Rac1
The role of Rac in dorsal closure
Two Drosophila genes DRacA and DRacB encode proteins with homology to mammalian
Rac1 and Rac2. In transgenic flies expressing dominant inhibitory N17DRacA, results in a high frequency of defects in dorsal
closure result, due to disruption of cell shape changes in the lateral epidermis. Embryonic
expression of N17DRacA also affects germband retraction and head involution. The epidermal cell
shape defects caused by expression of N17DRacA are accompanied by disruption of a localized
accumulation of actin and myosin thought to be driving epidermal cell shape change (Hardin, 1995).
canoe and polychaetoid genetically interact giving rise to a more severe dorsal closure phenotype than one resulting from canoe mutation alone. canoe3 is an embryonic lethal allele of cno and displays a typical dorsal open phenotype. cnomis1 is a weak hypomorph that yields adult flies with rough eyes and subtle changes in the bristle number. In a search for mutations that interact with cnomis1, polychaetoid was identified as an enhancer of cno phenotypes. Flies doubly homozygous for pydtam1 and cnomis1 die as embryos; this represents a synthetic lethal combination. Examination of embryonic cuticles demonstrates that the cnomis1;pydtam1 double mutant remains open dorsally. Comparisons of cell shape during dorsal closure reveal that cno3 embryos exhibit insufficient elongation of cells. This is most evident in the leading edge cells, which appear square in cno3, in contrast to the oblong cells of wild-type. cnomis1;pydtam1 double mutant embryos exhibit a more extreme phenotype than single mutants: the leading edge cells elongate even less than in cno3 mutants. These results suggest that cno and pyd are required for coordinated cell shape changes in the cells of the leading edge and the lateral ectodermal cells during dorsal closure (Takahashi, 1998).
There is compelling evidence that the small GTPase Drac1 functions in dorsal closure as an upstream (early acting) element of the JNK pathway, which is composed of hemipterous, basket and puckered. To determine if cno is further upstream of Drac1, puckered-lacZ expression was examined in cno3 homozygous embryos: the leading edge of the epidermis in these embryos is driven to express Drac1V12, a constitutively active form of Drac1. If Drac1 is upstream of cno, then the effect of Drac1V12 on puc-lacZ transcription should be blocked by the loss of cno function. Targeted expression of Drac1V12 in the leading edge cells restores puc-lacZ transcription in cno3 homozygotes to a level comparable to that of wild-type. This result is compatible with the hypothesis that cno is upstream of Drac1, or that cno functions in a pathway parallel to that of Drac1 (Takahashi, 1998).
Demonstration of a physical interaction between Cno and Pyd places Pyd similarly upstream of Rac in the dorsal closure pathway. Cno and Pyd exhibit a similar tissue distribution and appear to colocalize at junctional membrane sites within the cell. ZO-1 is a component of both tight junctions and adherens junctions in mammalian cells. Mammalian ZO-1 binds to alpha-spectrin, which cross-links with actin filaments, thereby affecting cell shape. Pyd and mammalin ZO-1 also interact with Drosophila Cortactin and mammalian cortactin respectively.
Mammalian Cortactin is known to be a filamentous actin cross-linking protein and a substrate of Src protein tyrosine kinase. Cortactin is phosphorylated at tyrosine
residues upon stimulation by extracellular signals. Filamentous actin cross-linking activity
of cortactin is attenuated by Src. The intracellular localization of mammalian cortactin is regulated by the activation
of Rac1. Cortactin redistributes from the cytoplasm into membrane ruffles as a result of growth factor-induced Rac1 activation, and
this translocation is blocked by expression of dominant negative Rac1N17. Thus in mammals, cortactin is a putative target of Rac1-induced
signal transduction events involved in membrane ruffling and lamellipodia formation. It would thus seem that Rac signaling is tied to actin dynamics and Polychaetoid/ZO-1 function both in Drosophila and mammals (Takahashi, 1998 and references).
In addition to the defects in myoblast fusion and CNS development,
Suppressor of (rac)1 alleles exhibit a dorsal closure defect
similar to that reported previously for mbc mutants and embryos expressing dominant-negative rac1 (Harden, 1995).
Su(rac)1 alleles were isolated based on their ability to
dominantly suppresses the GMR-rac1-induced rough eye surface as well as the underlying retinal morphology.
During dorsal closure, two symmetric epithelial monolayers coordinately migrate from their lateral position to fuse
along the dorsal midline. The row of cells along the dorsal apical
edge, known as the leading up edge (LE) cells, elongate first and
remain morphologically distinct from the more ventral cells until the
two sheets have nearly met at the midline. Recently, a Rac-mediated signaling pathway thatregulates this process has been elucidated. Rac1 appears to activate
the c-Jun amino (N)-terminal kinase (JNK) pathway, which leads to
decapentaplegic (dpp) expression in the LE cells of
the dorsal epidermis, and several JNK pathway mutants associated with
reduced dpp expression exhibit similar dorsal closure defects, including hemipterous (hep; Jun kinase kinase),
basket (Jun kinase), Djun, and kayak (c-Fos). To determine whether Mbc mediates the activity of Rac in the activation
of JNK during dorsal closure, the expression of
DPP mRNA was examined in mbc mutant embryos. In wild-type embryos,
dpp is expressed predominantly in the visceral mesoderm and
the LE of the dorsal epidermis. In mbc mutant embryos,
50% of which exhibit dorsal closure defects, dpp is expressed
at normal levels in the majority of embryos but appears to be mildly
reduced specifically in the leading edge cells of some of these embryos. This is
in contrast to hep mutant embryos, in which dpp
expression in leading edge cells is clearly absent. This result
suggests that Mbc is not absolutely required for JNK pathway activation
and may play a distinct role in dorsal closure. However, the possiblity cannot be excluded that Mbc contributes to the activation of JNK in
the leading edge cells, but the effects of its absence are masked by a redundant
function of Cdc42, which is also capable of activation of JNK in the leading edge
cells (Nolan, 1998 and references).
The mammalian protein DOCK180, has been demonstrated to interact directly with Rac, but it is unlikely to act as a RacGEF, that is, it is unlikely that DOCK180 functions directly as a Rac activator.
There are two models that most
simply explain the role of Mbc in dorsal closure. Possibly, Mbc is required
for Rac activation in the leading edge cells during dorsal closure, but some functional
redundancy for JNK regulation, which takes place in mbc mutants, is provided by Cdc42. In this scenario, Mbc is
still required for Rac-dependent cytoskeletal changes, reflecting a more
stringent requirement for Rac activity in regulating cell morphology
than in regulating transcription. Consistent with such a possibility, it is found that only a small fraction of mbc mutant embryos that exhibit a dorsal closure defect exhibit any detectable reduction in
dpp expression. Although this result suggests a lesser role for Mbc in JNK activation than in cytoskeletal regulation, it is observed
that overexpression of DOCK180 leads to activation of JNK in
transfected mammalian cells, suggesting that Mbc can potentially play a
role in activating JNK in vivo. Alternatively, two separate pools of
Rac, with different subcellular localizations, may be utilized for
distinct biological processes. In this scenario, Mbc promotes
activation of a pool of Rac that regulates reorganization of the actin
cytoskeleton but does not substantially affect the pool of Rac required
for JNK activation. Consistent with this model, it is found that DOCK180
colocalizes with Rac in membrane ruffles, raising the
possibility that Rac, and perhaps other GTPases, can regulate
distinct biological processes within a single cell by virtue of
subcellularly localized activation (Nolan, 1998).
The Rho subfamily of Ras-related small GTPases participates in a variety of cellular events including organization of the actin cytoskeleton and signaling by c-Jun N-terminal kinase and p38 kinase cascades. These functions of the Rho subfamily are likely to be required in many developmental events. The participation of the RHO subfamily in dorsal closure of the Drosophila embryo, a process involving morphogenesis of the epidermis, has been studied. Drac1, a Rho subfamily protein, is required for the presence of an actomyosin contractile apparatus believed to be driving the cell shape changes essential to dorsal closure. Expression of a dominant negative Drac1 transgene causes a loss of this contractile apparatus from the leading edge of the advancing epidermis, and consequently, dorsal closure fails. Two other Rho subfamily proteins, Dcdc42 and RhoA, as well as Ras1 are also required for dorsal closure. Dcdc42 appears to have conflicting roles during dorsal closure: establishment and/or maintenance of the leading edge cytoskeleton versus its down regulation. Down regulation of the leading edge cytoskeleton may be controlled by the serine/threonine kinase DPAK, a potential Drac1/Dcdc42 effector. RhoA is required for the integrity of the leading edge cytoskeleton specifically in cells flanking the segment borders. The interactions of the various small GTPases in regulating dorsal closure have been characterized and no evidence is found for the hierarchy of Rho subfamily activity described in some mammalian cell types. Rather, the results suggest that while all Rho subfamily p21s tested are required for dorsal closure, they act largely in parallel (Harden, 1999).
A model is given of the control of DC by the Drac1/JNK and Dcdc42/Dpp pathways. Drac1/JNK signaling, initiated by an as yet unknown factor, assembles cytoskeletal components (F-actin, myosin and focal complexes) and other proteins (Dpp, Puckered and Pak) in the leading edge cells and initiates the cellular migration that characterizes DC. Dpp-activated signaling controls the dynamics of epidermal migration, via Dcdc42 and the Dpp pathway, through the serine/threonine kinase Pak, which transiently downregulates the leading edge cytoskeleton at the segmental borders. Transient downregulation of the actin cytoskeleton and focal contacts near the segment border cells is likely to cause local relaxation of the anterior-posterior tension along the LE. Such transient relief of tension may then limit excessive migration of leading edge cells toward each other and prevent the bunching and shearing of epidermal segments that occurs following impairment of Dpp/Dcdc42 signaling. Segment borders cells are potential regions of highest Dpp signaling, because they are adjacent to the highest local concentrations of Dpp protein, and they have high levels of Pak protein and transcripts for the Tkv receptor. Segmental border cells are the only places where transient downregulation of the leading edge cytoskeleton is ever seen in wild-type embryos during DC. As such, it is proposed that the role of Dcdc42/Dpp signaling is the induction of Pak to downregulate the leading edge cytoskeleton at the segment borders, introducing a degree of flexibility to the leading edge during the dorsal closure process (Ricos, 1999).
In the process of dorsal closure, Rac1 appears to be a primary
upstream activator of JNK signaling. To position slipper
function in the JNK pathway relative to the GTPase,
additional genetic epistasis tests were performed. Due to the difficulty in following all relevant chromosomes in the embryo, the adult
Drosophila eye was used to evaluate a possible genetic interaction.
Expression of wild-type dRac1 in the eye, under regulation by
the glass promotor, causes a rough, glazed appearance. This phenotype is
dominantly suppressed by 50% reduction in the levels of JNK signal
transducers, msn, slpr, hep, and
bsk. Heterozygosity at the puc locus, encoding a
negative regulator of JNK signaling acting in opposition to
bsk, dominantly enhances the Rac1-induced rough eye.
To assess whether other putative JNKKKs in Drosophila can
interact in this assay, Drosophila TGF-ß activated kinase 1
(Tak1) was included in this analysis. Unlike slpr, removal
of one copy of Tak1 has no effect on the
Rac1-induced eye phenotype. These data suggest that there is
a dosage-sensitive interaction between the JNK pathway and Rac1
function in this tissue whereby increased Rac1 activity can be
suppressed by reduction in downstream components, including
slpr. Although it is not known whether Tak1 is
expressed in the eye or at what level, Tak1 shows no genetic
interaction with GMR-dRac1. Thus, slpr appears to be
a relevant JNKKK in this assay. Taken together, these epistasis tests are
consistent with slpr function being required downstream of
Rac1 and upstream of bsk (Stronach, 2002).
Dorsal closure of the Drosophila embryo involves morphological changes in two epithelia, the epidermis and the amnioserosa, and is a
popular system for studying the regulation of epithelial morphogenesis. The small GTPase Rac1 has been implicated in the assembly
of an actomyosin contractile apparatus, contributing to cell shape change in the epidermis during dorsal closure. Evidence is presented that Rac1 and Crumbs, a determinant of epithelial polarity, are involved in setting up an actomyosin contractile apparatus that
drives amnioserosa morphogenesis by inducing apical cell constriction. Expression of constitutively active Rac1 causes excessive
constriction of amnioserosa cells and contraction of the tissue, whereas expression of dominant-negative Rac1 impairs amnioserosa morphogenesis. These Rac1
transgenes may be acting through their effects on the amnioserosa cytoskeleton, since constitutively active Rac1 causes increased staining for F-actin and myosin,
whereas dominant-negative Rac1 reduces F-actin levels. Overexpression of Crumbs causes premature cell constriction in the amnioserosa, and dorsal closure
defects are seen in embryos homozygous for hypomorphic crumbs alleles. The ability of constitutively active Rac1 to cause contraction of the amnioserosa is
impaired in a crumbs mutant background. It is proposed that amnioserosa morphogenesis is a useful system for studying the regulation of epithelial morphogenesis by Rac1 (Hardin, 2002).
Expression of dominant negative Drac1N17 in the amnioserosa slows
morphogenesis of this tissue which remains as a squamous epithelium for a
longer period than in wild-type embryos. In Drac1N17-expressing embryos, where
amnioserosa morphogenesis is lagging, the movement of the epidermis is also
slowed, and the embryos have a larger dorsal hole than wild-type embryos of
similar age. It is thought that the impaired movement of the epidermis in such
embryos is caused by lack of morphogenesis in the amnioserosa. These results
are strong evidence that active cell shape changes in the amnioserosa are
required for normal dorsal closure. Examination of wild-type embryos has
shown that this cell shape change in the amnioserosa begins with apical
constriction of cells at the anterior and posterior ends of the amnioserosa.
These cells have elevated levels of myosin, F-actin and phosphotyrosine,
suggesting that an apically localized actomyosin contractile apparatus is
driving their constriction. Early in dorsal closure, the middle cells in
between the two clusters of apically constricted cells do not show elevated
levels of F-actin or myosin but do change shape, losing their original
elongation perpendicular to the A-P axis of the embryo. The middle cells may
be stretching passively, in response to tension from the cell constrictions
occurring at both ends of the amnioserosa. By the end of dorsal closure, the
middle cells are both elongated along the A-P axis and apically constricted,
and it is conceivable that late in dorsal closure they undergo an active cell
shape change as their neighbors did earlier (Hardin, 2002).
Excessive Drac1 activity induces a dramatic contraction of the amnioserosa
such that it shrinks to occupy less than half the dorsal hole, and this is
accompanied by elevated levels of myosin, F-actin, and phosphotyrosine in this
tissue. It is thought that Drac1V12 is driving premature and excessive
amnioserosa cell constriction through its effects on the cytoskeleton. It is
proposed that Drac1 participates in amnioserosa morphogenesis by driving the
assembly of an apical actomyosin contractile apparatus that constricts the
amnioserosa cells, first at the ends of the tissue and possibly later in the
middle. Contraction of an apical actomyosin belt has been implicated in
diverse types of epithelial morphogenesis including Drosophila gastrulation, which shows
some similarity to amnioserosa morphogenesis in that both processes involve
apical construction of a monolayer of cells that then invaginates (Hardin, 2002).
Cell ablation has been used to address the
contributions of the epidermis and amnioserosa to dorsal closure. This work has demonstrated that the amnioserosa is under tension, since ablation of cells in
the amnioserosa causes the tissue to recoil away from the wound site, and the
leading edge is pushed back away from the dorsal midline. It is concluded that there is active cell shape change in the amnioserosa that contributes
to dorsal closure, rather than the tissue being simply compressed by the
movement of the leading edge. The finding that the recoiling of the
amnioserosa after wounding pushes back the leading edge is consistent with the
result that impairing amnioserosa morphogenesis through Drac1N17 expression
hinders leading edge migration (Hardin, 2002).
Overexpression of Crb in the amnioserosa leads to contraction of the tissue
and failure of dorsal closure. This phenotype was examined in more detail; excessive Crb activity induces a premature constriction of cells at the
ends of the amnioserosa. Five P-element-induced
crb alleles were identified that are hypomorphic mutations, causing defects in dorsal
closure and germband retraction. One of these
crb mutations, crbS010409, was characterized in detail. Embryos
homozygous for crbS010409 show a dorsal closure defect
similar to that seen with expression of Drac1N17 in the amnioserosa:
amnioserosa morphogenesis is impaired, but the leading edge cytoskeleton is
intact. In contrast to amorphic crb alleles, the epidermis is not
disorganized in crbS010409 mutants and it secretes
cuticle. Amnioserosa morphogenesis and germband retraction may be particularly
sensitive to the level of Crb activity. It is thought that Crb
activity in the amnioserosa is required for amnioserosa morphogenesis,
although the possibility cannot be excluded that loss of Crb activity elsewhere
in the embryo is affecting this process (Hardin, 2002).
Drac1 may act through Crb in regulating the cytoskeleton, since the constitutively active Drac1V12-induced phenotype of excessive contraction of the amnioserosa is weakened in a crbS010409 mutant background. This weaker Drac1V12 phenotype of premature constriction of the end cells of the
amnioserosa is very similar to that caused by Crb overexpression. There may be
sufficient Crb in the crbS010409 mutant embryos for
Drac1V12 to be able to prematurely constrict cells at the ends of the
amnioserosa but not to excessively contract the tissue. Crb overexpression
does not appear to require Drac1 to cause premature constriction of
amnioserosa cells, since it can achieve this in the presence of Drac1N17. The
excessive contraction of the amnioserosa caused by Drac1V12 expression in
embryos with wild-type Crb activity, and the dumbbell-shaped amnioserosa
induced by Crb overexpression, could both result from excessive constriction
of amnioserosa cells to produce a tissue that only occupies a fraction of the
dorsal hole. Such excessive constriction may be driven by ectopic accumulation
of a normally apically localized actomyosin contractile apparatus. A role for
Crb in defining the location of the actomyosin contractile apparatus is
consistent with the idea that Crb defines the range of the apical membrane
cytoskeleton. The actin-crosslinking protein
ßHeavy(ßH)-spectrin normally has an
apicolateral distribution, but upon overexpression of Crb is also found at the
basolateral membrane, indicating a redistribution of the membrane cytoskeleton.
ßH-spectrin is required for apical constriction of follicle
cells during Drosophila oogenesis and may participate in organization
of an actomyosin contractile apparatus.
It is conceivable that the ectopic localization of
(ßH)-spectrin domain following Crb overexpression could be
accompanied by an ectopic accumulation of F-actin and myosin. Future goals in
studying Drac1-Crb function in amnioserosa morphogenesis will include
addressing the nature of the interaction between the two proteins and defining
which portion(s) of the Crb protein are required. The short cytoplasmic domain
of Crb appears sufficient to execute all Crb functions studied to date. No
definitive role has been found for the large extracellular domain, although
there is evidence that the Drosophila and human Crb proteins have
non-cell-autonomous functions (Hardin, 2002).
Although Drac1 and Crb both generate premature contraction of the
amnioserosa when their activity is experimentally upregulated in this tissue,
their phenotypic effects are not identical. Drac1V12 expression drives
constriction of all amnioserosa cells early in closure, whereas, at the same
stage, Crb overexpression only promotes constriction of the end cells. A
plausible explanation for this is that constriction of the middle cells
requires Drac1 to activate Crb-independent processes and that Crb function is
necessary but not sufficient for middle cell constriction. Crb overexpression
in the amnioserosa causes disruption of the leading edge cytoskeleton and a
failure of cell shape change in the epidermis, suggesting that a signal from
the amnioserosa required for dorsal closure is disrupted. That communication
between the amnioserosa and the epidermis is a component of dorsal closure is
demonstrated by the observation that JNK signaling in the
amnioserosa is required for phosphotyrosine accumulation at the leading edge
and dorsalward migration of the epidermis and by the observation that leading edge cells are specified wherever an interface of amnioserosa and dorsal epidermis occurs.
Drac1V12 expression in the amnioserosa does not disrupt the leading edge
cytoskeleton or prevent closure of the epidermis, and this result suggests
that Drac1V12 cannot activate a function of Crb that influences communication
between the amnioserosa and the epidermis (Hardin, 2002).
The Pak kinases are effectors for the small GTPases Rac and Cdc42 and are divided into two subfamilies. Group I Paks possess an autoinhibitory domain that can suppress their kinase activity in trans. In Drosophila, two Group I kinases have been identified, dPak and Pak3. Rac and Cdc42 participate in dorsal closure (DC) of the embryo, a process in which a hole in the dorsal epidermis is sealed through migration of the epidermal flanks over a tissue called the amnioserosa. DC is driven in part by an actomyosin contractile apparatus at the leading edge (LE) of the epidermis, and is regulated by a Jun amino terminal kinase (JNK) cascade. Impairment of dPak function using either loss-of-function mutations or expression of a transgene encoding the autoinhibitory domain (AID) of dPak leads to disruption of the LE cytoskeleton and defects in DC does not affect the JNK cascade. Group I Pak kinase activity in the amnioserosa is required for correct morphogenesis of the epidermis, and may be a component of the signaling known to occur between these two tissues. It is concluded that DC requires Group I Pak function in both the amnioserosa and the epidermis (Conder, 2004).
Three results indicate that Group I Pak kinase activity in the amnioserosa does not make a major contribution to amnioserosa morphogenesis: (1) amnioserosa cell shape change still occurs in embryos lacking maternal and zygotic dPak; (2) inhibition of Group I Pak kinase activity in the amnioserosa does not prevent apical constriction of amnioserosa cells; (3) Rac1V12 is still capable of inducing excessive contraction of the amnioserosa in a dpak6 mutant background. Presumably, Rac uses cytoskeletal effectors other than dPak to drive cell shape change in the amnioserosa (Conder, 2004).
The finding that expression of dPak-AID with either the LE or amnioserosa GAL4 drivers causes a high frequency of failures to form cuticle is curious. These GAL4 drivers do not show expression in the lateral epidermis with the exception of GAL4332.3, which shows scattered epidermal expression after completion of dorsal closure. One possibility is that many embryos are dying in late embryogenesis before completion of cuticle secretion. Another, perhaps less likely explanation, is that Group I Pak kinase activity in the amnioserosa and DME cells is required non-autonomously for cuticle secretion in the epidermis through cell-cell signaling. Whatever the mechanism, a further indication that alterations in Rho family small GTPase/Pak signaling have consequences with regard to cuticle secretion is demonstrated by the finding that the disruption of cuticle secretion by Rac1V12 expression is suppressed by zygotic loss of dPak (Conder, 2004).
Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested that Rac is a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. The tracheal system of Drosophila was used to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. A reduced Rac activity leads to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibits dynamic epithelial cell rearrangement. In contrast, hyperactivation of Rac inhibits assembly of newly synthesized E-Cadherin into cell junctions and causes loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules (Chihara, 2003).
Cadherin-based cell adhesions are vital to maintain the morphological and
functional features of the epithelium of multicellular organisms. During
morphogenesis of the epithelia, cell adhesions must be disrupted and
re-assembled in a regulated manner to allow movement of individual cells in
the epithelia. In vivo analyses have demonstrated that a reduction in Rac
activity prevents cell rearrangement. This phenotype is associated with an
increase in the level of E-Cadherin and its associated molecules, and
expansion of E-Cadherin localization to the basolateral membrane. It is inferred that increased E-Cadherin expression consolidates cell adhesiveness.
Hyperactivation of Rac prevents incorporation of newly synthesized E-Cadherin
into cell junctions and reduces cell adhesiveness, transforming the tracheal
epithelium into mesenchyme. It is suggested that switching of Rac between active
and inactive states promotes turnover of the complex containing E-Cadherin at
the cell junction, and maintains the plasticity of the tracheal epithelium to
allow branching morphogenesis (Chihara, 2003).
Expression of a dominant-negative form of Rac 1
greatly reduces cell rearrangement required for partitioning cells into the
stalk of the dorsal branch. Overproduction of this form, Rac 1N17, would shift the
cellular pool of Rac toward the inactive GDP-bound state. It is suggested that
turnover of E-Cadherin at a proper level requires a high level of Rac
activity. However, since highly active movement of cell extensions in the cells at
the tip is still visible, it is suggested that the ability of those cells to move
toward their target is 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 (Chihara, 2003).
Time-lapse analysis demonstrates that reduced Rac activity inhibits
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 is different from the phenotype of E-Cadherin-GFP
overexpression, which does not inhibit cell rearrangement. It is
suggested that a reduction in Rac promotes the association of cadherin-catenin
complexes with the cell membrane and stabilization of these complexes.
Activation of Rac results 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. It is suggested that the inhibitory action on the cadherin cell
adhesion system is a general property of Rac in the Drosophila
embryo (Chihara, 2003).
In addition to the defects in cell adhesion and rearrangement, Rac1, 2 mutant embryos show various defects in tracheal cell
migration and differentiation. A wide range of tracheal defects are observed
in Rac1, 2 mutants. In embryos showing the weak class phenotype, misrouting of the dorsal branch toward the anteroposterior direction is often observed. In
intermediate-class embryos, the number of truncated dorsal trunks increases and the
germband does not retract completely. The severity of the defects and their frequency increases when the gene dose of rac is progressively reduced. When the
maternal expression of Rac1 and Rac2 was reduced by half, the tracheal defects
occurred in 25% of the embryos. Rac1 Rac2 Mtl triple
mutants laid by Rac1 Rac2 Mtl heterozygous mothers were also analyzed; the
mutants showed higher frequency of severe tracheal defects similar to those
found in Rac1, 2 mutants. These results suggest that
a change in Rac activity within the physiological range significantly affects
morphogenetic movement of the tracheal system (Chihara, 2003).
p21-activated kinase (Pak) is known as a mediator of the activity of Rac
GTPase. Tracheal defects similar to those of Rac1, 2 mutants
are found in pak mutants. Furthermore,
Rac1, 2 and Pak mutations synergistically enhance tracheal
defects. Such results suggest that Rac and Pak are required for directed movement of tracheal branches (Chihara, 2003).
The loss of Rac activity also causes a defect in cell differentiation. Tips
of dorsal branch 1-9 are normally capped with terminal cells that extend terminal branch
in the ventral direction. In Rac 1, 2 mutant embryos, the loss of terminal
branches was observed with high penetrance. Consistently, serum response
factor (SRF), a marker protein for the terminal cell, also disappears, suggesting that terminal cell differentiation does not occur (Chihara, 2003).
Since directed cell migration and terminal cell differentiation are processes
requiring FGF signaling, it was asked whether Rac is involved in FGF signaling (a strong genetic interaction). Although tracheal patterning is only
mildly affected by half dose reductions of bnl (ligand), btl
(receptor) and dof (intracellular effector), the
phenotype is strongly enhanced by introducing one copy of Rac1, 2
mutant chromosome from mothers. A
similar genetic interaction was found between pak and bnl. These genetic
interactions suggest that Rac and Pak are required for the migration of
tracheal branches in response to FGF signaling (Chihara, 2003).
To determine the epistatic relationship between Rac and FGF signaling, the effect of constitutive activation of Rac was tested in btl mutants.
In the btl mutant, tracheal branching does not proceed beyond the
invagination at stage 11, and MAP kinase activation is absent (Chihara, 2003).
Expression of Rac1V12 partially restores the movement of tracheal cells, and
activates MAP kinase, as revealed by staining with the antibody against the
diphosphorylated form of MAP kinase (dp-MAPK). These results suggest
that Rac activation is an essential downstream event of tracheal cell motility
induced by FGF signaling (Chihara, 2003).
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 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, it was found that activated Rac 1 partially rescues tracheal cell motility
and MAP kinase activation in btl mutants. Involvement of Rac
in FGF-dependent events may not be limited to cell motility. Expression of SRF, the product of one of the target genes activated by FGF
signaling in the tracheal system, is lost in the mutant trachea with reduced
Rac activity because of Rac 1, 2 mutation or Rac 1N17. This result suggests that Rac also regulates transcription (Chihara, 2003).
Several lines of evidence suggest that FGF signaling is activated locally
at the tip of branches, and activation of FGF signaling in all tracheal cells prevents branching, 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? Since the effect of Rac 1N17 is
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 (Chihara, 2003).
The role of tip and rear cells in collective migration is still a matter of debate and their differences at the cytoskeletal level are poorly understood. This study analysed these issues in the Drosophila trachea, an organ that develops from the collective migration of clusters of cells that respond to Branchless (Bnl), a FGF homologue expressed in surrounding tissues. Individual cells in the migratory cluster were tracked and their features were characterized; two prototypical types of cytoskeletal organization were unveiled that account for tip and rear cells respectively. Indeed, once the former are specified, they remain as such throughout migration. Furthermore, it was shown that FGF signalling in a single tip cell can trigger the migration of the cells in the branch. Finally, specific Rac activation was found at the tip cells, and how FGF-independent cell features such as adhesion and motility act on coupling the behaviour of trailing and tip cells was analyzed. Thus, the combined effect of FGF promoting leading cell behaviour and the modulation of cell properties in a cluster can account for the wide range of migratory events driven by FGF (Lebreton, 2013).
Among the tracheal branches from each placode, two grow towards the ventral side of
the embryo, one in the anterior and the other in the posterior region of the segment,
the lateral trunk anterior (LTa) and the lateral trunk posterior (LTp) respectively. By a combination of migration, intercalation and elongation, the tip cell
of the LTp migrates towards the central nervous system (CNS), and the resulting
ganglionic branch (GB) connects the CNS to the main tracheal tube. Another cell from the LTp migrates towards the LTa of the adjacent
posterior metamere and makes a fusion branch that connects the two LT branches. This study focused on this branch (LTp/GB) because its complex morphology and pattern of migration make it
particularly appropriate for analysing the morphology and behaviour of the tip and
trailing cell during tracheal collective migration (Lebreton, 2013).
The FGF signalling pathway is involved in many
morphogenetic events requiring collective migration of cell clusters. However, it is
not entirely clear whether in these events FGF signalling is directly involved in
triggering cell migration, or alternatively if it is required for other processes such as
cell determination which only affect cell migration indirectly. Moreover, while FGF
might be required it is not clear either whether all the cells or just a subset of those
need to directly receive the signal to sustain the migration of the entire cluster. One
well-studied case is the role of FGF in the development of the zebra fish lateral line.
In that case, FGF appears to be produced by the leading cells which signal to the
trailing cells, the cells where FGF signalling is active. Restriction of FGF signalling is
thereafter required for the asymmetric expression of the receptors for the chemokines
that guide migration (Lebreton, 2013).
A very different scenario applies in the case of Drosophila tracheal migration. On the
one hand, FGF is expressed in groups of cells outside the migrating cluster. On the other hand the results in the LTp/GB
indicate that FGF signalling is required and sufficient in the leading cells, and not in
the trailing cells, for the migration of the whole cell cluster. Therefore, in spite of its
widespread involvement, the mechanisms triggered by FGF signalling in collective
migration appear to be quite different (Lebreton, 2013).
Rho inactivation produced breaks and
detachment in the LTp/GB cluster while its constitutive activation led these cells to
hold together impairing migration. Likewise, upon Cdc42 inactivation LTp/GB cells
were associated by thin extensions associated in some cases with breaks, while upon
its constitutive activation, the LTp/GB transient pyramidal organisation did not
evolve, or evolved much more slowly, towards branch elongation. However, the
phenotypes from each RhoGTPase mutants don't look alike and the detailed analysis
suggests that Rho impinges primarily on cell adhesion while Cdc42 does so on cell
motility (Lebreton, 2013).
These results are consistent with previous findings that show a role for Rho in
regulating adherens junctions stability and for Cdc42 as the main mediator of
filopodia formation. It is noted, however, that Cdc42 was found to exert in the LTp/GB an
opposite effect to the one identified in other systems, as Cdc42DN mutants showed
more protrusions and were more actin-enriched basally than wild-type cells and
Cdc42ACT mutants showed a reduced the motility of LTp/GB (Lebreton, 2013).
There is an increasing amount of data pointing to the different effect of RhoGTPases
in vitro versus in vivo models and also among various cell types. A unidirectional assignment between a
specific cellular process in vivo and a single RhoGTPase is probably an
oversimplification and this was not the aim of the current study. Rather the study relied on mutant
forms of the RhoGTPases to modulate cell features, either individually or collectively,
to assess their role in the overall behaviour of the cell cluster. In doing so, the results
point to a critical role for a balance between cell adhesion and cell motility for the
collective migration of a cell cluster (Lebreton, 2013).
The results support the following model for the specification, features and behaviour
of leading cells in the migration of the LTp/GB branch. Upon signalling
from the FGF pathway, tip cells reorganise their cytoskeleton features (actin
enrichment at the basal membrane, small apical surface and an apicobasal polarity
along the proximo-distal axis), thereby enabling them to acquire leading behaviour.
Indeed, FGF can induce migratory capacity to the whole cluster by signalling only the
tip cells, where a dynamic transition between states of Rac activity is needed to
acquire a leading role. How the behaviour of tip cells leads collective migration
thereafter depends on the features of the cells in the cluster, which are determined by
various regulators (among these, the RhoGTPases) which act, at least in part,
in an FGF-independent manner. Ultimately, the balance between individual cell
properties such as cell adhesion, motility and apicobasal polarity will (1) determine the
net movement of the overall cell bodies or alternatively changes in cell shape in terms
of elongation, (2) control the migratory speed and (3) define whether cells will migrate
individually or in clusters and whether clusters will bifurcate in different paths. The
combined effect of the changes promoting leading cell behaviour and modulation of
cell features is likely to be a widely exploited mechanism in collective migration. In
particular, the actual balance between these cell features may dictate the specifics of
each migratory process and, consequently, the final shape of the tissues and organs
they contribute to generate (Lebreton, 2013).
Peripheral glial cells in both vertebrates and insects are born centrally and travel large distances to ensheathe axons in the periphery. There is very little known about how this migration is carried out. In other cells, it is known that rearrangement of the Actin cytoskeleton is an integral part of cell motility, yet the distribution of Actin in peripheral glial cell migration in vivo has not been previously characterized. To gain an understanding of how glia migrate, the peripheral glia of Drosophila were labelled using an Actin-GFP marker and their development in the embryonic PNS was analyzed. It was found that Actin cytoskeleton is dynamically rearranged during glial cell migration. The peripheral glia were observed to migrate as a continuous chain of cells, with the leading glial cells appearing to participate to the greatest extent in exploring the extracellular surroundings with filopodia-like Actin containing projections. It is hypothesized that the small GTPases Rho, Rac and Cdc42 are involved in Actin cytoskeletal rearrangements that underlie peripheral glial migration and nerve ensheathement. To test this, transgenic forms of the GTPases were ectopically expressed specifically in the peripheral glia during their migration and wrapping phases. The effects on glial Actin-GFP distribution and the overall effects on glial cell migration and morphological development were assessed. It as found that RhoA and Rac1 have distinct roles in peripheral glial cell migration and nerve ensheathement; however, Cdc42 does not have a significant role in peripheral glial development. RhoA and Rac1 gain-of-function and loss-of-function mutants had both disruption of glial cell development and secondary effects on sensory axon fasciculation. Together, Actin cytoskeletal dynamics is an integral part of peripheral glial migration and nerve ensheathement, and is mediated by RhoA and Rac1 (Sepp, 2003).
The data suggest that RhoA and Rac1 are both involved in peripheral glial cell migration and nerve ensheathement, and have distinct effects on Actin rearrangement. For example, constitutively active Rac1 (V12) and RhoA (V14) expression results in halted migration of cell bodies as well as disrupted cytoplasmic process extension. The phenotypes of the two mutants are very different from one another. Rac1 (V12) mutants show ball-shaped collapsed glia, while RhoA (V14) mutants have very long, spike-shaped actin processes emanating from the cell bodies. The distinct and extreme phenotypes from these mutants suggest that there is a balance of RhoA and Rac1 activity in wild-type peripheral glia to generate normal migration and cytoplasmic process extension. The concept of a balance of GTPase function being necessary for glial cell migration is also supported by observations that glial cell migration is stalled in both the gain-of-function and loss-of-function mutations. These observations are interpreted as suggesting that there is a balance of GTPase activities that is necessary for glial cell migration. In other words, anything that affects this balance either through a loss of function or gain of function, affects the ability of glial cells to migrate (Sepp, 2003).
The well-characterized cultured fibroblast model has shown that Rac is involved in lamellipodia formation, while Rho mediates stress fiber polymerization and Cdc42 is involved in the extension of filopodia. It is possible that Rac1 and RhoA mediate the assembly of similar structures in peripheral glia. The long, straight actin fibers seen in constitutively active RhoA (V14) mutants could represent overextended stress fibers. Furthermore, the massive glial lamellar-like structures that are stimulated by Rac1L89 expression appear very similar to the lamellipodia of cultured fibroblasts. The biochemical activity of the Rac1L89 mutation is not known, and can act as either a dominant-negative or constitutively active form, depending on the cell type. The Rac1L89 phenotype in peripheral glia is most similar to overexpression of wild-type Rac1, suggesting that the ectopic lamellar structures are a result of moderate increase in Rac1 activity. Thus, it is possible that the Rac1L89 mutation causes Rac1 to be overactive but not as much as in the Rac1V12 mutation (Sepp, 2003).
It was interesting to note that the ectopic actin-containing projections of RhoAV14 and Rac1L89 mutants did not always reach over axon tracts, which are the normal peripheral glial migrational substrates in the wild type. For the steering of a migrating cell, large amounts of actin polymerization occur at the contact between the leading edge of the cell and the attractive migrational substrate. Perhaps the hyperactivity of the mutant GTPases enable the peripheral glia to extend processes out on less adhesive substrates compared with axons. It was also interesting to note that ectopic projections of peripheral glia (in the RhoAV14 and Rac1L89 mutants) do not interfere with axon pathfinding in the periphery. The ectopic glial projections could be a result of failed glial pathfinding instead. Interestingly, axons are capable of correctly migrating in the absence of glial sheaths (in the RhoAV14 and Rac1V12 mutants). Peripheral glia are know to be able to mediate sensory axon guidance to the CNS. Thus, peripheral glia most probably mediate sensory axon migration to the CNS using secreted cues (Sepp, 2003).
Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 gene. How these mutations affect neuronal development and function remains largely elusive. Specific point mutations or small deletions have been generated in the Drosophila fragile X-related (Fmr1) gene, and the roles of Fmr1 in dendritic development of dendritic arborization (DA) neurons have been examined in Drosophila larvae. Fmr1 can be detected in the cell bodies and proximal dendrites of DA neurons and Fmr1 loss-of-function mutations increase the number of higher-order dendritic branches. Conversely, overexpression of Fmr1 in DA neurons dramatically decreases dendritic branching. In dissecting the mechanisms underlying Fmr1 function in dendrite development, it was found that the mRNA encoding small GTPase Rac1 is present in the Fmr1-messenger ribonucleoprotein complexes in vivo. Mosaic analysis with a repressor cell marker (MARCM) and overexpression studies reveals that Rac1 has a cell-autonomous function in promoting dendritic branching of DA neurons. Furthermore, Fmr1 and Rac1 genetically interact with each other in controlling the formation of fine dendritic branches. These findings demonstrate that Fmr1 affects dendritic development and that Rac1 is partially responsible for mediating this effect (Lee, 2003).
Each abdominal hemisegment of Drosophila contains 44 sensory
neurons that can be grouped into dorsal, lateral and ventral clusters.
To test whether Fmr1 affects the dendritic growth of DA neurons,
the expression of Fmr1 in these neurons was confirmed. Fmr1 mRNA is expressed at high levels in the embryonic nervous system and in body wall muscles. To examine
the subcellular localization of Fmr1 in DA neurons of live larvae, a UAS-Fmr1-GFP transgenic fly line was generated. When
Fmr1-GFP was expressed in DA neurons driven by Gal4 109(2)80, Fmr1
expression was observed in the cytoplasm of DA neurons and in particle-like
structures in dendrites. To further confirm that the endogenous Fmr1 is expressed in DA neurons, immunostaining analysis was performed on dissected larvae using a
monoclonal antibody raised against Fmr1. Fmr1 is present in DA neurons and is expressed predominantly in the cytoplasm. The
expression of Fmr1 in the proximal dendrites of DA neurons and in body wall
muscle fibers was also detectable. Owing to the high level of Fmr1 expression in muscles, the
localization of endogenous Fmr1 in distal dendrites was barely visible with
confocal microscopy. The subcellular localization of Fmr1 in DA
neurons is consistent with the subcellular localization of FMR1 in mammalian
neurons (Lee, 2003).
Because Fmr1 mutants are viable, it is possible to directly examine
the effects of Fmr1 mutations on dendritic development of specific
neurons in a large number of live flies. To label all dendritic processes, UAS-mCD8::GFP, which targets to the cell membrane, was expressed in all DA
neurons. Third instar larvae were selected 4-5 days after egg laying (AEL) and the images of dendrites of ventral DA neurons were recorded from segments 5 and 6 in live animals. The Fmr1 mutant larvae exhibit more
dendritic processes than wild-type larvae. To quantify the
difference, the number of ends of all dendritic terminal processes were counted. On average, Fmr1 mutations increase the number of terminal dendritic processes of ventral DA neurons by 25%. To demonstrate that
the increased number of terminal dendritic processes in Fmr1 mutants
are indeed due to the absence of Fmr1 activity, one
copy of the wild-type Fmr1 gene was introduced into the Fmr14 mutant background; the transgene rescues the dendritic defects in Fmr1 mutants. A large
number of segments in wild-type and Fmr1 mutant larvae exhibit a
similar number of terminal dendritic processes, indicating that there is a
large variation among individual larvae of a given genotype and that
Fmr1 mutations cause subtle changes in neuronal morphology (Lee, 2003)
To further understand the function of Fmr1 in regulating dendritic
growth, Fmr1 was overexpressed in all DA neurons of wild-type
wandering larvae. To do so, UAS-Fmr1 flies were crossed with
Gal4 109(2)80 flies and the third-instar larvae were examined 4 days AEL. The numbers of terminal dendritic processes were dramatically
reduced in both ventral and dorsal DA neurons when Fmr1 was overexpressed. The length of remaining terminal processes was also greatly reduced. This
phenotype caused by Fmr1 overexpression is 100% penetrant (Lee, 2003)
Drosophila larvae increase their body surface over 50-fold from
the first to the third instar larval stages. Correspondingly, the dendritic
fields of DA neurons increase substantially during this period of development.
In larvae overexpressing Fmr1, the major dendritic branches are still capable
of extending more than fivefold during larval development. However, most
terminal processes fail to form or fully extend even at the first instar
stage. This demonstrates that overexpression of Fmr1
blocks the formation of higher-order dendritic branches and reduces the
complexity of DA neuron dendrites during development (Lee, 2003)
The KH domains of Fmr1 share more than 70% identity with the mammalian FMR1 proteins. Indeed, Fmr1 and human FMR1 have similar RNA-binding properties in vitro. A
number of studies have identified a large number of mRNAs that are
associated with FMR1 in mammalian systems.
However, systematic identification of Fmr1-binding targets in flies has not
been carried out. To gain mechanistic insights into Fmr1 function in
controlling dendritic growth in flies, co-immunoprecipitation
experiments were carried out to identify mRNAs that are associated with the Fmr1-mRNP complex in vivo. In this study, using primers specific for genes encoding small GTPase
Rac1, alpha-tubulin, and the voltage-gated K+ channel molecule
Hyperkinetic, RT-PCR analyses was performed on either total RNAs or the RNAs
that were immunoprecipitated by an anti-Fmr1 monoclonal antibody, from lysates derived from third instar larvae. All three mRNAs could be readily detected from total RNAs, while only the Rac1 mRNA was associated with Fmr1 in lysates derived from wild-type larvae as shown by coimmunoprecipitation experiments. These studies demonstrate that Rac1 mRNA is associated with Fmr1-mRNP complexes in vivo (Lee, 2003)
Based on the finding that Rac1 mRNA is present in Fmr1-mRNP
complexes in vivo, it was hypothesized that the effect of Fmr1 on dendritic
development in DA neurons may be partially mediated by Rac1. This hypothesis was tested genetically. First, the function of Rac1 in dendritic growth and branching of DA neurons was examined in Drosophila embryos. A null allele, Rac1J11, was tested. Gal4 109(2)80 was used to drive the expression of GFP in DA neurons in Rac1J11 mutant embryos and no gross defects were observed in dendritic branching patterns in later embryogenesis stages. DA neuron dendrites develop in discrete phases from the embryonic to larval stages. In embryos, dorsal dendrites of DA neurons extend from cell bodies first, and stop elongation 16-17 hours AEL, falling short of the dorsal midline. The lateral dendrites start to extend toward adjacent segment boundaries and cover the hemisegment before hatching (22-23 hours AEL). These findings in Rac1J11 mutant embryos suggest that Rac1 is not required for the initial growth of dorsal dendrites during embryogenesis (Lee, 2003)
During larval stages, the dendritic fields of DA neurons expand many-fold
in accordance with the increase of larval body size. Higher-order dendritic
branches further develop to cover the whole epidermal surface of each
hemisegment. The MARCM technique was used to examine
the role of endogenous Rac1 in dendritic growth in the third instar larval
stage. Single GFP-labeled wild-type or Rac1 mutant DA
neurons were generated in abdominal segments and the number of terminal dendritic branches was counted. Rac1 mutant ddaC neurons fewer dendritic branches than wild-type neurons,
a phenotype similar to that caused by Fmr1 overexpression. Different
Rac1J11 mutant ddaC neurons exhibit varying severities
of dendritic defects. On average, there was a 23% reduction in the number of
dendritic branches due to the Rac1 mutation. Similar dendritic
defects were also found in other DA neurons. These findings
demonstrate that Rac1 is required for normal dendritic branching of DA neurons in vivo, consistent studies that rely on the ectopic
expression of dominant mutant forms of Rac1 (Lee, 2003)
To support the notion further that Rac1 is partially responsible for the
effect of Fmr1 on dendritic development, Rac1 was overexpressed in DA neurons
in third instar larvae with the UAS-Gal4 system.
Consistent with the finding that Rac1 loss-of-function results in a decreased number of terminal dendritic branches, overexpression of Rac1 promotes
dendritic branching of DA neurons with 100% penetrance. This result is
also in line with previous studies that ectopic expression of the
constitutively active form of Rac1 promotes dendritic branching. The enhanced
dendritic branching caused by Rac1 overexpression is much more dramatic than
that caused by Fmr1 loss-of-function, and this is presumably due to
the high level of ectopic expression of Rac1 (Lee, 2003)
Because Fmr1 (or its mammalian homolog FMR1) can function as a
translation inhibitor, it was of interest to enquire whether the elevated Rac1 expression obtained by using the UAS-Gal4 system would partially rescue the dendritic phenotype caused by Fmr1 overexpression. To test this hypothesis, Fmr1 and Rac1 were expressed simultaneously in DA neurons driven by Gal4 109(2)80. Overexpression of Fmr1 decreases the number of higher-order dendritic branches, but could be partially rescued by co-expression of Rac1. In addition, the number of terminal dendritic branches in Fmr14 mutants with a reduced rac1 dosage was
significantly lower that that in Fmr14 mutants. These findings support the notion that Rac1 is one of the downstream components of Fmr1 function in controlling dendritic development (Lee, 2003)
Condensation is a process whereby a tissue undergoes a coordinated decrease in size and increase in cellular density during development. Although it occurs in many developmental contexts, the mechanisms underlying this process are largely unknown. This study investigated condensation in the embryonic Drosophila ventral nerve cord (VNC). Two major events coincide with condensation during embryogenesis: the deposition of extracellular matrix by hemocytes, and the onset of central nervous system activity. Preventing hemocyte migration by removing the function of the Drosophila VEGF receptor homologue, Pvr, or by disrupting Rac1 function in these cells, inhibits condensation. In the absence of hemocytes migrating adjacent to the developing VNC, the extracellular matrix components Collagen IV, Viking and Peroxidasin are not deposited around this tissue. Blocking neural activity by targeted expression of tetanus toxin light chain or an inwardly rectifying potassium channel also inhibits condensation. Disrupting Rac1 function in either glia or neurons, including those located in the nerve cord, causes a similar phenotype. These data suggest that condensation of the VNC during Drosophila embryogenesis depends on both hemocyte-deposited extracellular matrix and neural activity, and suggest a mechanism whereby these processes work together to shape the developing central nervous system (Olofsson, 2005).
Thus, disrupting hemocyte migration inhibits VNC condensation in the embryo. Lack of hemocyte migration is associated with a severe reduction of ECM components (Collagen IV and Peroxidasin) throughout the embryo and more particularly a loss of these components around the VNC. This leads to a proposal that correct assembly of the ECM depends on hemocytes, and that basement membrane is required for condensation. Supporting a role for ECM in VNC condensation, defects are observed in loss-of-function mutants of integrins, which are ECM receptors and appear themselves to be required for correct assembly of basement membranes. Mutants in integrins or the ECM component Laminin A share at least one other phenotype with embryos in which hemocyte migration has been inhibited: gut morphogenesis is impaired. Thus, a dysfunctional ECM may explain several of the morphogenetic defects seen in embryos with defective hemocyte migration (Olofsson, 2005).
How might basement membrane contribute to VNC condensation? Basement membrane may serve as a substrate for cellular movements involved in condensation and/or regulate signaling events relevant to condensation. Basement membrane is also required for normal neuromuscular junction development, and might be part of the functional blood-brain barrier in Drosophila. Hence, neural function may be disrupted when basement membrane formation is inhibited. However, condensation phenotypes in embryos with impeded hemocyte migration are more severe than in embryos in which neural activity has been blocked. This argues that the condensation phenotype seen in hemocyte migration-blocked embryos cannot be explained simply by a loss of neural activity (Olofsson, 2005).
Although animals in which hemocyte migration is blocked fail to deposit Collagen IV appropriately, it has not been demonstrated that Collagen IV function is required for condensation. However, embryos expressing a dominant negative form of Collagen IV under the control of a heatshock promoter fail to condense their nerve cord. While these data point towards a functional role of Collagen IV in condensation, further studies will be necessary to clarify the specific role of Collagen IV during condensation (Olofsson, 2005).
This study has not investigated whether phagocytosis of cells within the VNC contributes to condensation. pvr mutants show a perdurance of unengulfed cells at the ventral surface of the CNS at stage 14. The majority of these cells seem to disappear later, possibly engulfed by epidermal cells. pvr mutants also maintain some very restricted points of attachment between the epidermis and the VNC. This phenotype is not observed when hemocyte migration is blocked using mutant Rac1 expressed by crq-GAL4. This likely reflects failure of hemocyte migration at a later stage, after the two tissues have separated (Olofsson, 2005).
The major cell type that engulfs apoptotic corpses within the CNS is the subperineural glia. In the absence of macrophages (in the Bic-D mutants), apoptotic cells are still expelled from the CNS but accumulate at the ventral surface, similar to the observations in the pvr mutant. Hemocytes are required for normal CNS morphogenesis: at stage 16, pvr mutants and Crq RNAi treated embryos have mispositioned glia and minor axon scaffolding defects. These data were interpreted to reflect a failure of engulfment of cell corpses. In the context of these findings, an additional cause for glial mispositioning in pvr mutant embryos could be a loss of basement membrane components and the failure to condense (Olofsson, 2005).
VNC condensation correlates with the onset of neural activity in the CNS, and it is found that expressing tetanus toxin light chain or the inwardly rectifying K+ channel Kir2.1 pan-neuronally impairs condensation. This suggests that neural activity influences normal condensation. Neural activity could contribute to condensation in multiple ways. It could directly regulate cellular events relevant to condensation, such as adhesion or actin-based motility, or activity could influence the transcription of genes relevant to such events. Alternatively, neural activity could maintain synaptic connectivity among cells necessary for condensation, rather than directing changes in cellular behavior leading to condensation. Some condensation occurs before neural activity begins, and the condensation phenotypes resulting from impeding hemocyte migration are more severe than those resulting from blocking neural activity. This suggests that there may be multiple stages of condensation, including an earlier activity-independent stage and a later stage that is influenced by activity (Olofsson, 2005).
VNC condensation can be inhibited by expressing mutant Rac1 in lateral glia or
neurons. In glia, migration and ensheathing behaviors require cytoskeletal integrity. When mutant Rac1 is expressed in peripheral glia, the formation of cellular extensions is disrupted, and this is accompanied by glia migration and axon ensheathment defects. Similarly, ensheathment of longitudinal axon tracts by longitudinal glia is disrupted in htl loss of function embryos. The VNC condensation phenotype in these embryos is interpreted as indication that glia need dynamic actin cytoskeleton to generate a condensing force. Two types of VNC glia are particularly well placed to generate such a force: longitudinal glia associated with VNC longitudinal connectives, and perineural glia, which ensheath the cortex of the VNC. Cell-cell contacts and cell-ECM contacts among these cells accompanied by remodeling of extracellular matrix could help generate a condensing force within and across neuromeres through changes in cell shape, adhesion or migration. A similar process occurs during mesenchymal condensation (Olofsson, 2005).
In neurons, neurite extension requires normal Rac GTPase activity. Expressing mutant Rac1 in these cells causes defects in axonal outgrowth. In wild type animals, VNC axons are arranged into longitudinal connectives that extend along the length of the nerve cord, and these are well placed to generate an anteroposterior condensing force. This could happen through differential cell adhesion of neurites within the longitudinal connectives or overall shortening of the axons. The observation that axons in VNC longitudinal connectives loop out during condensation in metamorphic insects is consistent with this idea. It is interesting to note that condensation is inhibited in embryos in which mutant Rac1 is expressed in glia, but longitudinal axon tracts appear normal in these animals. This suggests that if axons help generate a condensing force, they likely do this with the help of glia, possibly using these cells as a substrate (Olofsson, 2005).
It is also possible that at least part of the force required for condensation may come from outside the VNC. Somatic muscles connect to the VNC during embryogenesis, and embryonic muscle activity toward the end of embryogenesis is well timed for generating such a force. Also, the methods used to manipulate glia or neuron development in this study may affect neuromuscular activity by disrupting blood-brain barrier formation, or by affecting the Rac-dependent formation of synaptic structures. However, the observation that the CNS can condense in mutants in which muscles do not form normally argues against a major contribution from muscle activity (Olofsson, 2005).
These data identify several areas for further investigation. By following the behavior of small populations of cells in the VNC it may possible to analyze in vivo changes associated with the condensation process and get insight into how changes in organ shape are generated and coordinated. It will also be interesting to examine the contributions made by components of the ECM to normal blood-brain barrier function. Finally, it may be possible to use VNC condensation in embryonic Drosophila to investigate the molecular and cellular basis of how neural activity is translated into a morphogenetic event (Olofsson, 2005).
Nervous system injury or disease leads to activation of glia, which govern postinjury responses in the nervous system. Axonal injury in Drosophila results in transcriptional upregulation of the glial engulfment receptor Draper; there is extension of glial membranes to the injury site (termed activation), and then axonal debris is internalized and degraded. Loss of the small GTPase Rac1 from glia completely suppresses glial responses to injury, but upstream activators remain poorly defined. Loss of the Rac guanine nucleotide exchange factor (GEF) Crk/myoblast city (Mbc)/dCed-12 has no effect on glial activation, but blocks internalization and degradation of debris. This study shows that the signaling molecules Downstream of receptor kinase (DRK) and Daughter of sevenless (DOS) (mammalian homologs, Grb2 and Gab2, respectively) and the GEF Son of sevenless (SOS) (mammalian homolog, mSOS) are required for efficient activation of glia after axotomy and internalization/degradation of axonal debris. At the earliest steps of glial activation, DRK/DOS/SOS function in a partially redundant manner with Crk/Mbc/dCed-12, with blockade of both complexes strongly suppressing all glial responses, similar to loss of Rac1. This work identifies DRK/DOS/SOS as the upstream Rac GEF complex required for glial responses to axonal injury, and demonstrates a critical requirement for multiple GEFs in efficient glial activation after injury and internalization/degradation of axonal debris (Lu, 2014).
Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. This study used the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. Inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/ß-catenin complex, and shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating ß-catenin localization during gland morphogenesis. These results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis (Pirraglia, 2006).
This study shows that the Rac GTPases regulate salivary gland morphogenesis through modulation of cadherin/catenin-based cell–cell adhesion, likely by dynamin-mediated endocytosis. The characterization of the Rac mutant phenotypes suggests a model where Rac normally regulates cadherin-mediated cell–cell adhesion in salivary gland cells to allow enough plasticity for its invagination and migration yet keep the cells of the tube adhered to one another so that the gland can migrate as a cohesive tube. One mechanism by which cell surface cadherin levels are regulated is through selective endocytosis of E-cadherin from the apical–lateral membrane in a dynamin-mediated process. When Rac function is compromised through loss-of-function mutations or expression of dominant-negative mutations, the balance between E-cadherin at the plasma membrane and internalized E-cadherin appears to be abrogated so that more E-cadherin remains at the plasma membrane resulting in increased cell–cell adhesion and causing the gland to sever. These studies reveal the importance of precise regulation of adherens junction remodeling during cell migration in the context of a developing organ (Pirraglia, 2006).
In all stage 14 Rac1L89 mutant embryos examined, the salivary gland broke apart close to its approximate mid-point. Reduction in cadherin levels rescues the mutant Rac severing phenotype, suggesting that severing occurs because loss of Rac leads to an increase in cadherin-mediated cell–cell adhesion. At least two possible explanations for the midpoint severing phenotype are envisioned. In the first scenario, levels of cadherin remodeling may differ throughout the gland such that in Rac1L89 embryos the cells in the distal tip are least affected and cells in the mid-region of the gland are most affected by the increase in cadherin function. In this situation, when the distal cells begin to migrate posteriorly, the increased adhesivity of the mid-region cells prevents their migration and causes the gland to sever in the middle. In the second scenario, movement of the mid-region and the distal region of the gland may occur through different mechanisms. It is possible that while the distal most cells migrate by undergoing cell shape change and extending prominent protrusions in the direction of migration, cells in the middle of the gland may follow the distal cells by rearranging their positions along the gland, such as occurs during the convergence extension movements observed in epithelial morphogenesis. Dynamic remodeling of E-cadherin may be particularly important for proper rearrangement of the mid-region cells and an inability to rearrange when E-cadherin adhesion is increased may cause severing of the gland and subsequent separation of the migrating distal portion from the rest of the gland. Alternatively, it is possible that both of these scenarios are at play during normal salivary gland migration. Currently it is not possible to distinguish between these possibilities. In the developing tracheal tubes, Rac1 is required for cell rearrangements; in tracheal cells expressing a dominant-negative Rac1 mutation, the dorsal branch was shorter than that of wild-type embryos. Therefore, it will be important to determine whether cell rearrangement plays a role during salivary gland migration and to further elucidate the role of the Rac genes in this process (Pirraglia, 2006).
When Rac1 function is over-activate, dynamin-mediated endocytosis of E-cadherin may be increased, resulting in decreased cadherin at the plasma membrane, and decreased cell–cell adhesion. The loss of adhesion leads to the dispersal of salivary gland cells and ultimately cell death. Preventing Rac1V12-induced cell death led to the formation of abnormally shaped glands demonstrating that the Rac1V12 salivary gland phenotype is primarily due to abrogation of gland morphogenesis and not to activation of the apoptotic pathway. Moreover, since wild-type full-length E-cadherin is sufficient to rescue the Rac1V12 salivary gland phenotype, loss of cadherin function appears to be the primary cause for salivary gland defects. Thus, the Rac genes function in salivary gland cells to regulate E-cadherin-mediated cell–cell adhesion during tube morphogenesis (Pirraglia, 2006).
During salivary gland morphogenesis, gland integrity is kept intact while cells perform extensive cell shape changes and movements. Rac-regulated endocytosis of E-cadherin is one mechanism by which cell–cell adhesion is likely to be downregulated temporarily. After E-cadherin is endocytosed, it can be recycled back to the cell surface, sequestered transiently inside the cell or routed to late endosomes and lysosomes for degradation. Once salivary gland migration is complete and the gland has reached its final position, cell–cell adhesion may then need to be strengthened again in the mature gland and Rac activity may be downregulated to promote increase in surface cadherins (Pirraglia, 2006).
In addition to endocytosis, studies in mammalian cultured cells have shown that Rac can regulate levels of cell surface E-cadherin by other mechanisms, such as cleavage by presenilins and metalloproteinases, or tyrosine phosphorylation of the cadherin adhesion complex in a process involving reactive oxygen species. Thus, it will be interesting to determine whether additional mechanisms of E-cadherin regulation exist in salivary gland cells during gland morphogenesis (Pirraglia, 2006).
Numerous studies in cell culture have demonstrated that recycling of E-cadherin occurs in both a clathrin-dependent and caveolin-dependent manner. Since dynamin mediates both clathrin- and caveolin-dependent endocytosis, these studies do not allow distinguishing which type is involved in cadherin endocytosis during salivary gland migration. Alternatively, both types of endocytosis may mediate Rac1 regulation of E-cadherin in salivary gland cells (Pirraglia, 2006).
Expression of the Rac1V12 mutation in salivary gland cells leads to loss of expression of salivary gland specific proteins, apical–basal polarity proteins and E-cadherin/β-catenin. Concomitant with changes in gene expression, Rac1V12 mutant salivary gland cells lose adhesion to each other and subsequently migrate away or die by apoptosis. The data suggest that overactivation of Rac1 primarily affects E-cadherin/β-catenin-mediated adhesion and salivary gland cell fate and that the observed cell death is a secondary consequence of these earlier changes. When cell death was prevented in Rac1V12 embryos by expressing p35, more cells expressed the salivary gland specific protein PH4αSG1 than in Rac1V12 embryos; however, the expression level was drastically reduced compared to wild-type, suggesting that even in the Rac1V12p35 cells, cell differentiation was still mostly altered. Moreover, Rac1V12p35 salivary gland cells did not form a normal gland, demonstrating a role for Rac1 in gland morphogenesis. It is possible that apoptosis of Rac1V12 cells is brought about by the loss or reduction of Forkhead (Fkh) function. Fkh is expressed early in the salivary gland placode and its expression is maintained throughout embryogenesis. In the absence of fkh function, salivary gland cells die by apoptosis during the invagination stage. Since expression of dCREB-A and PH4αSG1 is reduced in Rac1V12 mutant salivary gland cells, it is possible that Fkh expression is also similarly reduced, thereby, causing the cells to undergo apoptotic cell death (Pirraglia, 2006).
Many human cancers are due to epithelial-derived tumors. When epithelial cells metastasize, they first undergo an epithelial to mesenchymal transition (EMT) before migrating away from the primary tumor to invade surrounding tissues. EMT is characterized by the loss of epithelial polarity and cell–cell adhesion. When Rac1V12 was expressed in salivary gland cells, expression of apical membrane proteins, Crumbs and aPKC and adherens junction proteins E-cadherin and β-catenin, was either lost or mislocalized. Based on these criteria, activation of Rac1 function induces features characteristic of early changes in EMT and metastasis. Interestingly, the expression levels of Rho GTPases are found to be elevated in a number of human cancers. For example, increased Rac protein levels and fast-cycling Rac mutations have been correlated with colorectal and breast tumors. Expression of constitutively active Rac1 causes some salivary gland cells to lose polarity and adhesion to neighboring cells and migrate away in a manner similar to EMT. These findings suggest that Rac1-regulated endocytosis of E-cadherin in the Drosophila salivary glands may be critical in maintaining epithelial character and preventing the loss of cell–cell adhesion and cell polarity. The Drosophila salivary gland might thus be powerful as a simple system to identify and characterize mechanisms that regulate cadherin-based cell–cell adhesion and certain aspects of EMT (Pirraglia, 2006).
Trace conditioning is valued as a simple experimental model to assess how the brain associates events that are discrete in time. This study adapted an olfactory trace conditioning procedure in Drosophila by training fruit flies to avoid an odor that is followed by foot shock many seconds later. The molecular underpinnings of the learning are distinct from the well-characterized simultaneous conditioning, where odor and punishment temporally overlap. First, Rutabaga adenylyl cyclase (Rut-AC), a putative molecular coincidence detector vital for simultaneous conditioning, is dispensable in trace conditioning. Second, dominant-negative Rac expression, thought to sustain early labile memory, significantly enhances learning of trace conditioning, but leaves simultaneous conditioning unaffected. It was further shown that targeting Rac inhibition to the mushroom body (MB) but not the antennal lobe (AL) suffices to achieve the enhancement effect. Moreover, the absence of trace conditioning learning in D1 dopamine receptor mutants is rescued by restoration of expression specifically in the adult MB. These results suggest the MB as a crucial neuroanatomical locus for trace conditioning, which may harbor a Rac activity-sensitive olfactory 'sensory buffer' that later converges with the punishment signal carried by dopamine signaling. The distinct molecular signature of trace conditioning revealed in this study should contribute to the understanding of how the brain overcomes a temporal gap in potentially related events (Shuai, 2011).
In trace conditioning, the conditional stimulus (CS) and the
unconditional stimulus (US) are separated in time by a stimulus-
free interval. This so-called 'trace interval' can last for
a fraction of a second in eyeblink conditioning but many seconds
in fear conditioning, which poses a challenging question: how
does the brain overcome this temporal gap to form the association
between the CS and US? Intriguingly, trace conditioning
in mammals engages neural substrates fundamentally different from delay conditioning, where the CS precedes but also temporally overlaps with the US. Early evidence comes from lesion studies with experimental animals showing that acquisition of trace conditioning requires intact hippocampal formation and medial prefrontal cortex, whereas delay conditioning can occur even with the entire forebrain removed. Later studies involving human subjects further validate the
involvement of different brain circuits in these two conditioning
variants and even suggest, more surprisingly, that conscious awareness might be a prerequisite for trace but not delay conditioning. It is then hypothesized that the participation of hippocampus and neocortex, as well as the associated higher
cognitive function, is necessary in trace conditioning to maintain
a representation of the CS or CS/US contingency so as to bridge
the temporal gap. However, little is known about what form this representation takes and how it eventually converges with the US (Shuai, 2011 and references therein).
This study characterized trace conditioning in the fruit fly and used mutant analyses to show that it is distinct from the well-characterized simultaneous conditioning at the molecular level. These data complement the mammalian circuit-level studies and, more importantly, open up a molecular understanding
of the internal trace that the brain uses to bridge the temporal gap (Shuai, 2011).
Odor footshock pairing elicits robust learning in fruit flies. The current
study adapted this assay to study trace conditioning simply
by modifying the timing relationship between the CS+ odor and
the US punishment. To mimic the widely used simultaneous
conditioning paradigm, CS- presentation is kept at 45 s
after the punishment. Single-trial training is sufficient to elicit considerable learning performance; the learning index for OCT and 4-methycyclohexanol (MCH) is ~35 for trace conditioning at a trace interval of 30 s. Although a portion of the score (~10) might be attributed to attraction to the CS- via backward conditioning, the behavioral results clearly indicate a marked ability of fruit flies to associate events that are temporally discrete (Shuai, 2011).
One remarkable finding of the current study is that
flies devoid of Rut-AC perform normally in trace conditioning.
This result is interesting in view of the belief that dually regulated
adenylyl cyclase plays a central role in invertebrate associative
learning. The function of Rut-AC is best described as a molecular coincidence detector that is synergistically activated by the CS-evoked calcium entry and the US-evoked G protein-coupled receptor activation. It has
been hypothesized that the stimulus-free gap in trace conditioning
can be bridged by the temporal integration property of
Rut-AC. However, the current results disagree with this hypothesis.
The normal or even higher performance of rut-deficient
mutants suggests that CS-US association in trace conditioning
may recruit separate molecular machineries or occur in a distinct
group of neurons. Also pertinent to this study is that
cAMP levels in the prefrontal cortex negatively influence working
memory performance. Therefore, whereas cAMP signaling
is essential for some learning tasks, it is dispensable or
even detrimental for others (Shuai, 2011).
Another intriguing finding is that induced expression of
dominant-negative Rac enhances the learning of trace but not
simultaneous conditioning. Notably, no learning enhancement
was observed in a number of simultaneous conditioning variants
with altered training parameters, including lowered odor concentration
and conditioned intensity discrimination in the current
work, as well as reduced shock pulses and lowered shock
voltage in a previous report. Thus, the differential effects
are not explained by a ceiling effect or other ancillary factors.
Trace conditioning testing was performed almost immediately (within 3 min) after the training, rendering a better retention of the acquired associative memory also unlikely. Trace conditioning becomes less efficient as trace interval increases, indicating that an inner trace of the odor gradually degrades with
time. It is therefore speculated that inhibition of Rac activity
might preserve this transient 'sensory buffer' so as to facilitate
trace conditioning. In the learning of simultaneous conditioning,
the co-occurrence of odor and shock makes it possible to process
the CS and US information automatically, e.g., via simple convergence
on coincidence detection molecules like Rut-AC; hence the requirement of an olfactory sensory buffer is superfluous, which explains the lack of enhancement from Rac inhibition. The above speculation is particularly attractive considering a recently established role of Rac in the forgetting of a cold-shock sensitive
early associative memory. It appears that the perdurance of
two short-lived memory forms, one registered after a passive olfactory
experience and lasting tens of seconds and the other
registered after an associative reinforcement and lasting several
hours, are both sensitive to Rac signaling manipulation (Shuai, 2011).
Drac1(N17) takes effect in the MB, the center for olfactory learning and
sensory integration in insects. The localization of the Drac1
(N17) effect, combined with the full rescue of the dDA1 mutant
phenotype in the MB, implies a possible trace conditioning
model in which the MB bridges the temporal gap by holding a
short-term sensory buffer of the odor, which later converges with
the reinforcement signal carried by dopamine signaling. In accordance
with this model, two recent studies in fruit fly and
honey bee found no correlation between trace conditioning
behavior and the postodor calcium response patterns in olfactory
sensory neurons and projection neurons of the AL. Both studies
pointed out the likelihood that the sensory buffer relevant to
trace conditioning is in neurons downstream of the AL, most
likely in the MB. Nonetheless, the AL may still retain odor information
in biochemical signals other than calcium or in shortterm
synaptic plasticityThe rapidly evolving molecular imaging techniques in fruit flies may help to delineate the nature of the putative sensory buffer and how it interacts later with a biologically significant stimulus (Shuai, 2011).
Another remaining puzzle is that both simultaneous and trace
conditioning, although recruiting different molecular mechanisms,
rely on the MB as a mutual crucial site. This seems at variance with the view from mammalian studies, where trace conditioning recruits neural circuits distinct from delay conditioning. Species or paradigm differences might explain the discrepancy,
but it awaits to be fully addressed by future studies
exploring whether brain regions outside the MB are additionally engaged in trace conditioning in fruit flies and, more importantly, whether various MB subdivisions contribute differentially to these two conditioning variants (Shuai, 2011).
back to Rac1 Effects of mutation part 1/2
Rac1:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| References
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