1 Department of Genetics, Graduate University for Advanced Studies, National
Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken 411-8540 Japan
2 Genetic Strain Research Center, National Institute of Genetics, 1111 Yata,
Mishima, Shizuoka-ken 411-8540 Japan
3 Morphogenetic Signaling Group, Riken Center for Developmental Biology, 2-2-3,
Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047 Japan
4 Department of Biological Sciences, Stanford University, Stanford, CA 94305,
USA
* Present address: Department of Biological Sciences, Stanford University,
Stanford, CA 94305, USA
Author for correspondence (e-mail:
shayashi{at}cdb.riken.go.jp)
Accepted 30 December 2002
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SUMMARY |
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Key words: Morphogenesis, Cadherin, Cell adhesion, Drosophila
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INTRODUCTION |
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Junctional structures are essential because epithelial breakdown causes internal fluid to leak, which is lethal to organisms. However, during organogenesis, epithelia undergo drastic remodeling, as is observed in vertebrate neurulation, in which the neuroectoderm forms the neural tube, and in angiogenesis, where a meshwork of blood vessels forms. During this process, cells must transiently loosen their adhesions and rearrange their relative positions to reshape tissues and organs. It is therefore important to understand the mechanism that regulates turnover of junctional structures during epithelial morphogenesis. However, little is known about how the turnover of cell adhesion is regulated.
A Rho GTPase family member, Rac is likely to fulfill the task of
coordinating cell adhesion and actin-based cytoskeletal motility because it
has been strongly implicated in the regulation of both processes
(Hall, 1998;
Kaibuchi et al., 1999
;
Van Aelst and D'Souza-Schorey,
1997
). The roles of Rac in developmental events involving
extensive cell-shape changes, such as axon and dendrite development in the
nervous systems, closure of dorsal epidermis and muscle development, are well
documented (Hakeda-Suzuki et al.,
2002
; Luo et al.,
1994
; Ng et al.,
2002
). Rac has also been implicated in the regulation of
cadherin-dependent cell adhesion in cultured epithelial cells, but its exact
role is controversial. For example, whereas several investigators have
reported that Rac promotes E-Cadherin-dependent cell adhesion
(Kuroda et al., 1998
;
Takaishi et al., 1997
), others
have reported that Rac inhibits it (Braga
et al., 2000
; Gimond et al.,
1999
; Potempa and Ridley,
1998
). It appears that the results have been influenced greatly by
the experimental settings and the way in which Rac activity was manipulated.
To clarify the in vivo roles of Rac in the context of epithelial morphogenesis
during development, we have been studying the phenotypes of
Drosophila embryos with mutations in Rac genes.
We chose the Drosophila tracheal system as a model to study the
roles of Rac in epithelial cell rearrangement. The tracheal system is derived
from segmentally repeated epithelial cell clusters consisting of 80
ectodermal cells that undergo branching and migration processes to form a
network of tubular epithelium (Samakovlis
et al., 1996
). Tracheal cell migration and differentiation are
triggered by Branchless (Bnl)/FGF, which is expressed in the positions to
which tracheal cells will migrate
(Sutherland et al., 1996
)
(Fig. 2A). Bnl/FGF activates
Breathless (Btl) receptor tyrosine kinase
(Klambt et al., 1992
), which
is expressed in all tracheal cells. Localized activation of Btl induces
primary branching that transforms sac-like tracheal primordia into tubules
consisting of multiple cells surrounding the circumference (multicellular
tubules). While the primary branches are extended toward target sites, cell
rearrangement takes place to change these branches into thin unicellular
tubules consisting of cells with autocellular junctions. Because these
processes take place without cell division, cell-shape changes and cell
rearrangement play major roles in the formation of the tracheal network.
Tracheal branch migration requires zygotically expressed E-Cadherin
(Tanaka-Matakatsu et al.,
1996
; Uemura et al.,
1996
), which suggests that a de novo supply of E-Cadherin is
essential for tracheal cell rearrangement. However, little is known about how
cell adhesion and cell rearrangement are coordinated in this process.
In this study, we found that Rac was required for cell rearrangement in the tracheal epithelium. Reduced Rac activity was associated with the phenotype of high accumulation of cadherins and its associated molecules. Activation of Rac, however, transformed the tracheal epithelium into mesenchyme, suggesting an essential role of Rac in controlling cell adhesion.
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MATERIALS AND METHODS |
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Time-lapse observation
Dechorionated embryos were mounted on a glass coverslip with rubber cement
and were covered with halocarbon oil 700 (Sigma). GFP fluorescence was
captured by a confocal microscope (Olympus, FV-300) attached to an upright
microscope (Olympus BX51) with a 60x oil immersion lens (NA 1.4). To
minimize phototoxicity, we reduced the Ar laser intensity (488 nm, 10 mW) to
1% and opened the pinhole to its maximum size (300 µm). Typically, 13
x 2 µm z stacks were taken every 5 minutes over a period of
6 hours. Images of the stacks were projected and were displayed as MPEG format
movies.
Immunohistochemistry
Primary antibodies used were the following: rabbit
anti-ß-galactosidase (Cappel), rabbit anti-GFP (MBL), mouse anti-actin
(Sigma), mouse anti-SRF (provided by M. Gilman), rat anti-E-cadherin
(Oda et al., 1994), rat
anti-N-cadherin (Iwai et al.,
1997
), rat anti-
-catenin
(Oda et al., 1993
), anti-rat
PP2AA (Uemura et al., 1993
),
mouse 2A12, mouse anti-Armadillo N2 7A1, rabbit anti-Rac1
(Eaton et al., 1995
), mouse
anti-Crumb Cq4 and mouse anti-Fasciclin 3 7910 (Developmental Studies of
Hybridoma Bank, University of Iowa). Fluorescent images were captured with a
confocal microscope (Olympus, FV-500).
Western blotting
Protein extracted from 15 embryos was separated by SDS-PAGE (9%
polyacrylamide) and transferred to a PVDF membrane. After the blots had been
incubated with primary antibodies and HRP-linked secondary antibodies,
antibody signals were detected by use of Super Signal West Pico (PIERCE).
Blots were repeatedly probed according to the manufacturer's instructions.
Intensities of the bands were quantified by densitometric scanning of the film
exposed to chemiluminescence.
mRNA quantification
Poly A+ mRNA was isolated from approximately 30 embryos with a QuickPrep
Micro mRNA purification kit (Pharmacia Biotech). The rac1, rac2
double mutant chromosome was balanced over the TM3, Kr-Gal4 UAS-gfp
chromosome (Casso et al.,
2000), and mutant homozygotes were identified by the lack of GFP
fluorescence. An amount of mRNA equivalent to three embryos was reverse
transcribed by AMV reverse transcriptase (Life Science) with random hexamers
used as primers. mRNA was quantified by using a Prism 7000 Sequence Detection
System (ABI) with a SYBRE Green PCR Master mix according to the manufacturer's
instructions. Control reactions without reverse transcriptase for each mRNA
preparation revealed no significant genomic DNA contamination. PCR primers
were designed by using Primer Express Version 2.0 software (ABI), the
sequences of which are available upon request. Specificity of PCR reactions
was confirmed by measuring dissociation temperature, agarose gel
electrophoresis and DNA sequencing of the PCR products.
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RESULTS |
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To analyze the state of epithelial cell adhesion in Rac1, 2
mutants, we first observed the subcellular localization of Drosophila
epithelial-cadherin (E-Cadherin) in the embryonic epidermis. E-Cadherin is a
key component of adherence junctions (Oda
et al., 1994; Tepass et al.,
1996
; Uemura et al.,
1996
). Its intracellular domain binds to ß-catenin, which
associates with
-catenin. This cadherin-catenin complex is concentrated
at the apical side of the cell-cell junction of columnar epithelial cells and
is essential for cell adhesion (Tepass et
al., 2000
; Yagi and Takeichi,
2000
). Double labeling of the normal embryonic epidermis with the
septate junction marker Fasciclin 3 (Patel
et al., 1987
) revealed that the apical domain of the cell-cell
contact sites was uniquely labeled with E-Cadherin and that the basolateral
domain had accumulated Fasciclin 3 and a low level of E-Cadherin
(Fig. 1A). However, in the
Rac1, 2 mutant embryos at stage 15, E-Cadherin was highly and more
broadly accumulated; and the distinction between the apical and basolateral
domains of the cell contact site was lost in a subset of the epidermis
(Fig. 1B). In stage 16 embryos,
the columnar cells became shorter, and parts of the epidermis became
multilayered, as revealed by F-actin staining
(Fig. 1C,D).
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We also examined the effects of the constitutively active form of Rac1 (Rac1V12). Although the mRNA levels of cadherins and catenins increased to some extent, their protein levels did not change (Fig. 1F), suggesting that Rac activation does not directly leads to a decrease of the amount of E-Cadherin. The role of Rac on E-Cadherin expression will be further discussed below.
Rac is required for tracheal morphogenesis
Drosophila tracheal development provides a valuable model for
studying epithelial cell rearrangement. At stage 12, tracheal cells have
already stopped their cell division; and their cell number is about 80
(Fig. 2A). To achieve the
elaborate pattern of the tracheal network
(Fig. 2C), tracheal cells
drastically change their relative location, together with cell differentiation
and cell-shape changes (Fig.
2A,B). It is worth noting that the trachea consists of highly
polarized epithelial cells/tubules, as shown by the localization of Crumbs,
which is an apical marker of epithelial cells
(Fig. 2A,B).
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As shown in Figs 1, 2, Rac1, 2 mutants had defects in several tissues. To remove the secondary effects of defects in tissues other than trachea, we inhibited the activity of Rac by expressing Rac1N17 specifically in tracheal cells. In those embryos, we found that E-Cadherin was sometimes localized at the lateral and basal membranes, as in the case of Rac1, 2 mutant embryos (Fig. 2I). Such embryos also showed phenotypes of a truncated and/or zigzagged dorsal trunk (DT), misguided dorsal branch (DB) and no terminal branches (Fig. 2D; data not shown) that were similar to the mild class of defects observed in Rac1, 2 or Pak mutants (see Fig. 6). Thus, autonomous Rac activity may be required for apical localization of E-Cadherin in tracheal cells and may contribute to tracheal morphogenesis.
Time-lapse analysis revealed a role for Rac in tracheal cell
rearrangement
Tracheal branching is a coordinated process of cell migration and cell
rearrangement. To investigate the role of Rac in the dynamic aspects of
tracheal branching, we performed time-lapse analysis of tracheal cells labeled
with GFP-moesin. Primary branching of tracheal primordia generated six
multicellular branches consisting of cells joined by intercellular junctions
(Fig. 3A-E, see Movie 1 at
http://dev.biologists.org/supplemental/).
While the DB, consisting of five to seven cells, extended toward the dorsal
midline, the tracheal cells changed their position relative to each other to
form thinner unicellular tubules with intracellular junctions
(Fig. 3D,E). Such cell
rearrangement was greatly inhibited by Rac1N17
(Fig. 3F-J; see Movie 2 at
http://dev.biologists.org/supplemental/).
Although paired cells with extensive movement of cell protrusions were
attempting to migrate out of the DT, the DB was shorter than that of the
control embryos (Fig. 3F-H).
The number of cells incorporated into the stalk of the DB was reduced to 0 or
1, when compared with three to five cells in wild-type embryos, leaving only
thin cytoplasmic extensions (Fig.
3I,J). Occasionally, there was no sprout of the DB at all
(Fig. 3F,G; asterisk), although
cytoplasmic extensions from the site of DB outgrowth were still visible.
Reduced Rac activity may block the cell rearrangement needed to allocate
tracheal cells into the DB. Our observations also suggest that the rapid
movement of cell protrusions of tracheal tip cells is insensitive to this
level of change in Rac activity.
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Rac signaling genetically interacts with FGF signaling
In addition to the defects in cell adhesion and rearrangement described
above, Rac1, 2 mutant embryos showed various defects in tracheal cell
migration and differentiation. A wide range of tracheal defects was observed
in Rac1, 2 mutants (Fig.
6A,B, see figure legends for criteria to classify tracheal
phenotypes). In embryos showing the weak class phenotype, misrouting of the DB
toward the anteroposterior direction was often observed
(Fig. 6A). In
intermediate-class embryos, the number of truncated DTs increased and the
germband did not retract completely (Fig.
6B). The severity of the defects and their frequency increased
when the gene dose of rac was progressively reduced
(Fig. 6F, lanes 1-5). When the
maternal expression of Rac1 and Rac2 was reduced by half, the tracheal defects
occurred in 25% of the embryos (Fig.
6F, lane 3). We also analyzed Rac1 Rac2 Mtl triple
mutants laid by Rac1 Rac2 Mtl heterozygous mother and found that the
mutants showed higher frequency of severe tracheal defects similar to those
found in Rac1, 2 mutants (data not shown). Those results suggest that
a change in Rac activity within the physiological range significantly affects
morphogenetic movement of the tracheal system.
p21-activated kinase (Pak) is known as a mediator of the activity of Rac GTPase. We found tracheal defects similar to those of Rac1, 2 mutants in pak mutants (data not shown and lanes 6-8 in Fig. 6F). Furthermore, Rac1, 2 and Pak mutations synergistically enhanced tracheal defects (compare lane 9 with lanes 2 and 7 in Fig. 6F). Such results suggest that Rac and Pak are required for directed movement of tracheal branches.
The loss of Rac activity also caused a defect in cell differentiation. Tips
of DB 1-9 are normally capped with terminal cells that extend terminal branch
in the ventral direction (Guillemin et al.,
1996). In Rac 1, 2 mutant embryos, the loss of terminal
branches was observed with high penetrance. Consistently, serum response
factor (SRF) (Guillemin et al.,
1996
), a marker protein for the terminal cell, also disappeared
(data not shown), suggesting that terminal cell differentiation did not
occur.
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To determine the epistatic relationship between Rac and FGF signaling, we
tested the effect of constitutive activation of Rac in btl mutants.
In the btl mutant, tracheal branching does not proceed beyond the
invagination at stage 11 (Fig.
6D), and MAP kinase activation is absent
(Gabay et al., 1997).
Expression of Rac1V12 partially restored the movement of tracheal cells, and
activated MAP kinase, as revealed by staining with the antibody against the
diphosphorylated form of MAP kinase (dp-MAPK,
Fig. 6E). These results suggest
that Rac activation is an essential downstream event of tracheal cell motility
induced by FGF signaling.
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DISCUSSION |
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We demonstrated that expression of a dominant-negative form of Rac 1
greatly reduced cell rearrangement required for partitioning cells into the
stalk of the DB. Overproduction of this form, Rac 1N17, would shift the
cellular pool of Rac toward the inactive GDP-bound state. We suggest that
turnover of E-Cadherin at a proper level requires a high level of Rac
activity. However, highly active movement of cell extensions in the cells at
the tip was still visible, suggesting that the ability of those cells to move
toward their target was mostly intact. A stronger reduction in Rac activity
might be required to demonstrate a role for Rac in promoting cell extensions,
as proposed from studies on tissue culture cells
(Hall, 1998;
Kaibuchi et al., 1999
;
Van Aelst and D'Souza-Schorey,
1997
).
Post-transcriptional control of E-Cadherin by Rac
Our time-lapse analysis demonstrated that reduced Rac activity inhibited
cell rearrangement during branching of tracheal tubules. Under this condition,
the amounts of cadherins and catenins were increased and filled the cell
membrane. This phenotype was different from the phenotype of E-Cadherin-GFP
overexpression, which does not inhibit cell rearrangement
(Oda and Tsukita, 1999). We
suggest that a reduction in Rac promotes the association of cadherin-catenin
complexes with the cell membrane and stabilization of these complexes.
Activation of Rac resulted in an opposite phenotype characterized by the loss
of E-Cadherin and cell dissociation, and in prevention of E-Cadherin-GFP from
accumulating at apical cell junctions. All of these observations are
consistent with a hypothesis that Rac regulates the formation of
cadherin-catenin complexes at the cell junction. Incorporation of a
cadherin-catenin complex into the cellular junction would explain
stabilization of the complex when Rac activity is reduced. Possible modes of
Rac action on cadherin include apical transport and assembly/stabilization of
the complex. We suggest that the inhibitory action on the cadherin cell
adhesion system is a general property of Rac in the Drosophila
embryo.
Relationship between Rac and FGF signaling
Extracellular signals that promote tracheal branching are good candidates
for regulators of Rac in tracheal cells. In this regard, the strong genetic
interaction between Rac and FGF signaling components that we observed suggests
an intriguing possibility that FGF signaling activates Rac within tracheal
cells to promote both cell motility and cell rearrangement. In support of this
idea, we found that activated Rac 1 partially rescued tracheal cell motility
and MAP kinase activation in btl mutants
(Fig. 6E). Involvement of Rac
in FGF-dependent events may not be limited to cell motility. We found that
expression of SRF, the product of one of the target genes activated by FGF
signaling in the tracheal system, was lost in the mutant trachea with reduced
Rac activity because of Rac 1, 2 mutation or Rac 1N17 (data not
shown). This result suggests that Rac also regulates transcription.
Several lines of evidence suggest that FGF signaling is activated locally
at the tip of branches (Ikeya and Hayashi,
1999; Ohshiro et al.,
2002
), and activation of FGF signaling in all tracheal cells was
shown to prevent branching (Ikeya and
Hayashi, 1999
), suggesting that localized activation of FGF
signaling is essential for branching. Therefore the proposed function of Rac
in transducing FGF signaling must be localized at the tip of branches. How
does the proposed function of Rac in transducing FGF signaling relate to the
Rac function in regulating cell rearrangement? As the effect of Rac 1N17 was
most clearly observed in cells destined to become tracheal stalk cells, the
location of tracheal cells requiring two of the Rac functions appears to be
different. One idea is that FGF signaling activated at the tracheal tip is
transmitted to tracheal stalk cells by a secondary signal that activates Rac
to promote cell rearrangement. It will be important to identify the upstream
signal regulating Rac in stalk cells.
Use of the Drosophila tracheal system to investigate the
mechanism of cell signaling and motility
This study provided in vivo evidence for the role of Rac in epithelial
morphogenesis, which role was suggested previously from work in tissue culture
systems. The tracheal system seems to be particularly sensitive to alteration
of Rac activity compared with another well-studied system of
Drosophila epithelial morphogenesis, i.e. closure of the dorsal
epidermis. Dorsal closure is driven by extensive cell stretching and
accumulation of F-actin, but does not involve much cell rearrangement. Studies
on tracheal branching and dorsal closure would complement each other to reveal
the rich spectrum of Rac functions.
We have shown that filopodial movement remains active in tracheal cells
with reduced Rac activity, indicating that the control of cytoplasmic
extension and cell adhesion appears to involve two distinct processes that
require different levels of Rac activity. Recently, it was revealed that FGF
signaling affected the formation of dynamics of filopodia at the tip of
migrating branches (Ribeiro et al.,
2002). How FGF signaling regulates filopodial formation and cell
rearrangement through regulation of Rac and MAP kinase is still not clear at
the moment. Further investigation using the tracheal system should provide
useful insight into functions of intracellular signaling molecules involved in
regulating cell adhesion previously indicated from studies in vitro.
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ACKNOWLEDGMENTS |
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Footnotes |
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