its receptors in embryonic
stem cell differentiation. J. Cell Biol.
157,1279
-1290.
Martin-Bermudo, M. D., Alvarez-Garcia, I. and Brown, N. H.
(1999). Migration of the Drosophila primordial midgut cells
requires coordination of diverse PS integrin functions.
Development 126,5161
-5169.
Murray, P. and Edgar, D. (2000). Regulation of
programmed cell death by basement membranes in embryonic development.
J. Cell Biol. 150,1215
-1221.
Niewiadomska, P., Godt, D. and Tepass, U.
(1999). DE-Cadherin is required for intercellular motility during
Drosophila oogenesis. J. Cell Biol.
144,533
-547.
Ojakian, G. K. and Schwimmer, R. (1994).
Regulation of epithelial cell surface polarity reversal by beta1 integrins.
J. Cell Sci. 107,561
-576.
Raghavan, S., Bauer, C., Mundschau, G., Li, Q. and Fuchs, E.
(2000). Conditional ablation of beta1 integrin in skin. Severe
defects in epidermal proliferation, basement membrane formation, and hair
follicle invagination. J. Cell Biol.
150,1149
-1160.
Reuter, R., Grunewald, B. and Leptin, M.
(1993). A role for the mesoderm in endodermal migration and
morphogenesis in Drosophila. Development
119,1135
-1145.
Roote, C. E. and Zusman, S. (1995). Functions for PS integrins in tissue adhesion, migration, and shape changes during early embryonic development in Drosophila. Dev. Biol. 169,322 -336.[CrossRef][Medline]
Rorth, P. (2002). Initiating and guiding migration: lessons from border cells. Trends Cell Biol. 12,325 -331.[CrossRef][Medline]
Schwander, M., Leu, M., Stumm, M., Dorchies, O. M., Ruegg, U. T., Schittny, J. and Muller, U. (2003). Beta1 integrins regulate myoblast fusion and sarcomere assembly. Dev. Cell 4,673 -685.[Medline]
Sheppard, D. (2003). Functions of pulmonary
epithelial integrins: from development to disease. Physiol.
Rev. 83,673
-686.
Sinclair, D. A. R., Moore, G. D. and Grigliatti, T. A. (1980). Isolation and preliminary characterization of putative histone gene deficiencies in Drosophila melanogaster. Genetics 94,S96 -S97.
Sokol, N. S. and Cooley, L. (1999). Drosophila filamin encoded by the cheerio locus is a component of ovarian ring canals. Curr. Biol. 9,1221 -1230.[CrossRef][Medline]
Starz-Gaiano, M. and Lehmann, R. (2001). Moving towards the next generation. Mech. Dev. 105, 5-18.[CrossRef][Medline]
Tanentzapf, G., Smith, C., McGlade, J. and Tepass, U.
(2000). Apical, lateral, and basal polarization cues contribute
to the development of the follicular epithelium during Drosophila oogenesis.
J. Cell Biol. 151,891
-904.
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Tepass, U. and Hartenstein, V. (1994a). The development of cellular junctions in the Drosophila embryo. Dev. Biol. 161,563 -596.[CrossRef][Medline]
Tepass, U. and Hartenstein, V. (1994b).
Epithelium formation in the Drosophila midgut depends on the interaction of
endoderm and mesoderm. Development
120,579
-590.
Uemura, T., Oda, H., Kraut, R., Hayashi, S., Kotaoka, Y. and Takeichi, M. (1996). Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Genes Dev. 10,659 -671.[Abstract]
Wang, A. Z., Ojakian, G. K. and Nelson, W. J. (1990). Steps in the morphogenesis of a polarized epithelium. I. Uncoupling the roles of cell-cell and cell-substratum contact in establishing plasma membrane polarity in multicellular epithelial (MDCK) cysts. J. Cell Sci. 95,137 -151.[Abstract]
Wieschaus, E. and Noell, E. F. (1986). Specificity of embryonic lethal mutations in Drosophila analyzed in germ line clones. Rouxs Arch. Dev. Biol. 195, 63-73.
Wright, T. R. (1960). The phenogenetics of the embryonic mutant, lethal myospheroid, in Drosophila melanogaster. J. Exp. Zool. 143,77 -99.
Yee, G. H. and Hynes, R. O. (1993). A novel,
tissue-specific integrin subunit, beta nu, expressed in the midgut of
Drosophila melanogaster. Development
118,845
-858.
Zegers, M. M., O'Brien, L. E., Yu, W., Datta, A. and Mostov, K. E. (2003). Epithelial polarity and tubulogenesis in vitro. Trends Cell Biol. 13,169 -176.[CrossRef][Medline]
Zusman, S., Patel-King, R. S., ffrench-Constant, C. and Hynes, R. O. (1990). Requirements for integrins during Drosophila development. Development 108,391 -402.[Abstract]