Rac1
Strong and ubiquitous expression of Rac1 mRNA is evident in precellular as well as cellular blastoderm stages. The transcript is concentrated at the basal part of the cellular blastoderm. After gastrulation, Rac1 transcripts become highly enriched in mesoderm between stages 10 and 12. At stage 13, Rac1 transcripts start to appear in the nervous system and the gut. Later in development, somatic mesoderm expression vanishes, whereas the nervous system and gut expression persists. The expression pattern of Cdc42, another Rho family GTPase, is qualitatively similar to that of Rac1 in all of the stages described for Rac1 (Luo, 1994).
The Rho GTPases Rac1 and Cdc42 have been implicated in the regulation of axon outgrowth and guidance. However, the downstream effector pathways through which these GTPases exert their effects on axon development are not well characterized. Axon outgrowth defects within specific subsets of motoneurons expressing constitutively active Drosophila Rac1 largely persist even with the addition of an effector-loop mutation to Rac1 that disrupts its ability to bind to p21-activated kinase (Pak) and other Cdc42/Rac1 interactive-binding (CRIB)-motif effector proteins. While hyperactivation of Pak itself does not lead to axon outgrowth defects as when Rac1 is constitutively activated, live analysis reveals that Pak can alter filopodial activity within specific subsets of neurons similar to constitutive activation of Cdc42. Moreover, the axon guidance defects induced by constitutive activation of Cdc42 persist even in the absence of Pak activity. These results suggest that (1) Rac1 controls axon outgrowth through downstream effector pathways distinct from Pak, (2) Cdc42 controls axon guidance through both Pak and other CRIB effectors, and (3) Pak's primary contribution to in vivo axon development is to regulate filopodial dynamics that influence growth cone guidance (Kim, 2003).
These studies support the idea that Rac1 mediates axon outgrowth through downstream effector pathways that are distinct from those that mediate Cdc42-dependent guidance. This is based on the following: (1) by using mutants of Rac1 and Cdc42 that render them constitutively active, the downstream cellular events that each is responsible for during axon development can be effectively isolated. Rac1 and Cdc42 likely work through different effector pathways, since constitutive activation of each leads to distinct phenotypes. (2) An effector-loop mutation that disrupts Cdc42's ability to bind CRIB-motif proteins can effectively suppress axonal defects resulting from constitutively active Cdc42. In contrast, the parallel mutation in Rac1 only partially suppresses defects resulting from constitutive activation of Rac1. Finally, activation of Pak, a CRIB effector for both Rac1 and Cdc42, leads to guidance defects and increased filopodial activities similar to those seen in activated Cdc42 mutants, but not activated Rac1 mutants (Kim, 2003).
A model is favored in which Rac1 mediates axon outgrowth mainly through effector proteins that do not possess the CRIB-motif, while Cdc42 mediates growth cone guidance primarily through CRIB proteins. Since activation of the CRIB protein Pak does not lead to axon outgrowth, but rather, guidance defects, Pak does not seem to play a direct role in Rac1-dependent outgrowth. This model is consistent with reports demonstrating that Pak is not required for axon outgrowth, at least during its initial phase, but is required during proper guidance and targeting of axons in a later phase. This, however, does not necessarily exclude Pak from playing a significant role in other Rac signaling pathways that mediate axon development (Kim, 2003).
Closer examination of growth cone behavior begins to reveal nonlinear signaling events. The ability of activated Pak to enhance filopodial activity similar to what activated Cdc42 achieves suggests that Pak likely plays a role in Cdc42-mediated growth cone guidance that depends on filopodial activity control. In this context, however, Pak's contribution to filopodial regulation is thought necessary, but not entirely sufficient, to dictate proper guidance. Also, Pak may not be the sole effector responsible for Cdc42-dependent filopodial activity. CRIB proteins affected by the Y40C mutation in constitutively active Cdc42 do not play a role in filopodial regulation, suggesting that other effectors are likely playing redundant roles in mediating filopodial activity. In addition, the persistence of guidance defects in motoneurons expressing Dcdc42V12 in a Pak null genetic background further suggests that Cdc42 is likely working through other CRIB effectors, in addition to Pak, to mediate its effects on growth cone behavior and guidance (Kim, 2003).
By isolating cellular events downstream of Rac1 and Cdc42 through the use of constitutively active mutants, the effector pathways responsible for axon development have become amenable to dissection. Since constitutively active mutants lock the GTPases in an active GTP-bound state, downstream events can be examined irrespective of GTPase activation. Furthermore, by coupling effector-loop mutations with the constitutively active mutation, a role for specific effectors becomes more evident. More studies that isolate effector pathways are necessary to complement existing studies (Kim, 2003).
In conclusion, studies using in situ analysis support the general view that cytoskeletal dynamics, outgrowth, and guidance are not necessarily coupled directly under the activation of Rac1 or Cdc42. Instead, these studies imply that a complex repertoire of growth cone behaviors are being mediated in parallel as a meshwork of events, and that the workings of Pak and other Rac1 and Cdc42 effector proteins, many of which are yet to be examined in situ, collectively contribute to proper axon development (Kim, 2003).
Although bilateral animals appear to have left–right (LR) symmetry from the outside, their internal organs often show directional and stereotypical LR asymmetry. The mechanisms by which the LR axis is established in vertebrates have been extensively studied. However, how each organ develops its LR asymmetric morphology with respect to the LR axis is still unclear. This study showed that Drosophila Jun N-terminal kinase (JNK) signaling is involved in the LR asymmetric looping of the anterior-midgut (AMG) in Drosophila. Mutant embryos of puckered (puc), which encodes a JNK phosphatase, show random laterality of the AMG. Directional LR looping of the AMG requires JNK signaling to be down-regulated by puc in the trunk visceral mesoderm. Not only the down-regulation, but also the activation of JNK signaling is required for the LR asymmetric looping. It was also found that the LR asymmetric cell rearrangement in the circular visceral muscle (CVM) is regulated by JNK signaling and required for the LR asymmetric looping of the AMG. Rac1, a Rho family small GTPase, augments JNK signaling in this process. These results also suggest that a basic mechanism for eliciting LR asymmetric gut looping may be conserved between vertebrates and invertebrates (Taniguchi, 2007).
This report, demonstrates that JNK signaling activity must be controlled properly in CVM cells for normal LR asymmetric development of the AMG. This idea is supported by the following observations. First, in the puc mutant, which has a hyperactivated JNK signal, the LR asymmetry of this organ is random, and the LR defects are rescued by puc expression in the trunk visceral mesoderm. Second, these LR defects are suppressed by the down-regulation of bsk, a Drosophila JNK homolog. Third, a stage-specific suppression of JNK signaling by the overexpression of puc results in laterality defects in the AMG. Importantly, however, there was no LR asymmetry in the expression pattern of the JNK signal or puc before or during the LR asymmetric development of this organ. It is therefore speculated that JNK signaling has a permissive role for LR asymmetric development of the AMG, rather than being an instructive cue for this process. For example, bilateral JNK signaling could have some influence on an instructive LR signaling molecule that has a LR differences in its activity or distribution. In this case, hyperactivation of JNK signaling may bring this instructive LR signal up or down across a threshold for the LR difference, triggering the LR asymmetric development of this organ (Taniguchi, 2007).
In Drosophila, several downstream targets of JNK signaling have been identified and studied extensively, and decapentaplegic and Delta are known downstream target genes of JNK signaling. However, the curret studies, including gene expression and genetic interaction analyses, demonstrate that these genes are unlikely to be involved in the JNK signal-driven LR asymmetric development of the AMG. In addition, previous report showed that the overexpression of Myo31DF inversed the laterality of the AMG. However, it was also found that puc did not influence the laterality defects induced by expressing Myo31DF, suggesting distinct roles for Myo31DF and JNK signaling in this process. In contrast to these observations, the expression of Fas3, which encodes an adhesion molecule localized to septate junctions, is significantly decreased in the CVM of the puc mutant. However, Fas3 is not required for the normal LR asymmetric development of the AMG. Therefore, it is presently unknown which target gene of JNK signaling is involved in the LR asymmetric development of the AMG (Taniguchi, 2007).
In contrast, this study demonstrates that the up-regulation of Rac1 activity in CVM cells results in LR defects of the AMG. In addition, Rac1 functions upstream of JNK in the CVM during LR asymmetric development of the AMG. Thus, the cytoskeletal rearrangement that is modulated by Rac1 upstream of JNK could be important in the LR asymmetric rearrangement of CVM cells. It is known that JNK activates head involution defective (hid) to induce apoptosis in and the clockwise rotation of the terminalia, which arises from the genital disc. In contrast to this organ's requirement for apoptosis for normal LR asymmetric development, this study found that the overexpression of p35, a potent suppressor of hid-dependent apoptosis, in CVM cells does not affect the LR asymmetric development of the AMG. In addition, the increased apoptosis of CVM cells was not observed in pucGS16811 mutant embryos. These results suggest that, unlike its role in the genital disc, apoptosis is not involved in the LR asymmetric rearrangement of CVM cells, although JNK signaling plays essential roles in the LR asymmetric development of both organs (Taniguchi, 2007).
puc was found to be required for normal LR asymmetric development of the AMG at some time during stages 11 to 14. In addition, the LR asymmetric rearrangement of CVM cells is required for the normal laterality of the AMG. However, the LR asymmetric rearrangement of these cells occurs after stage 16. These results suggest that the down-regulation of JNK signaling by puc is required before, rather than during, the LR asymmetric cell rearrangement in the CVM. A possible explanation is that the down-regulation of JNK signaling is required to change the states of these cells to allow them to respond to the LR signal. It is speculated that a small LR bias at the initial stage, as found in CVM cells at late stage 15, is probably sufficient to introduce stereotypic LR asymmetry into the subsequent events of AMG morphogenesis. According to this model, the subsequent rotation of the AMG, which mostly occurs in the dorsoventral direction, augments the initial LR bias. Therefore, if the small bias is not introduced initially, the AMG shows nondirectional LR asymmetry, as was found in the puc mutant embryos (Taniguchi, 2007).
The Drosophila gut consists of two layers of cells, the epidermis and the visceral musculature. It was previously demonstrated that Myo31DF, which is essential for normal laterality of the hindgut, is required in the epithelium of this organ (Hozumi, 2006). This finding suggests that rearrangement of the epithelial cells is responsible for the LR asymmetric development of the embryonic hindgut. In contrast, for the normal LR asymmetric development of the AMG, the surrounding CVM cells play a crucial role (Taniguchi, 2007).
In zebrafish, the looping of the LR asymmetric gut is elicited as a secondary consequence of the LR asymmetric migration of the lateral plate mesoderm. The embryonic gut of this organism is located along the midline between the lateral plate mesoderm of each lateral half. The shape of the space between the left and right lateral plate mesodermal tissue becomes stereotypically LR asymmetrical, as a consequence of LR asymmetric changes in the configuration of these mesodermal tissues. Because the gut lies in the vacant space delineated by these tissues, the morphology of this organ also develops directional LR asymmetry. This study has demonstrated that the mesodermal tissue also plays an essential role in the LR asymmetric development of the Drosophila AMG. Although it is not known whether JNK signaling is involved in the LR asymmetric development of the lateral plate mesoderm in zebrafish, the roles of the mesodermal cells in the LR asymmetric development of the gut may be evolutionarily conserved between vertebrates and insects, at least to some extent (Taniguchi, 2007).
The p21-activated kinases (PAKs) play essential roles in diverse cellular processes and are required for cell proliferation, apoptosis, polarity establishment, migration, and cell shape changes. This study has identified a novel function for the group I PAKs in cell-cell fusion. The two Drosophila group I PAKs, DPak3 and DPak1, have partially redundant functions in myoblast fusion in vivo, with DPak3 playing a major role. DPak3 is enriched at the site of fusion colocalizing with the F-actin focus within a podosome-like structure (PLS), and promotes actin filament assembly during PLS invasion. Although the small GTPase Rac is involved in DPak3 activation and recruitment to the PLS, the kinase activity of DPak3 is required for effective PLS invasion. A model is proposed whereby group I PAKs act downstream of Rac to organize the actin filaments within the PLS into a dense focus, which in turn promotes PLS invasion and fusion pore initiation during myoblast fusion (Duan, 2012).
The PAK family of Ser/Thr kinases have been implicated in
many biological processes, including cell migration, invasion,
proliferation, and survival, as well as regulation of neuronal
outgrowth, hormone signaling, and gene transcription. However, a role for PAKs in muscle development and cell-cell
fusion has not been previously uncovered. This study reveals
an essential function for Drosophila group I PAKs during
myoblast fusion in vivo. Specifically, it was shown that
the two group I PAKs in Drosophila, DPak3 (a close homologue
of mammalian PAK2) and DPak1 (a close homologue of
mammalian PAK1), have partially redundant functions in myoblast
fusion, based on the following lines of evidence. First,
double and single mutants of dpak3 and dpak1 exhibited a range
of fusion defects, dependent on the residual endogenous
protein level. Clearly, DPak3 plays a more significant role than
DPak1, and the minor role of DPak1 can only be revealed in the
context of the dpak1,dpak3 double mutant. Second, DPak3 is
enriched in the F-actin foci in wild-type embryos. In contrast, DPak1 only accumulates in the F-actin foci in the absence
of DPak3, consistent with its compensatory function in the fusion
process. Third, overexpression of DPak1 in the dpak3zyg
mutant leads to a slight but reproducible rescue of fusion.
Finally, overexpression of a kinase-inactive form of DPak3
(DPak3K322A) in dpak3zyg mutant embryos significantly enhances
the fusion defect, presumably by forming nonproductive
DPak3K322A-substrate complexes that exclude DPak1 (Duan, 2012).
What accounts for the differential effects of DPak3 and
DPak1 in myoblast fusion? One possibility is that DPak3 is recruited
to the PLS at a higher level than DPak1 in wild-type embryos.
However, the different recruitment levels cannot solely
account for the differential effects of these two proteins because
DPak1 overexpression in dpak3zyg mutant embryos does not
completely rescue the fusion defect. A second possibility is that
DPak3 and DPak1 may have different interacting partner(s) in
the PLS, and thus may respond differently to upstream Rac signaling
and/or transduce different downstream signals. In this
regard, it has been reported that human PAK2, but not PAK1,
can interact with MYO18A, which is involved in actin filament
organization and cell migration. A third possibility
is that these two kinases may have intrinsic differences in
substrate binding affinity and/or kinase activity. For example,
DPak3 may preferentially bind and activate specific substrates
in wild-type embryos and DPak1 could only access and/or inefficiently
activate these substrates in the absence of DPak3. In
support of this hypothesis, expressing the kinase-inactive from
of DPak3 (DPak3K322A) in the dpak3zyg mutant abolishes the
functional compensation by DPak1, suggesting that DPak3K322A
may efficiently compete with DPak1 for substrate binding by
forming high-affinity DPak3K322A-substrate complexes. Obviously,
identification of the preferred substrates of these group I
PAKs in vivo will be required to further test this hypothesis (Duan, 2012).
Previous studies have shown that the activity of group I PAKs
is regulated by the small GTPases Rac/Cdc42. The subcellular
localization of group I PAKs, on the other hand, is thought to be
controlled by SH2-SH3 domain-containing small adaptor proteins
Nck and Grb. Although the expression of a
dominant-negative
form of Rac resulted in a loss of DPak1 localization
at the leading edge during dorsal closure in Drosophila
embryos, it was unclear if Rac directly regulates
DPak1 recruitment to the leading edge. This study provides
evidence that the localization of DPak3 to a specific subcellular
structure, the F-actin focus within the podosome-like
structure (PLS), is directly controlled
by Rac. First, Rac colocalizes with DPak3 within the F-actin
foci during myoblast fusion. Second, DPak3 is no longer localized
to the F-actin foci in rac1,rac2 double mutant embryos.
Third, DPak3 carrying mutations in the Rac-binding domain
(DPak3H29,31L) fails to localize to the F-actin foci or rescue the
dpak3zyg mutant phenotype, despite its constitutive kinase activity (Duan, 2012).
It is noted that although the subcellular localization of group II
PAKs has been shown to be controlled by Cdc42 in cultured
mammalian cells and in Drosophila photoreceptor
cells, this study reveals,
for the first time, such a localization mechanism for a group I
PAK. Moreover, this study has positioned group I PAKs in a new
signaling branch downstream of the Rac GTPase during myoblast
fusion, in addition to the previously known branch involving the Scar complex (Duan, 2012).
Mammalian group I PAKs have been implicated in regulating
podosome formation, size, and number in cultured cells. However,
the function of PAKs in individual podosomes, especially in
intact organisms, remained completely unknown. This
study demonstrates that group I PAKs are required for regulating
the invasive behavior of individual podosomes in an intact
organism. DPak3 is required specifically in the
FCMs and colocalizes with the F-actin foci within the PLS. It was also shown
also show that in dpak3
mutants, the F-actin foci persisting to late developmental stages
appear dispersed and fail to invade into the apposing founder
cells/myotubes. As a result, fusion pores fail to form between
these defective FCMs and their apposing founder cells/myotubes.
Thus, the current study not only strongly supports the
model that PLS invasion is required for fusion pore formation,
but also reveals, for the first time, that group I PAKs are important
regulators of podosome invasion in vivo (Duan, 2012).
How do group I PAKs regulate PLS invasion? The dispersed
morphology of the F-actin foci in dpak3 mutants suggests that group I PAKs may be involved
in organizing branched actin filaments into a dense
focal structure within the PLS. Because the kinase activity of
DPak3 is required for its function during myoblast fusion, DPak3
may regulate actin cytoskeletal remodeling by phosphorylating
downstream substrates associated with the actin cytoskeleton,
such as regulators of actin polymerization, depolymerization,
and/or actin filament bundling/cross-linking. Genetic
and immunohistochemical analyses suggest that DPak3 is
unlikely to promote actin polymerization via the Arp2/3 NPFs
WASP and Scar, because DPak3 functions in parallel with the
WASP and Scar complexes and the amount of F-actin in each
PLS is not markedly reduced in dpak3zyg mutant embryos. In
addition, DPak3 is unlikely to suppress actin depolymerization
via PAK’s well-characterized substrate, LIM kinase (LIMK),
because loss-of-function mutants of LIMK and its substrate, the
actin depolymerization factor cofilin, did not have a myoblast
fusion defect, and DPak3 did not show genetic interactions with
LIMK or cofilin during myoblast fusion.
Therefore, it is conceivable that the group I PAKs may regulate
actin bundling and/or cross-linking proteins, which, in turn,
organize the assembly of branched actin filaments into tightly
packed bundles to promote PLS invasion. In this regard,
it has been shown that a tight intermolecular packing of
the actin filaments mediated by actin cross-linkers leads to the
formation of highly stiff actin bundles that exert large protrusive
forces against the cell membrane. Future experiments are required to identify the bona fide downstream
substrate(s) of DPak3 in regulating PLS invasion during myoblast fusion (Duan, 2012).
Interestingly, mammalian group I PAKs have been associated
with cellular invasion of other cell types, such as cancer cells
during metastasis. Elevated expression and hyperactivity
of PAK1 and PAK2 are seen in several types of tumors. Overexpression of constitutively active PAK1
promotes cancer cell migration and invasion, whereas inhibiting
PAK1 suppresses these phenotypes. It is well known that cancer cell invasion is mediated
by invadopodia, which are podosome-like structures with larger
F-actin-enriched cores and less dynamic actin polymerization. A role of PAK1 and PAK2 in invadopodia
formation in an invasive metastatic human melanoma cell line
has been revealed. Thus, further studies of PAK function in podosome invasion in Drosophila myoblast
fusion will not only provide additional insights into muscle differentiation,
but also cancer cell invasion during tumorigenesis (Duan, 2012).
Cells heal disruptions in their plasma membrane using a sophisticated, efficient, and conserved response involving the formation of a membrane plug and assembly of an actomyosin ring. This paper describes how Rho family GTPases modulate the cytoskeleton machinery during single cell wound repair in the genetically amenable Drosophila embryo model. Rho, Rac, and Cdc42 were found to rapidly accumulate around the wound and segregate into dynamic, partially overlapping zones. Genetic and pharmacological assays show that each GTPase makes specific contributions to the repair process. Rho1 is necessary for myosin II activation, leading to its association with actin. Rho1, along with Cdc42, is necessary for actin filament formation and subsequent actomyosin ring stabilization. Rac is necessary for actin mobilization toward the wound. These GTPase contributions are subject to crosstalk among the GTPases themselves and with the cytoskeleton. Rho1 GTPase was found to use several downstream effectors, including Diaphanous, Rok, and Pkn, simultaneously to mediate its functions. These results reveal that the three Rho GTPases are necessary to control and coordinate actin and myosin dynamics during single-cell wound repair in the Drosophila embryo. Wounding triggers the formation of arrays of Rho GTPases that act as signaling centers that modulate the cytoskeleton. In turn, coordinated crosstalk among the Rho GTPases themselves, as well as with the cytoskeleton, is required for assembly/disassembly and translocation of the actomyosin ring. The cell wound repair response is an example of how specific pathways can be activated locally in response to the cell's needs (Abreu-Blanco, 2013).
Actin and myosin II recruitment and organization at the wound
edge are key and conserved elements of the single cell wound
repair process in different organisms. In epithelial cells and
Xenopus oocytes, wounding induces a strong cytoskeletal
response dependent on calcium and Rho family GTPase
signaling. In particular, the contractile actomyosin
ring formed in Xenopus oocyte wounds is accompanied by
the flow of cortical F-actin filaments toward the wound from
neighboring regions. Myosin II also accumulates as foci
at the wound edge, and its recruitment is independent of
F-actin and cortical flow, although its subsequent assembly
into a continuous ring does require an intact F-actin network. Interestingly, this study found that actin and myosin II are both
actively mobilized toward the wound in the Drosophila
syncytial embryo, in a process dependent on cortical flow (Abreu-Blanco, 2013).
This mobilization of myosin II is necessary for proper actomyosin
ring assembly and function. In the context of
Drosophila cell wound repair, Rac coordinates the actin and
microtubule network that influences actin and myosin recruitment,
consistent with its known functions in regulating the
polymerization of both actin and microtubules. The sharp
actomyosin ring formed at the leading edge of the wound, as
well as the accompanying halo of dynamic actin, provides
the contractile force driving rapid wound closure. This study found
that Rho1 is necessary for myosin II activation, leading to its
association with actin: actin fails to accumulate as a ring at
the wound edge when myosin is disrupted. This is consistent
with Rho's known role in increasing the phosphorylation of
the myosin II regulatory light chain via its downstream effector
Rok. Rho1, along with Cdc42, also functions upstream of
the assembly and constriction of the actomyosin ring, where
they likely trigger actin filament formation and subsequent
actomyosin ring stabilization (Abreu-Blanco, 2013).
In addition to Rho family GTPases regulating actomyosin
ring assembly, the actin and myosin II cytoskeleton reciprocally
mediates GTPase function. For example, the assembly/
disassembly of the actin network in Xenopus oocyte wounds
has been shown to disrupt the local activation and inactivation
patterns of Rho and Cdc42, which are required for wound healing
progression. In the Drosophila model, actin is required
for the translocation, refinement, and maintenance of the Rho
family GTPase arrays, while myosin II is required for their
recruitment and organization. The actomyosin ring acts as
scaffold for signaling molecules that, in turn, are responsible
for the polymerization of actin and activation of myosin II.
Stabilization of the actin network by Jasplakinolide treatment
provides strong evidence that the actomyosin array and the
underlying signals translocate together. A striking observation
from this model is that Rho1, the first Rho family GTPase to be
recruited, accumulates at the wound site even when the actin
and myosin II cytoskeleton is severely disrupted, indicating
that its recruitment is independent of cytoskeleton integrity.
Cdc42 and Rac1, which accumulate after Rho1, are sensitive
to disruption of actin and myosin II. Thus, Rho
family GTPases are required in Drosophila syncytial embryos
for proper wound healing through reciprocal regulation with
the dynamic and integrated actin and myosin II networks (Abreu-Blanco, 2013).
Rho family GTPases localize at the wound edge with a precise
and characteristic organization pattern. This study shows the recruitment of Rac GTPases to the wound: Rac1 and Rac2 accumulate as graded ring-like arrays at the wound
edge. By analyzing the distribution of the endogenous Rho
GTPases, it was found that Rho, Cdc42, and Rac (Rac1/2) form
partially overlapping concentric arrays that correlate with their
specific functions during wound repair. Another interesting
observation is that Rho is recruited to the wound before the
onset of actin and myosin II recruitment and followed by
Cdc42 and Rac (Rac1/2) with a 90 s delay. The localization of active Rho GTPases was analyzed using a combination
of activity biosensors and downstream effectors. In the
Drosophila model, active Rho1 localizes as a discrete array
internal to the actomyosin ring. This is in contrast to that
observed in Xenopus oocyte wounds, where active Rho and
Cdc42 arrays are formed as discrete concentric rings overlapping
with myosin II and actin, respectively. Although the
timing and configuration of the GTPase arrays is different in
the two cell wound models, they both achieve the same end
result: organization of a highly contractile actomyosin ring
and a dynamic surrounding zone of actin and myosin assembly/
disassembly, thereby ensuring that a region of highly
contractility enriched in myosin II is followed by one of low
contractility where actin can assemble. Indeed, the organization
of Rho GTPases as local arrays is not only restricted to
contractile ring structures such as those observed in wound
repair and cytokinesis, but is a common strategy in different
biological processes. In the context of cell migration, coordinated
zones of Rho family GTPases at the cell's leading
edge regulate the waves of cellular protrusion and retraction
necessary for migration. In this scenario, Rho activation
occurs first, followed by Cdc42 and Rac1 with a 40 s delay.
Rho accumulates at the front of the cell concomitant with protrusions
and its levels are reduced during retraction, while
Rac1 accumulates behind the Rho array with its levels remaining
high during the retraction phase. These patterns of activation
and organization correlate with their proposed activities:
Rho modulates contraction and polymerization, whereas
Cdc42 and Rac1 regulate adhesion dynamics (Abreu-Blanco, 2013).
Negative feedback among Rho GTPases is a conserved
theme in multiple cellular and developmental processes.
In the Xenopus wound model, Rho has been shown to negatively
modulate the integrity of the Cdc42 array, while inhibition
of Cdc42 activity strongly suppresses local Rho activation.
Moreover, Abr, a protein with Rho/Cdc42 GEF and Cdc42 GAP
activity has been shown to accumulate at the Rho zone where
its GAP activity is required to locally suppress Cdc42 activity,
allowing Rho and Cdc42 to segregate into their respective
zones. It was not possible to specifically deplete Cdc42 in
the Drosophila syncytial embryo wounding assays, so its effects on Rho1 and Rac were not specifically addressed in this
model. Nonetheless, the results support the idea of GTPase
crosstalk playing a role in the control of GTPase array organization,
segregation, and levels. A notable example of crosstalk
in this context comes from the expansion of the Rac1 wound-induced
array in embryos where Rho1 levels were depleted,
suggesting that high levels of Rho1 at the wound edge inhibit
Rac1 accumulation at the interior of the actomyosin ring,
thereby allowing Rac1 to control the dynamic actin halo
surrounding the wound. These intricate and highly regulated
interactions among Rho family GTPases allow them to efficiently
execute their multiple functions in the cell (Abreu-Blanco, 2013).
A current challenge in the field is to determine how Rho
GTPases are maintained locally at high levels that dynamically
adjust during wound closure. Recent studies in the Xenopus
model propose a mechanism of GTPase treadmilling wherein
Rho and Cdc42 are subject to rapid local activation and
inactivation, which is different for each GTPase: the Rho
activity zone is shaped by trailing edge inactivation, whereas
Cdc42 undergoes variable inactivation across the array.
The development of biologically active photoactivatable Rho
family GTPase reporters will be necessary to determine the
relevance of this option in the Drosophila cell wound model.
An alternate possibility is that Rho family GTPases and their
regulators are anchored at the plasma membrane in an actin-dependent
manner. Putative candidates for anchoring proteins
are the ERM family proteins, which are known to interact
and regulate Rho GTPases in different cellular process (Abreu-Blanco, 2013).
It was surprising to find that Rho1 utilizes multiple downstream
effectors simultaneously during wound repair, begging
the question of how this specificity is achieved, maintained,
and tweaked dynamically. Considering the complexity of
wound repair, cytoskeletal responses are likely regulated via
several signaling pathways that converge on the contractile
ring. These pathways require precise coordination to provide
and integrate the different components required for the
dynamic assembly and disassembly of contractile ring
machinery as the wound is drawn closed. Rho family GTPases,
rather than switching the behavior of the entire cell, would
need to be capable of locally modulating the dynamics of the
cytoskeleton from one part of the cell to another. This specificity
associated with simultaneous recruitment and function
of downstream effectors is likely to be a key regulatory feature
for dynamic orchestration of cell wound repair. Future challenges
include defining the molecular composition of these
signaling modules and delineating the combinations and specific
subcellular localizations of Rho GTPases, GEFs, GAPs,
upstream regulators, and downstream effectors that are
needed for the proper functioning of these pathways (Abreu-Blanco, 2013).
The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. This question was investigated in the developing adult brain of Drosophila. Extension and retraction is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern (Srahna, 2006).
Based on the observation that blocking Fz2 results in decreased numbers of dorsal cluster neuron (DCN) axons in the medulla, it was reasoned that Fz2 could be a receptor for a putative stabilization signal. Since Fz2 and Fz are partially redundant receptors for the canonical Wnt signaling pathway, expression of the canonical Wnt ligand Wingless (Wg) was investigated in the brain during pupation. However, no Wg expression was detected in the pupal optic lobes, suggesting that Wg is unlikely to be involved in regulating DCN axon extension. Therefore, the expression of Wnt5, which has been shown to be involved in axon repulsion and fasciculation in the embryonic CNS, was investigated. Anti-Wnt5 staining revealed widely distributed Wnt5 expression domains beginning at PF and lasting throughout pupal development and into adult life. Wnt5 is strongly expressed in the distal medulla and is also present on axonal bundles crossing the second optic chiasm.The number of DCN axons crossing to the medulla was examined in wnt5 mutant flies. The number of DCN axons crossing the optic chiasm is reduced from 11.7 to 7.9 in the absence of wnt5, suggesting that it may play a role in stabilizing DCN axons (Srahna, 2006).
Next, the requirement of the Wnt signaling adaptor protein Dsh was tested. In animals heterozygous for dsh6, a null allele of dsh, the average number of DCN axons crossing between the lobula and the medulla is reduced from 11.7 to 7.6 with 78.5% showing less than eight axons crossing. Signaling through Dsh is mediated by one of two domains. Signaling via the DIX (Disheveled and Axin) domain is thought to result in the activation of Armadillo/β-Catenin. DEP (Disheveled, Egl-10, Pleckstrin) domain-dependent signaling results in activation of the JNK signaling pathway by regulation of Rho family GTPase proteins during, for example, convergent extension movements in vertebrates. To uncover which of these two pathways is required for DCN axon extension the dsh1 mutant, deficient only in the activity of the DEP domain, was tested. Indeed, in brains from dsh1 heterozygous animals the number of extending axons was reduced from 11.7 to 7.4. In flies homozygous for the dsh1 allele the average number of axons crossing was further reduced to 4.7, with all the samples having less than six axons crossing. In contrast, the DCN-specific expression of Axin, a physiological inhibitor of the Wnt canonical pathway, did not affect the extension of DCN axons. Similarly, expression of a constitutively active form of the fly β-Catenin Armadillo also had no apparent effect on DCN extension. Finally, whether Wnt5 and Dsh interact synergistically was tested. To this end, wnt5, dsh1 trans-heterozygous animals were generated. These flies show the same phenotype as flies homozygous for dsh1, suggesting that Wnt5 signals through the Dsh DEP domain (Srahna, 2006).
To determine if dsh is expressed at times and places suggested by its genetic requirement in DCN axon outgrowth, the distribution of Dsh protein during brain development was examined. Dsh protein is ubiquitously expressed during brain development. High expression of Dsh is detected in the distal ends of DCN axons at about 15% PF shortly before they extend across the optic chiasm toward the medulla. In general, higher levels of Dsh were observed in the neuropil than in cell bodies (Srahna, 2006).
In summary, these data indicate that the stabilization of DCN axons is dependent on the Dsh protein acting non-canonically via its DEP domain. Importantly, the axons that do cross in dsh mutant brains do so along the correct paths. This suggests that, like JNK signaling, Wnt signaling regulates extension, but not guidance, of the DCN axons (Srahna, 2006).
Wnt signaling to Dsh requires the Fz receptors. To examine if the effect of Wnt5 on DCN axon extension is also mediated by Fz receptors, the number of DCN axons crossing the optic chiasm in was counted fz, fz2, and fz3 mutants. There was no significant change in the number of axons crossing in the brain of fz3 homozygous animals. In contrast, in brains heterozygous for fz and fz2, the number of the axons crossing was reduced from 11.7 to 6.6 (fz) and 6.9 (fz2), with 71% and 85.7%, respectively, showing less than eight axons crossing. These data suggest that DCN axons respond to Wnt5 using the Fz and Fz2 receptors, but not Fz3. To determine whether the Fz receptors act cell-autonomously in individual DCNs, single-cell clones doubly mutant for fz and fz2 were generated and the number of DCN axons crossing the optic chiasm was counted. In contrast to wild-type cells, where 37% of all DCN axons cross, none of the fz, fz2 mutant axons reach the medulla. To test whether wnt5, fz, and fz2 genetically interact in DCNs, flies trans-heterozygous for wnt5 and both receptors were examined. Flies heterozygous for both wnt5 and fz mutations show a strong synergistic loss of DCN axons (11.7 to 3.7) and in fact have a phenotype very similar to that of flies homozygous for dsh1. Flies doubly heterozygous for wnt5 and fz2 also show a significant decrease in DCN axons (5.7), compared with either wnt5 (~8) or fz2 (8.5) mutants. These data indicate that the genetic interaction between wnt5 and fz is stronger than the interaction between wnt5 and fz2 (Srahna, 2006).
Examination of the expression domains of Fz and Fz2 in the developing brain supports the possibility that they play roles in stabilizing DCN axons. Both Fz and Fz2 are widely expressed in the developing adult brain neuropil. In addition, Fz is expressed at higher levels in DCN cell bodies (Srahna, 2006).
The observation that the wnt5 null phenotype can be enhanced by reduction of Fz, Fz2, or Dsh suggests that another Wnt may be partially compensating for the loss of Wnt5. To test this possibility, flies heterozygous for either wnt2 or wnt4 were examined. wnt2 heterozygotes display a reduction of DCN axon crossing from 11.7 to 7.3, whereas no phenotype was observed for wnt4. Thus, wnt2 and wnt5 may act together to stabilize the subset of DCN axons that do not retract during development. In summary, these results support the model that Wnt signaling via the Fz receptors transmits a non-canonical signal through Dsh resulting in the stabilization of a subset of DCN axons (Srahna, 2006).
Data is provided that supports the hypothesis that the regulation of JNK by Rac1 modulates DCN axon extension. As such attempts were made to determine how Wnt signaling might interact with Rac1 and JNK. The opposite phenotypes of dsh and Rac1 loss-of-function suggest that they might act antagonistically. To determine if Rac1 is acting upstream of, downstream of, or in parallel to Dsh in DCN axon extension, dominant-negative Rac1 was expressed in dsh1 mutant flies. If Rac1 acts upstream of Dsh, the dsh1 phenotype (i.e., decreased numbers of axons crossing the optic chiasm) is expected. If Rac1 acts downstream of Dsh, the Rac1 mutant phenotype (i.e., increased number of axons crossing) would be expected If they act in parallel, an intermediate, relatively normal phenotype is expected. Increased numbers of axon crossing were observed, suggesting that Rac1 acts downstream of Dsh during DCN axon extension and that Dsh may repress Rac1 (Srahna, 2006).
Next, whether Dsh control of DCN axon extension is mediated by the JNK signaling pathway acting downstream of Wnt signaling was tested, as the similarity of their phenotypes suggests. If this were the case, activating JNK signaling should suppress the reduction in Dsh levels. Conversely, reducing both should show a synergistic effect. Therefore the JNKK hep was expressed in dsh1 heterozygous flies and it was found that the hep gain-of-function is epistatic to dsh loss-of-function. Furthermore, reducing JNK activity by one copy of BSK-DN in dsh1 mutant animals results in a synergistic reduction of extension to an average of 0.8 axons with 60% showing no axons crossing and no samples with more than three axons. In summary, the results of genetic analyses suggest that Wnt signaling via Dsh enhances JNK activity through the suppression of Rac1 (Srahna, 2006).
Dsh appears to promote JNK signaling and to be expressed in DCN axons prior to their extension toward the medulla early in pupal development. Since JNK signaling is required for this initial extension, it may be that Dsh also plays a role in the early extension of DCN axons. To test this possibility, DCN axon extension was examined at 30% pupal development in dsh1 mutant brains. In wild-type pupae, essentially all (~40) DCN axons extend toward the medulla. In contrast, in dsh1 mutant pupae, a strong reduction in the number of DCN axons crossing the optic chiasm between the lobula and the medulla was observed (Srahna, 2006).
Although the genetic data indicate that Dsh- and Rac-mediated signaling have sensitive and antagonistic effects on the JNK pathway, they do not establish whether the Dsh-Rac interaction modulates JNK's intrinsic activity. To test this, the amount of phosphorylated JNK relative to total JNK levels in fly brains was evaluated by Western blot analysis using phospho-JNK (P-JNK) and pan-JNK specific antibodies. Then it was determined if Dsh is indeed required for increased levels of JNK phosphorylation. Dsh1 mutant brains showed a 25% reduction in P-JNK consistent with a stimulatory role for Dsh on JNK signaling. The reduction caused by loss of Dsh function is reversed, when the amount of Rac is reduced by half, consistent with a negative effect of Rac on JNK signaling downstream of Dsh. These data support the conclusion that Dsh and Rac interact to regulate JNK signaling by modulating the phosphorylated active pool of JNK (Srahna, 2006).
Taken together, these data suggest that during brain development DCN axons extend under the influence of JNK signaling. A non-canonical Wnt signal acting via Fz and Dsh ensures that JNK signaling remains active by attenuating Rac activity. In contrast, activation of the FGFR activates Rac1 and suppresses JNK signaling. These data support a model whereby the balance of the Wnt and FGF signals is responsible for determining the number of DCN axons that stably cross the optic chiasm. To test this model, FGFR levels were reduced, using the dominant-negative btl transgene, in dsh1 heterozygous flies. It was found that simultaneous reduction of FGF and Wnt signaling restored the number of axons crossing the optic chiasm to almost wild-type levels (10.2, with 33% of the samples indistinguishable from wild-type, suggesting that the two signals in parallel, act to control the patterning of DCN axon connectivity (Srahna, 2006).
These data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types (Srahna, 2006).
Proper nerve connections form when growing axons terminate at the correct postsynaptic target. Transforming growth factor β (TGFβ) signals regulate axon growth. In most contexts, TGFβ signals are tightly linked to Smad transcriptional activity. Although known to exist, how Smad-independent pathways mediate TGFβ responses in vivo is unclear. In Drosophila mushroom body (MB) neurons, loss of the TGFβ receptor Baboon (Babo) results in axon overextension. Conversely, misexpression of constitutively active Babo results in premature axon termination. Smad activity is not required for these phenotypes. This study shows that Babo signals require the Rho GTPases Rho1 and Rac, and LIM kinase1 (LIMK1), which regulate the actin cytoskeleton. Contrary to the well-established receptor activation model, in which type 1 receptors act downstream of type 2 receptors, this study shows that the type 2 receptors Wishful thinking (Wit) and Punt act downstream of the Babo type 1 receptor. Wit and Punt regulate axon growth independently, and interchangeably, through LIMK1-dependent and -independent mechanisms. Thus, novel TGFβ receptor interactions control non-Smad signals and regulate multiple aspects of axonal development in vivo (Ng, 2008).
Once growing axons reach the correct postsynaptic target, axon outgrowth
terminates and synaptogenesis begins. These studies suggest that TGFβ
signals play a role. When Babo is inactivated, MB axon growth does not
terminate properly and overextends across the midline. Consistent with this,
CA Babo expression results in precocious termination, forming axon
truncations. How Babo is spatially and temporally regulated remains to be
determined. Analogous to the Drosophila NMJ, MB axon growth might be
terminated through retrograde signalling. Target-derived TGFβ ligands
could signal to Babo (on MB axon growth cones) and stop axons growing further.
In an alternative scenario, TGFβ ligands might act as a positional cue
that prevents MB axons from crossing the midline. Recent data have shown that
Babo acting through Smad2 restricts individual R7 photoreceptor axons to
single termini. Loss of Babo, Smad2, or the nuclear import regulator
Importin α3 (Karyopherin α3 - FlyBase), results in R7 mutant axons
invading neighbouring R7 terminal zones. With the phenotype described in this study,
Babo could similarly be restricting MB axons to appropriate termination zones,
its loss resulting in inappropriate terminations on the contralateral
side (Ng, 2008).
In contrast to MB neurons, Babo inactivation in AL and OL neurons resulted
in axon extension and targeting defects. This might reflect cell-intrinsic
differences in the response in different neurons to a common Babo signalling
program. This may be the case for MB axon pruning and DC axon extension,
which require Babo/Smad2 signals. Whether these differences derive from cell-intrinsic
properties, or from Babo signal transduction, they underline the importance of
Smad-independent signals in many aspects of axonal development (Ng, 2008).
The results suggest that Smad-independent signals involve Rho GTPases. One
caveat in genetic interaction experiments is that the loss of any given gene
might not be dosage-sensitive with a particular assay. Nevertheless, all the
manipulations together suggest that Babo-regulated axon growth requires Rho1,
Rac and LIMK1. How Babo signals involve Rho GTPases remains to be fully
determined. In addition to LIMK1, which binds to Wit, one possibility, as
demonstrated for many axon guidance receptors, is that the RhoGEFs, RhoGAPs and Rho proteins might be linked to the Babo receptor complex. Thus, ligand-mediated changes in receptor properties would lead to
spatiotemporal changes in Rho GTPase and LIMK1 activities (Ng, 2008).
The data suggest that a RhoGEF2/Rho1/Rok/LIMK1 pathway mediates Babo
responses. Whether Rac activators are required is unclear, as tested RacGEFs do not genetically
interact with babo. In this respect, rather than through GEFs, Babo
might regulate Rac through GAPs, by inhibiting Tumbleweed (Tum) activity (Ng, 2008).
Do mutations in Rho1 and Rac components phenocopy babo phenotypes?
β lobe overextensions are observed in Rok,
Rho1 and Rac mutant neurons. In
MB neurons, Rac GTPases also control axon outgrowth, guidance and branching. Rho1 also
has additional roles in MB neurons. Although Rho1 mutant neuroblasts have cell proliferation defects, single-cell αβ clones do show β lobe extensions. RhoGEF2 strong loss-of-function clones do not exhibit axon overextension. As there are 23 RhoGEFs in the Drosophila genome, there might well be redundancy in the way Rho1 is
activated. LIMK1 inactivation in MB neurons was reported previously. Axon
overextensions were not observed as LIMK1 loss results in axon outgrowth and
misguidance phenotypes. This suggests that LIMK1 mediates multiple axon
guidance signals, of which TGFβ is a subset in MB morphogenesis (Ng, 2008).
Although their phenotypes are similar, several lines of evidence indicate
that CA Babo does not simply reflect LIMK1 misregulation in MB neurons. First,
whereas LIMK1 genetically interacts with most Rho family members and
many Rho regulators, CA babo is dosage-sensitive only to Rho1
and Rac and specific Rho regulators, suggesting that
Babo regulates LIMK1 only through a subset of Rho signals (Ng, 2008).
Second, the LIMK1 misexpression phenotype is suppressed by
expression of wild-type cofilin (Twinstar Tsr), S3A Tsr, or the cofilin phosphatase Slingshot (Ssh). By
contrast, only wild-type Tsr, but not S3A Tsr or Ssh, suppresses CA Babo. The suppression by wild-type Tsr might reflect a restoration of the endogenous balance or spatial distribution of
cofilin-on (unphosphorylated) and -off (phosphorylated) states within neurons. Indeed, optimal axon outgrowth requires cofilin to undergo cycles of phosphorylation and dephosphorylation. Since S3A forms of cofilin cannot be inactivated and recycled from actin-bound complexes, wild-type cofilin is more potent in actin cytoskeletal regulation (Ng, 2008).
CA Babo might not simply misregulate LIMK1 but also additional cofilin
regulators. Recent data suggest that extracellular cues (including mammalian
BMPs) can regulate cofilin through Ssh phosphatase and phospholipase Cγ activities. In
different cell types, cofilin phosphorylation and phospholipid binding (which
also inhibits cofilin activity) states vary and potently affect cell motility
and cytoskeletal regulation. Whether a combination of LIMK1, Ssh and phospholipid regulation affects cofilin-dependent axon growth remains to be determined (Ng, 2008).
Third, by phalloidin staining, LIMK1, but not CA Babo, misexpression
results in a dramatic increase in F-actin in MB neurons. Thus, CA Babo does not in itself lead to actin
misregulation. Fourth, Babo also regulates axon growth independently of LIMK1 (Ng, 2008).
This study differs significantly from the canonical model of Smad
signalling, in which type 1 receptors function downstream of the
ligand-type 2 receptor complex. In this study, the gain- and loss-of-function results
suggest that type 2 receptors act downstream of type 1 signals. Since ectopic only Wit
and Put suppress the babo axon overextension phenotype, this
implies that Smad-dependent and -independent signals have distinct type 1/type
2 receptor interactions. How these interactions propagate Smad-independent
signals remains to be fully determined. Babo could act as a ligand-binding
co-receptor with Wit and Put. In addition, Babo kinase activity could regulate
type 2 receptor or Rho functions. The results suggest, however, that provided
that Wit or Put signals are sufficiently high, Babo is not required. Whatever
the mechanism(s), it is likely that Babo requires the Wit C-terminus-LIMK1
interaction to relay cofilin phosphoregulatory signals. How Put functions is
unclear. Since the put135 allele (used in this study) carries
a missense mutation within the kinase domain, this suggests that kinase
activity is essential. put does not genetically interact with
LIMK1. Since Put lacks the C-terminal extension of Wit that is necessary
for LIMK1 binding, this suggests that Put acts independently of LIMK1. One
potential effector is Rac, which, in the context of Babo signalling, also
appears to be Pak1- and thus LIMK1-independent (Ng, 2008).
In MB neurons, Wit and Put can function interchangeably. In other in vivo
paradigms, type 2 receptors are not interchangeable.
However, since the Wit C-terminal tail is required to substitute for Put, this
suggests that Wit axon growth signals are independent of its kinase activity. Together, this suggests that Smad-independent signals involve LIMK1-dependent and -independent mechanisms (Ng, 2008).
This study shows that Babo mediates two distinct responses in related MB populations. How do MB
neurons choose between axon pruning and axon growth? The babo rescue
studies suggest that whereas Baboa or Babob elicits
Smad-independent responses, only Baboa mediates Smad-dependent
responses. Since Babo isoforms differ only in the extracellular domain,
differences in ligand binding could determine Smad2 or Rho GTPase activation.
However, it is worth noting that in DC neurons, either isoform mediates axon
extension through Smad2 and Medea. In addition, although expressed in all MB neurons,
CA babo misexpression (which confers ligand-independent signals)
perturbs only αβ axons. Thus, cell-intrinsic properties might also be essential in determining Babo responses (Ng, 2008).
Many TGFβ ligands signal through Babo. For
example, Dawdle, an Activin-related ligand, patterns Drosophila motor
axons, whereas Activin (Activin-β, FlyBase) is required for MB axon pruning. Whether
these ligands regulate Babo MB, AL and OL axonal morphogenesis is unclear.
Taken together, the evidence suggests that Babo signalling is varied in vivo
and is involved in many aspects of neuronal development (Ng, 2008).
TGFβ signals are responsible for many aspects of development and
disease and, throughout different models, Smad pathways are closely involved.
Although Smad-independent pathways are known, their mechanisms and roles in
vivo are unclear. TGFβ signals often drive cell shape changes in vivo.
During epithelial-to-mesenchymal transition (EMT), cells lose their epithelial
structure and adopt a fibroblast-like structure that is essential for cell
migration during development and tumour invasion. TGFβ-mediated changes in the actin cytoskeleton and adherens junctions are necessary for EMT. Although Smads are crucial, TGFβ signals also
involve the Cdc42-Par6 complex, resulting in cell de-adhesion and F-actin
breakdown through Rho1 degradation. In
other studies, however, TGFβ-mediated EMT has been shown to require Rho1, which
can be regulated by Smad activity (Ng, 2008).
Many TGFβ-driven events in Drosophila are Smad-dependent. Whether Smad-independent roles exist beyond those identified in this study remains to be tested. This study therefore provides a framework to understand how non-Smad signals regulate cell morphogenesis during development (Ng, 2008).
Different actin-filament-based structures co-exist in many cells. This study characterises dynamic actin-based protrusions that form at distinct positions within columnar epithelial cells in the Drosophila pupal notum, focusing on basal filopodia and sheet-like intermediate-level protrusions that extend between surrounding epithelial cells. Using a genetic analysis, it was found that the form and distribution of these actin-filament-based structures depends on the activities of apical polarity determinants, not on basal integrin signalling. Bazooka/Par3 acts upstream of the RacGEF Sif/TIAM1 to limit filopodia to the basal domain, whereas Cdc42, aPKC and Par6 are required for normal protrusion morphology and dynamics. Downstream of these polarity regulators, Sif/TIAM1, Rac, SCAR and Arp2/3 complexes catalyse actin nucleation to generate lamellipodia and filopodia, whose form depends on the level of Rac activation. Taken together, these data reveal a role for Baz/Par3 in the establishment of an intercellular gradient of Rac inhibition, from apical to basal, and an intimate association between different apically concentrated Par proteins and Rho-family GTPases in the regulation of the distribution and structure of the polarised epithelial actin cytoskeleton (Georgiou, 2010).
Although many studies have used the segregation of apical, junctional and basolateral markers as a model of epithelial polarity, and a number of studies have reported the existence of cell protrusions in the notum and other epithelia, these structures and the genes regulating their formation have not been characterised in detail. This study used Neuralized-Gal4 to express GFP-fusion proteins in isolated epithelial cells to reveal the dynamic shape of cells within the dorsal thorax of the fly during pupal development. Using this method, distinct populations of protrusions were characterised based on their form, dynamics and location within the basolateral domain of columnar epithelial cells. The analysis reveals dynamic protrusions at three distinct locations within the epithelial cell: apical microvillus-like structures, intermediate-level sheet-like protrusions and basal-level lamellipodia and filopodia. Importantly, although these are all dependent on continued actin filament dynamics, these populations of protrusions rely on different gene activities for their formation (Georgiou, 2010).
Cdc42, Rac, SCAR/WAVE and the Arp2/3 complex are required for the formation of basal lamellipodia and filopodia, but not for the formation of the apical microvillus-like structures. This analysis also confirms that HSPC300 should be considered to be a functional component of the SCAR complex. Moreover, the SCAR and Arp2/3 complexes are required to induce the formation of both lamellipodia and filopodia in this system. Although many studies have suggested that Rac activates the SCAR complex to induce branched Arp2/3-dependent actin nucleation that underlies lamellipodial formation, whereas Cdc42 is required to induce filopodial formation, this analysis suggests that the macroscopic form of the protrusion in a tissue context is not dictated by the nucleator used. In this, the current results are in line with several recent studies in cell culture. Instead, the macroscopic structure generated depends on the local level of Rac activity, with high levels of Rac driving filopodial formation and low levels leading to lamellipodial formation. Since the forces required to distort the membrane to generate finger-like protrusions are likely to be greater than those required to generate the equivalent section of a sheet-like protrusion, protrusion morphology might be a product of a force balance between membrane tension, extracellular confinement and local actin-filament formation. Since wild-type cells have a graded distribution of protrusions, with lamellipodia predominating apically and filopodia basally, wild-type cell morphology might reflect a gradient in the level of Rac activation, from high basal levels to low apical levels (Georgiou, 2010).
Within this system, Cdc42-Par6-aPKC and Baz/Par3 appear to have antagonistic roles in the formation of basolateral protrusions. Cdc42-Par6-aPKC is required for actin filament formation and protrusion dynamics, whereas Baz/Par3 ensures the separation of basal and intermediate protrusions by limiting the extent of basal filopodia along the apical-basal axis. In this, the current analysis adds to
the growing body of evidence that Baz/Par3 and Par6-aPKC have distinct molecular targets. Moreover, the data confirm that Par6-aPKC act together with the Rho-family GTPase Cdc42. Significantly, the loss of Baz/Par3 phenocopies gain-of-function mutations in Rac and the overexpression of the Rac-GEF Sif/TIAM1, a Par3-interacting protein. Baz/Par3 might therefore serve as a cell-intrinsic cue to polarise the dynamic actin cytoskeleton along the epithelial apical-basal axis, giving epithelial cells their characteristic polarised morphology (Georgiou, 2010).
Baz/Par3 has previously been implicated in the restriction of actin polymerisation to specific subcompartments within a cell, allowing for the formation of distinct populations of protrusions. This has been studied most extensively in hippocampal neurons, in which Par3 was shown to interact with TIAM1 to regulate the activation of Rac within distinct domains of the cell during axon specification and dendritic spine morphogenesis. Indeed, it has been suggested that the formation of a Cdc42-Par6-Par3-TIAM1-Rac1 complex is required to establish neuronal polarity. The current study suggests that Baz/Par3 acts in a similar fashion in the morphogenesis and positioning of dynamic protrusions in epithelia. However, this analysis reveals an antagonistic relationship between Sif/TIAM1 and Baz/Par3 in protrusion formation. Baz/Par3 might sequester Sif/TIAM1 to prevent its association with Rac. Furthermore, because the loss of Cdc42, Par6 or aPKC results in the loss of basolateral protrusions and a marked reduction in the GFP:Moe reporter (a phenotype that can be rescued by the coexpression of RacV12 or Sif) Cdc42, Par6 and aPKC are probably required for the basal activation of Rac in epithelial cells in the Drosophila notum. Thus, signals from apically concentrated polarity determinants appear to be communicated and translated into local protrusion formation within the basolateral domain. Whether this occurs through the diffusion of an apically localised regulator or via long-range transmission of polarity information e.g. via microtubules, will be an important area of future research. An intriguing correlation is the largely apical localisation of Baz and its proximity to intermediate-level sheet-like protrusions. This would suggest a possible gradient of Baz/Par3-mediated Rac inhibition, allowing sheet-like protrusions at an intermediate level and restricting filopodial protrusions to the very base of the cell. Since Baz/Par3 has been shown to localise PTEN to apical junctions, it is possible that Baz recruits PTEN, which acts on PtdIns(3,4,5)P3 to generate a PtdIns(3,4,5)P3 gradient from high levels basally to low levels apically. PIP3 could then act to aid in the recruitment and activation of Rac at the membrane (Georgiou, 2010).
Taken together, these data demonstrate that different components of the apical determinants of cell polarity act in conjunction with the Rho-family GTPases Cdc42 and Rac to regulate the positioning of lamellipodial and filopodial protrusions over the entire span of the apical-basal cell axis. Significantly, in this tissue context, Rac, SCAR and Arp2/3 complexes promote the formation of both lamellipodia and filopodia, whose structure appears to depend on the level of Rac activation (Georgiou, 2010).
Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. DC and the follicular epithelium were used to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. It is proposed that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. It is concluded that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion (Bahri, 2010).
Some embryos lacking zygotic Pak function successfully bring the epidermal flanks together at the dorsal midline but fail to restore septate junctions and adherens junctions at the LE in the DME cells. Thus, Pak at the LE membrane of the dorsal-most epithelial cells (DME) is regulating establishment of apicobasal polarity during a mesenchymal-epithelial transition. It is suspected that Pak is acting through different routes in its regulation of adherens junction formation versus septate junction formation. This study has focused on Pak regulation of the Scrib complex in septate junction formation at the LE. The data indicate that Pak is a component of the Scrib complex at the lateral membrane. Although Pak might be associating with the Scrib complex throughout epithelia, it might only be required for recruitment of the Scrib complex in epithelia derived from a mesenchymal-like intermediate such as the follicular epithelium and the LE. With the exception of the LE, apicobasal polarity in the epidermis is determined much earlier in development with formation of the blastoderm by cellularization. The epidermis is therefore a primary epithelium that does not arise from a mesenchymal intermediate, and Pak function does not appear to be required for apicobasal polarity in primary epithelia (Bahri, 2010).
Localization of Pak at the lateral membrane in both the follicular epithelium and in the epidermis is integrin-dependent. Studies using organ culture of embryonic kidney mesenchyme and MDCK cells demonstrate a requirement for integrins in apicobasal polarity of epithelia derived from MET, and this study has shown that βPS-integrin is required for Scrib complex and septate junction protein recruitment at the LE and in the follicular epithelium. Furthermore, previous studies in the follicular epithelium and another Drosophila epithelium derived from MET, the midgut, have demonstrated a requirement for integrins in the maintenance of apicobasal polarity. It is proposed that, at the LE, the absence of the septate junction diffusion barrier allows the accumulation of integrin complexes along the lateral membrane. These lateral integrin complexes recruit Pak, around which the Scrib complex is assembled. Thus, the absence of septate junctions allows the recruitment of proteins needed for the assembly of septate junctions. The model suggests that there might be transient Pak-mediated links between integrin and the Scrib complex. Interestingly, Dlg and βPS-integrin have been shown to co-immunoprecipitate from fly head extracts, consistent with these proteins existing in a complex in the nervous system and/or in epithelia (Bahri, 2010).
The data and recent studies on the amnioserosa support the idea that septate junctions restrict the accumulation of lateral integrins. The amnioserosa is devoid of septate junction proteins such as FasIII, and this might be owing to absence in this tissue of the transcription factor Grainy head, which promotes expression of septate junction proteins. The wild-type amnioserosa has high levels of lateral βPS-integrin, but ectopic expression of Grainy head in the amnioserosa leads to an accumulation of septate junction proteins and an accompanying disruption of βPS-integrin localization. Similarly, at the completion of DC, septate junctions appear at the LE and this is accompanied by downregulation of LE lateral integrins. In pak14pak376A and cora14 embryos where LE septate junctions are deficient, lateral LE βPS-integrin persists (Bahri, 2010).
A recent study in mammalian cell culture indicates that Scrib recruits Pak to the LE (Nola, 2008), and this study has shown that Pak localization in the follicular epithelium is Scrib-dependent. This study of the LE at the end of DC demonstrates that the relationship between Cdc42/Rac signaling complexes and Scrib can act in the opposite direction: membrane-localized Pak recruits the Scrib complex. A bidirectional interaction between the Scrib complex and Cdc42/Rac signaling complexes, including Pak, might be a crucial regulator of events at the LE of closing epithelia during both wound healing and development in diverse systems. Scrib at the newly formed LE can lead to recruitment of the Cdc42/Rac signaling complex, allowing acquisition of mesenchymal characteristics and polarized cell migration. When the opposing epithelial flanks meet up, events can be reversed with Pak recruiting the Scrib complex to the lateral membrane, contributing to restoration of apicobasal polarity and cell adhesion at the LE during MET. The Scrib/Pak complex is believed to be a 'toggle switch', enabling the epithelial membrane to shift back and forth between a migratory state characterized by actin-based extensions and an apicobasal polarized state characterized by cell-cell adhesion (Bahri, 2010).
Establishment and maintenance of stable muscle attachments is essential for coordinated body movement. Studies in Drosophila have pioneered a molecular understanding of the morphological events in the conserved process of muscle attachment formation, including myofiber migration, muscle-tendon signaling, and stable junctional adhesion between muscle cells and their corresponding target insertion sites. In both Drosophila and vertebrate models, integrin complexes play a key role in the biogenesis and stability of muscle attachments through the interactions of integrins with extracellular matrix (ECM) ligands. This study shows that Drosophila Importin7 (Dim7) is an upstream regulator of the conserved Elmo-Mbc-->Rac signaling pathway in the formation of embryonic muscle attachment sites (MASs). Dim7 is encoded by the moleskin (msk) locus and was identified as an Elmo-interacting protein. Both Dim7 and Elmo localize to the ends of myofibers coincident with the timing of muscle-tendon attachment in late myogenesis. Phenotypic analysis of elmo mutants reveal muscle attachment defects similar to that previously described for integrin mutants. Furthermore, Elmo and Dim7 interact both biochemically and genetically in the developing musculature. The muscle detachment phenotype resulting from mutations in the msk locus can be rescued by components in the Elmo-signaling pathway, including the Elmo-Mbc complex, an activated Elmo variant, or a constitutively active form of Rac. In larval muscles, the localization of Dim7 and activated Elmo to the sites of muscle attachment is attenuated upon RNAi knockdown of integrin heterodimer complex components. These results show that integrins function as upstream signals to mediate Dim7-Elmo enrichment to the MASs (Liu, 2013).
Previous studies have shown that Dim7 localizes to developing muscle-tendon insertion sites
and removal of Dim7 has severe consequences in muscle attachment maintenance (Liu, 2011). The current studies extend these observations to elucidate the functional contribution of Dim7-Elmo in regulating Drosophila muscle attachment. The results show that Dim7 is an upstream adaptor protein that recruits Elmo in response to integrin adhesion and/or signaling. Thus, it is proposed that the spatial and temporal regulation Elmo-Mbc activity results in regulation of the Rac-mediated actin cytoskeleton changes at the MASs (Liu, 2013).
The 'myospheroid' phenotype in elmo or msk mutants resemble attachment defects first characterized in mutated genes that encode for integrins, ILK and Talin, and is not due to earlier developmental defects in myogenesis. A similar number of cells expressing the muscle differentiation factor DMef2 was present in elmo or msk mutants, indicating that muscle specification was not affected (Geisbrecht, 2008; Liu, 2011). Genes essential for muscle migration and targeting also lead to detached muscles. For example, in kon/perd or grip mutants, the early arrest of migrating myotubes resulting from defective migration eventually leads to a linkage failure between the muscle and tendon cells. In mutant embryos with reduced levels of Elmo or Dim7, the muscle detachment phenotype did not appear to result from muscle migration defects. First, the spatial-temporal accumulation of Elmo and Dim7 is developmentally regulated. Both proteins are not detected at the leading edges of migrating muscles, but begin to accumulate at MASs after stage 15. Second, failure of muscle ends contacting
their corresponding attachment sites was not observed in elmo or msk mutants at late stage 15, when muscle migration was almost complete (Liu and Geisbrecht, 2011) (Liu, 2013).
Both membrane localization and Rac-dependent cell spreading of the uninhibited, active
version of Elmo is enhanced compared to native WT Elmo in cultured mammalian cells
(Patel, 2010). These in vitro results are in agreement with the current in vivo analysis, where ElmoEDE (a mutation that prevents the autoinhibitory interaction of Elmo) is enriched at larval muscle ends compared to the poor accumulation of full-length Elmo-YFP. This may reflect a potential regulatory mechanism controlling the subcellular localization of Elmo from the cytoplasm to the muscle ends upon the release of Elmo autoinhibition. Within different cells or tissues, various proteins may regulate Elmo localization to the cell periphery, or other sites where active Elmo is needed. In cultured mammalian epithelial cells, membrane recruitment of the Elmo-Dock180 complex is dependent on active RhoG for cell spreading. Consistent with a functional role for membrane-targeted Elmo, active Elmo promotes cell elongation in Hela cells, when co-expressed with RhoG (Patel, 2010; Liu, 2013 and references therein).
The data argues that adaptor proteins may be required in muscle cells for activated Elmo membrane recruitment. Decreased levels of ElmoEDE are observed at the polarized ends of muscle insertion sites when Dim7 levels are decreased. It is still not clear if Dim7 binding is required for the conformational change that results in Elmo activation or if an activated Dim7-Elmo complex already exists within the cell and gets recruited as a complex upon integrin activation. Furthermore, complete loss of Elmo-EDE protein levels is not observed, suggesting that either Dim7 protein levels are not depleted enough or other proteins in addition to Dim7 play a role in Elmo membrane recruitment. Alternatively, post-translational modification(s), such as phosphorylation,
could be an additional mechanism for the relief of Elmo autoinhibition. Thus, it is concluded that in muscle, Dim 7 is an essential adaptor protein for the polarized membrane localization of active Elmo or the active Elmo-Mbc complex downstream of integrin signaling pathway (Liu, 2013).
What is the relationship between the integrin adhesome and the Dim7-Elmo complex? Two explanations are proposed that are not mutually exclusive. One possibility is that the Dim7-Elmo-Mbc complex assembles at MASs via integrin-mediated 'outside-in' signaling. Upon ligand binding to ECM molecules, integrin activation results in Dim7-Elmo-Mbc complex localization for the spatial-temporal regulation of Rac activity to maintain dynamic actin filament adhesion at the MASs. It is predicted that localization of activated Elmo to the MASs is a prerequisite regulatory mechanism for actin cytoskeleton remodeling via Rac to maintain stable attachments. This hypothesis is supported by three lines of evidence: (1) muscle attachment defects upon loss of Dim7 or Elmo are only observed after the establishment of the integrin adhesion complex and onset of muscle
contraction; (2) muscle detachment in msk mutants can be rescued by expressing low
levels of activated Rac; and (3) the enrichment of Dim7 and Elmo-EDE proteins at the ends of muscle fibers is greatly reduced in integrin-deficient larvae (Liu, 2013).
It is also possible that accumulation of the Dim7-Elmo complex to the ends of muscles regulates 'inside-out' signaling to dynamically regulate integrin affinity for strong ligand binding and stable muscle attachments. Previously, it was reported that Dim7 acts upstream of the Vein-Egfr signaling pathway in muscle to tendon cell signaling (Liu, 2011). Combined with previous results that muscle-specific Vein secretion is dependent on the adhesive role of βPS integrin, the Dim7-Elmo complex may be internally required for integrins to regulate Vein secretion. A decrease in Vein-Egfr signaling and loss of tendon cell terminal fate results in a reduction in ECM secretion and weakened integrin-ECM attachment. This is consistent with the observation that msk or elmo mutants phenocopy embryos with reduced or excessive amounts of the αPSβPS integrin complex, where
pointed muscle ends result in smaller muscle attachments. Future studies analyzing Dim7-Elmo-Mbc complex localization and function in the background of integrin deletion constructs which separate the 'inside-out' and 'outside-in' signaling pathways will be essential to uncover more detailed molecular mechanisms (Liu, 2013).
What is the relationship between the Dim7-Elmo-Mbc-->Rac signaling pathway and the integrin mediated adhesome complex assembly (including the Talin, IPP [integrin linked kinase (ILK)-PINCH-Parvin-α) complex]? It is proposed that actin filaments within the muscle cell are anchored to the muscle cell membrane via the IPP complex, while regulation of MAS-actin remodeling iscontrolled by the Dim7-Elmo-Mbc-->Rac pathway. The data suggests that these two complexes assemble independently at the muscle ends. In msk mutant embryos, both ILK and Talin properly accumulate at the MASs, suggesting that Dim7 is not responsible for their localization (Liu, 2011). Similarly, both MAS-enriched Dim7 and active Elmo can be detected at two ends of the muscles in ILK-deficient larva, even in fully detached muscles. In a vertebrate cell culture model, Elmo2 was found to physically interact with ILK for the establishment for cell polarity (Ho and Dagnino, 2012; Ho, 2009). Thus, it is possible that the current approaches have not fully knocked down Ilk levels or that the Dim7-Elmo recruitment by Ilk is redundant with another attachment site protein. Alternatively, an upstream scaffold protein may function to recruit both the IPP and
Dim7-Elmo complex to the MASs. It is likely that these two complexes are temporally regulated in embryogenesis, where the actin remodeling complex is not needed until initial muscle-tendon initiation has been established (Liu, 2013).
The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. This study demonstrates that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. Phenotypic similarities and genetic interactions were observed among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. It is proposed that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner (Abe, 2014).
During brain development, neurons undergo multiple morphological changes to form an elaborate neural network. The Drosophila mushroom body (MB), which forms bilaterally symmetric and dorsomedially bifurcated axonal lobe structures in the central brain, has been well studied as a model of neuronal development. Among various regulators of neuronal morphogenesis, ADF/Cofilin and Rac GTPase (Rac) are key molecules in controlling cytoskeletal remodeling in axonal development. Cofilin [Twinstar (Tsr) in Drosophila] plays an essential role as a regulator of axonal growth by severing and depolymerizing F-actin. Because Cofilin is activated by dephosphorylation by the Slingshot (Ssh) phosphatase and is inactivated by phosphorylation by LIMK, the loss of Ssh or excessive activation of LIMK results in an axonal growth defect. In Drosophila, Rac has been proposed to act as a bidirectional switch for signaling cascades. One signaling event is the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth. The overexpression of Pak, a downstream effector of Rac, induces axonal growth defects similar to those observed with LIMK overexpression. In addition, introducing one mutant copy of Rac or Pak suppresses the axonal defect induced by LIMK overexpression. Another signaling event is the Pak-independent non-canonical pathway to positively regulate axonal growth. Rac mutant animals show multiple MB axonal defects, but the axonal growth defect is alleviated by the exogenous expression of Rac1Y40C, which lacks the ability to activate Pak but does not affect lamellipodia formation. Furthermore, Rac1Y40C overexpression remarkably suppressed the LIMK overexpression phenotype (Abe, 2014).
Although several pieces of evidence have suggested the importance of the non-canonical pathway and predicted the existence of its mediator, whether this pathway finally converges upon the downstream Cofilin pathway and subsequent F-actin reorganization remains unclear. Moreover, many biochemical studies have assessed the regulation of Cofilin function and F-actin states using in vitro systems; the endogenous changes in F-actin and Cofilin phosphorylation appear not to have been analyzed simultaneously with an internal control in developing brain. To address these issues, a novel factor was sought that interacts with Rac-Cofilin signaling components and positively regulates MB axonal growth. It was observed that Sickie, which has a calponin homology (CH) actin-binding domain and shares structural similarities with the human microtubule-associated protein (MAP) neuron navigator 2 (NAV2), showed prominent expression in F-actin-rich newborn MB axons and genetically interacted with Rac-Cofilin signaling regulators. Although Sickie was originally identified by genome-wide RNAi screening in Drosophila S2 cells and the report proposed the involvement of Sickie in the innate immune response (Foley, 2004), in this report focus was placed on the function of Sickie in the regulation of Cofilin-mediated F-actin remodeling and propose an expanded model of regulatory mechanisms during axonal development (Abe, 2014).
By combining the MARCM technique with epistatic analysis, this study demonstrated that Sickie regulates the axonal growth of Drosophila MB neurons via the non-canonical Rac-Cofilin pathway. The following model is proposed. In wild type, Sickie relays the non-canonical pathway signal to Ssh to facilitate F-actin-mediated axonal growth by counteracting the canonical signal. In a sickie mutant, mediation of the non-canonical pathway is defective, which causes an imbalance in the regulation of Cofilin activity. Because neurons are morphologically polarized and the amount of actin is limited in each cell, the growing axons may efficiently control actin recycling by facilitating F-actin turnover by balancing between the non-canonical and canonical pathways. Consistently, a stronger axonal growth defect was found with increased P-Cofilin in the LIMKWTM6 ssh1-63 and sickieΔ LIMKKD double-mutant animals than in the single mutants ssh1-63, sickieΔ and LIMKKD. Cofilin activity might be decreased in the developing axons of these double mutants by the preponderance of the canonical pathway. If so, these results highlight an essential role of the non-canonical pathway to balance Cofilin activity in axonal growth (Abe, 2014).
Unlike the clear elevation of P-Cofilin levels in the ssh1-63 mutant, constitutive activation of LIMK did not result in a similar increase in P-Cofilin despite F-actin elevation. This apparent paradox might be explained by considering the positive regulation of Ssh by F-actin. The phosphatase activity of SSH-1L is F-actin dependent, and the addition of F-actin dramatically increases its phosphatase activity. In the LIMKKD mutant axons, endogenous Ssh may be activated by a large amount of F-actin and subsequently dephosphorylates Cofilin. Consistently, highly elevated signals of both F-actin and P-Cofilin were detected in the LIMKWTM6 ssh1-63 double-mutant clones. In this mutant, Cofilin activity was severely reduced by high phosphorylation levels due to constitutive LIMK activation and a lack of Ssh phosphatase activity, resulting in the posterior arrest severe axonal defect, similar to the cofilin knockdown mutant. In addition, relatively moderate increases in P-Cofilin signal were detected in the developing axons of the sickieΔ LIMKKD double mutant. These results also support the model that Sickie functions in the same pathway as Ssh to positively regulate Cofilin function by counteracting the canonical Rac-Pak-LIMK pathway. Ssh might be downregulated in the sickie mutant axons due to defects in the mediation of Pak-independent Rac1 function or in the interaction among Ssh and F-actin by the loss of Sickie. The similar increases in the P-Cofilin and F-actin signals and the similar posterior arrest phenotype in the LIMKWTM6 ssh1-63 double-mutant clone and those of the PakMyr mutant clone are also consistent with results of in vitro studies that showed that SSH-1L is inactivated by Pak4. Thus, in the current model, Pak concurrently inactivates Ssh and activates LIMK in axonal growth (Abe, 2014).
Whereas ssh or cofilin mutants are embryonic lethal and their mutant clones display developmental defects in non-neuronal tissues, sickie mutants are not embryonic lethal, and conspicuous phenotypes are found only in the substructures of the central brain, such as MB and EB, implying that more elaborate mechanisms involving Sickie function are required for ensuring their proper development. Given that MB neurons exhibit a densely bundled axonal morphology, the growing MB axons might require Sickie to smoothly extend their neurites within the lobe core region by coordinating the dynamics of actin and microtubules (MTs). Sickie and human neuron navigator proteins (NAVs) have conserved EB1-binding motifs, and Sickie shows a genetic interaction with MT components. Double RNAi of sickie and EB1 or β-tubulin both resulted in synergistic increases in the axonal defects. In addition, a recent cell biological study demonstrated a functional link between Cofilin and MTs. Through its interaction with EB1, Sickie might act as a navigator for the plus-end of MTs to link to the F-actin complex and thereby ensure elaborate neuronal wiring. To further elucidate the signaling mechanism, the relationships with other components of the Ssh-dependent Cofilin pathway need to be studied. Recent studies have revealed that PKD, 14-3-3 protein and Pak4 play key roles in suppressing Ssh function (Abe, 2014).
Finally, preliminary data suggest a post-developmental role for Sickie. Adult stage-specific knockdown of Sickie in MBs impairs olfactory memory. Moreover, recent mammalian studies have suggested the possible involvement of NAVs and Cofilin in neurodegenerative disease. In Alzheimer's disease brains, the NAV3 transcript level is elevated, and Cofilin-actin rod-shaped inclusions, which are formed by the hyperactivation of Cofilin, are enriched. Further studies are required to understand the wide variety of contributions of sickie and the general importance of this evolutionarily conserved gene in brain development and function (Abe, 2014).
Similar to the Drosophila Egfr and to the mammalian PDGFR family, stimulation of PDGF- and VEGF-receptor related (Pvr) activates the MAP-kinase pathway in Schneider cells as well as in border cells. However, it has been shown, by loss-of-function and gain-of-function experiments, that MAP-kinase signaling does not affect border cell migration. In addition, no effect of phospholipase C-gamma (PLC-gamma) or phosphatidylinositol 3' kinase (PI3K) has been demonstrated on this migration, using loss-of-function mutants (PLC-gamma) or border cell expression of dominant negative and dominant activated forms (PI3K). This was somewhat unexpected, since PLC-gamma and PI3K have been implicated in motility and guidance effects of RTKs (in particular PDGFR) in tissue culture cells. To address how Pvr signaling might be affecting cell migration in vivo, the effect of Pvr signaling on cell morphology and cytoskeleton was tested. In border cells as well as in other follicle cells, expression of lambda-Pvr has a dramatic effect on the actin cytoskeleton. Massive F-actin accumulation, actin-rich extensions, and changes in cell shape were produced in lambda-Pvr expressing follicle cells. The normal cells have modest cortical F-actin accumulation. This result was likely to be relevant to the guidance function of Pvr, because direct control of F-actin accumulation would allow receptor activation to control cell migration (Duchek, 2001).
The actin cytoskeleton has been shown to be affected by small GTPases of the Rho superfamily in many systems, with the exact effects depending on the cellular context. In the border cell migration system, Rac is an attractive candidate for mediating the effect of activated Pvr, since dominant negative Rac (RacN17) has been shown to inhibit border cell migration. Epistasis experiments could not be done by quantifying border cell migration because activated Pvr and dominant negative Rac have the same effect. Instead, whether Rac is required for the effect of Pvr on the actin cytoskeleton in follicle cells was tested. Coexpression of dominant negative Rac suppresses the effect of activated Pvr on the actin cytoskeleton. In addition, follicle cells expressing activated Rac (RacV12) have dramatic accumulation of F-actin, resembling that caused by activated Pvr. Finally, if Rac were directly downstream of Pvr, one would expect activated Rac to inhibit border cell migration, as observed for the activated receptor. Although a previous study reported that activated Rac does not affect border cell migration (Murphy, 1996), this was reexamined using the slboGal4 driver and it was found that activated Rac completely blocks border cell migration. These results are consistent with a role of Rac in the guidance pathway downstream of Pvr (Duchek, 2001).
In mammalian tissue culture cells, PDGF stimulation can cause Rac-dependent F-actin accumulation, suggesting that the effect observed in follicle cells may reflect a conserved pathway. PI3K has been implicated as a mediator of the effect of PDGFR on Rac in Swiss 3T3 cells. However, PI3K does not appear to play a key role in guidance of border cell migration as discussed above. To investigate how Pvr might lead to activation of Rac, two groups of Drosophila mutants were tested for their effect on border cell migration: mutants in genes shown to be downstream of receptor tyrosine kinases in other contexts, and mutants linked to Rac activation. Most mutations were homozygous lethal, so their effect in border cells was tested by generating mutant clones in a heterozygous animal (mosaic analysis). Of the 8 genes tested, only myoblast city (mbc) has a detectable effect on border cell migration. Mbc is homologous to mammalian DOCK180 and C. elegans CED-5. Mbc/DOCK180/CED-5 acts as an activator of Rac (Duchek, 2001 and references therein).
mbc has been independently identified in a screen for gain-of-function suppressors of the slbo mutant phenotype. slbo mutant border cells migrate poorly. Increased expression of mbc in slbo mutant border cells improves their migration, suggesting that mbc has a positive role in promoting border cell migration. Mbc protein is detected in follicle cells, including border cells, and is overexpressed upon induction of the EP element EPg36390 located upstream of mbc. Removing mbc function from border cells by generating mutant clones causes severe delays in their migration. At stage 10, when 100% of control (GFP) clones have reached the oocyte, only 10% of mbc mutant border cell clusters have done so, and these are the oldest egg chambers. Thus, mbc is not absolutely required for border cell migration, but, contrary to the other genes implicated in RTK and Rac signaling, loss of mbc function severely impairs this cell migration (Duchek, 2001).
During collective migration, guidance receptors signal downstream to result in a polarized distribution of molecules, including cytoskeletal regulators and guidance receptors themselves, in response to an extracellular gradient of chemotactic factors. However, the underlying mechanism of asymmetry generation in the context of the migration of a group of cells is not well understood. Using border cells in the Drosophila ovary as a model system for collective migration, this study found that the receptor tyrosine kinase (RTK) PDGF/VEGF receptor (PVR) is required for a polarized distribution of recycling endosome and exocyst in the leading cells of the border cell cluster. Interestingly, PVR signaled through the small GTPase Rac to positively affect the levels of Rab11-labeled recycling endosomes, probably in an F-actin-dependent manner. Conversely, the exocyst complex component Sec3 was required for the asymmetric localization of RTK activity and F-actin, similar to that previously reported for the function of Rab11. Together, these results suggested a positive-feedback loop in border cells, in which RTKs such as PVR act to induce a higher level of vesicle recycling and tethering activity in the leading cells, which in turn enables RTK activity to be distributed in a more polarized fashion at the front. Evidence is also provided that E-cadherin, the major adhesion molecule for border cell migration, is a specific cargo in the Rab11-labeled recycling endosomes and that Sec3 is required for the delivery of the E-cadherin-containing vesicles to the membrane (Wan, 2013).
It has been proposed that repeated cycles of endocytosis of RTKs
(or active RTKs) and recycling of them back to the membrane
would effectively concentrate active RTK in the front of the
migrating border cells. However, if the levels of endocytic recycling
remain uniform in all the outer border cells during migration, a fast
amplification of RTK activity levels between front and back would
be difficult to achieve. This study shows that there is a polarized
endogenous distribution of the recycling endosome and exocyst in
the leading border cells within the migrating cluster, which could
conceivably make such amplification faster and more efficient in the
leading cells. It was also shown previously that Sec15-GFP has an
asymmetric localization at the front, when it is overexpressed in
border cells. Along their migrating route, the
border cells often tumble or rotate as a cluster, resulting in position
changes such as front cells becoming lateral and back cells and vice
versa. In such a scenario, a fast and robust
amplification process would be essential to relocalize active RTKs.
Indeed, this study found that overexpressing Sec3 or Rab11-GFP, but not
Sec5-GFP, in a single cell clone within a
mosaic border cell cluster significantly promotes the likelihood of
such a cell being positioned at the leading position, suggesting that this cell utilizes its increased
recycling and tethering to amplify and relocalize active RTKs faster
and more efficiently than other wild-type neighbor cells. The
difference in promoting effect from Sec3 and Sec5 is interesting,
suggesting that when overexpressed the Sec3 subunit is more able
to enhance the overall exocyst function than Sec5. This is consistent
with a Sec3 study in budding yeast, which shows that as a unique
subunit of exocyst Sec3 serves as a spatial landmark on the bud tip
to recruit a subcomplex (comprising seven subunits) of exocyst
containing all subunits but Sec3. Only when the subcomplex along with the associated vesicle
arrives at the bud tip, can Sec3 be joined with it to form a fully
functional tethering complex (Wan, 2013).
The next question is how the polarized distribution of recycling
and tethering activity is initiated in border cells. This study demonstrated
that this was likely to be induced by the guidance receptors in
response to the external gradient of guidance cues, as removing
guidance signaling by DN-PVR and DN-EGFR expression
abolished Rab11 and Sec5 polarized distribution, and DN-PVR
expression alone markedly reduced the polarization. These data
suggested the presence of a positive feedback loop of active
RTKs-endocytic recycling-active RTKs in border cells, as Rab11
and exocyst components (Sec3 and Sec15) were shown to be
conversely required for polarized pTyr or active RTK localization at
the front.
Interestingly, this study found that PVR signals downstream through Rac and
then polymerized actin to promote recycling endosome levels,
providing mechanistic details to this feedback loop.
Interestingly, it was recently shown that Rab11 interacts with Rac
and actin cytoskeleton regulator moesin during border cell migration. Furthermore, this study
found that strong Rab11
stainings were proximal to or partially overlapping with strong F-actin
staining in the leading edge of wild-type border cells and
around the ectopic F-actin regions in the λ-PVR, RacV12 or twinstar-
RNAi expressing follicle cells and border cells. F-actin appears to
be the direct cause rather than the effect of recycling endosome
accumulation, because manipulating its levels by Lat-A or twinstar RNAi
leads to either up- or downregulation of the levels of recycling
endosome. However, the possibility cannot be ruled out that Rac can
somehow act on recycling endosome-associated regulators directly
(independently of F-actin) to affect their function. It was previously
shown that actin polymerization is required for recycling of cargo
back to plasma membrane, possibly through F-actin serving as a
track for the movement of vesicles. However,
how F-actin induces recycling endosome formation and organization
is not clear and remains to be elucidated (Wan, 2013).
It was previously proposed that recycling of active RTKs needs
to be directional (toward the front) to achieve polarized RTK activity. If
active RTKs in the leading edge are endocytosed and then recycled
to new regions in the membrane, RTK activity would be delocalized.
What causes the recycling to be directed toward the front membrane
is not clear. The proposed feedback loop via F-actin suggests that
the active PVR (RTK) in the leading edge could locally induce
higher levels of recycling endosome through Rac and enhanced actin
polymerization (by Rac). As a result, the directional recycling could
be achieved with the localized actin filaments serving both as a
recycling endosome inducing agent and as tracks for movement of
vesicles (carrying active RTKs) toward the front membrane, which
prevents the active RTKs from being recycled to elsewhere and
becoming delocalized. Indeed, inhibiting actin polymerization in the
border cells by Lat-A treatment abolished both the polarized F-actin
and the elevated Rab11 stainings proximal to F-actin, which are
normally present in the leading edge of the wild-type cluster.
Lastly, this work also provides some insight into the kinds of
cargo that are recycled during border cell migration.
E-cadherin is a specific cargo. E-cadherin is the major adhesion
molecule required for border cell migration, whereas integrin plays only a minor role and is not required
in border cells (Wan, 2013).
These finds suggests that cycles of endocytosis and recycling of E-cadherin
could promote the dynamic assembly and disassembly of
E-cadherin-mediated adhesion on the substrate (nurse cell E-cadherin),
similar to how the turnover of integrin at the focal
adhesion is regulated by endocytic recycling in mammalian cells. Interestingly,
elevated intracellular E-cad stainings tended to be localized below the cell
membrane that juxtaposes nurse cell membrane, suggesting that E-cadherin is normally
delivered to or recycled back to this membrane region by Rab11 and
exocyst during adhesion and migration. Another important candidate
cargo to be determined is PVR. However, no significant colocalization was detected
between Rab11 with PVR or active PVR with the previously reported PVR or pPVR antibody. Therefore, the definitive role of PVR or active PVR as a cargo for recycling still awaits further
determination (Wan, 2013).
Cell motility is essential for embryonic development and physiological processes such as the immune response, but also contributes to pathological conditions such as tumor progression and inflammation. However, understanding of the mechanisms underlying migratory processes is incomplete. Drosophila border cells provide a powerful genetic model to identify the roles of genes that contribute to cell migration. Members of the Hedgehog signaling pathway were uncovered in two independent screens for interactions with the small GTPase Rac and the polarity protein Par-1 in border cell migration. Consistent with a role in migration, multiple Hh signaling components were enriched in the migratory border cells. Interference with Hh signaling by several different methods resulted in incomplete cell migration. Moreover, the polarized distribution of E-Cadherin and a marker of tyrosine kinase activity were altered when Hh signaling was disrupted. Conservation of Hh-Rac and Hh-Par-1 signaling was illustrated in the wing, in which Hh-dependent phenotypes were enhanced by loss of Rac or par-1. This study has identified a pathway by which Hh signaling connects to Rac and Par-1 in cell migration. These results further highlight the importance of modifier screens in the identification of new genes that function in developmental pathways (Geisbrecht, 2013).
A role for the Hh signaling pathway in collective migration of the border cells was
uncovered in two independent genetic screens. Previous genetic mosaic screens in border cells
identified a role for cos2 in polar cell differentiation, but had yet to
reveal a role for Hh signaling components in border cell migration. It has long been recognized
that alternative screening methods are advantageous in uncovering genes that may be required
earlier in development and/or for those genes with redundant functions. Both of these
explanations are supported by published literature and the data presented in this study. First, ectopic Hh
signaling, either by overexpression of hh itself or loss of the downstream components ptc, cos2,
or Pka-C1, produces early ovarian phenotypes that include oocyte mis-positioning and excess
polar cells. These events occur prior to border cell recruitment and migration and thus may
complicate analyses of Hh signaling in subsequent oogenic processes. This potential
issue was bypassed by inducing downregulation of the Hh pathway specifically in border cells just prior to their migration. Second, the migration defects due to loss of Hh pathway components appear to
be incompletely penetrant. Despite considerable reduction of Hh signaling due to overexpression
of ptc, most border cells were able to complete their migration. However, the significant
suppression of RacN17 motility defects by overexpression of Hh particularly indicates an
important functional role for this pathway in border cells. The data are thus consistent with other,
at present unknown, signaling pathways functioning in concert with Hh for proper cell migration (Geisbrecht, 2013).
The Hh pathway is capable of regulating a wide variety of cellular responses through
transcriptional regulation of downstream target genes. In most tissues, Hh is
secreted from a local source, but the downstream effects occur only in ptc-receiving cells that
may reside up to ten cell diameters away. In migrating border
cells, the results presented in this study suggest an autocrine mechanism where Hh is both produced and
received by the same cells. Both the hh-lacZ and multiple ptc-lacZ enhancer traps/reporters
reveal transcriptional activity in the outer, migratory cells of the cluster. Furthermore, this study has shown
that hh expression is regulated by JAK/STAT signaling and is independent of Slbo regulation. It remains a distinct possibility that the Hh signal is relayed between border cells within
the cluster. Nonetheless, the data favor a role for Hh specifically in border cells rather than
receiving Hh signal from other cells in the ovary. This idea is supported by findings that
migration was impaired when Hh and proteins required for its signal reception and transduction
were knocked down by RNAi selectively in the border cells using slbo-GAL4. Furthermore,
border cells mutant for disp did not complete their migration. As Disp is required in Hh-secreting cells for release of lipid-modified active Hh this further indicates that border
cells produce Hh signal. It is still unclear whether paracrine versus autocrine Hh signaling is
biologically important. However, a number of studies have reported roles for autocrine Hh
activity in the Drosophila wing disc and optic primordium in the embryo and the salivary gland in the larva, as well as autocrine Sonic hedgehog (Shh), a vertebrate Hh homolog, in neural stem cells, B-cell lymphoma, and interferon-stimulated cerebellar dysplasia during brain development. This type of Hh signaling mechanism also occurs in a variety of human tumors, where abnormal Hh pathway activation in an autocrine fashion increases cell proliferation and invasion (Geisbrecht, 2013).
A question raised by this study is what role the Hh signaling pathway plays in border cell
migration. As depicted in a proposed model, border cells with reduced Hh activation
exhibited altered localization of E-cad and depolarized p-Tyr, either of which could affect border cell motility. E-cad is required for border cell migration by promoting proper adhesion with the nurse cell substrate. Importantly, disruption of E-cad localization contributes to migration defects
caused by loss of steroid hormone signaling and cell polarity genes. Loss of one of the guidance ligands, pvf1, disrupts
E-cad localization in border cells similar to what was observed when Hh activity was impaired. Given that PVF1 signals to the receptor PVR on border cells, this
suggests a connection between Hh and RTK signaling. Indeed, wild-type border cells exhibit
polarized activation of the RTKs PVR and EGFR prior to migration as assayed by global
tyrosine phosphorylation and specific phosphorylation of PVR (Tyr-1428). Disruption of this polarized RTK by several mechanisms, as shown by
reduced or mislocalized p-Tyr, impairs border cell migration. The data suggest that Hh signaling restricts polarized p-Tyr to the
front of the border cell cluster. Interestingly, overexpression of vertebrate Shh in keratinocytes increased activation of EGFR and invasion through matrix. Moreover,
there is evidence for synergism between Hh and EGFR signaling to activate Gli (Ci homolog)
transcription targets in human cells. Nonetheless, proteins other than RTKs
(and/or their targets) can be phosphorylated on tyrosines and thus recognized by the p-Tyr
antibody; thus, the role for Hh signaling in border cells may be independent of the RTK
pathways. Regulators of endocytosis are also required for localized, high levels of p-Tyr in
border cells. However, in contrast to loss of hh, loss of
endocytic pathway members do not significantly impair E-cad levels or localization. Thus, Hh likely regulates border cell migration by a distinct mechanism. Further
experiments will be needed to determine if Hh signaling is required for the proper levels or
distribution of an unknown protein(s) that affects tyrosine phosphorylation and E-cad
localization during border cell migration (Geisbrecht, 2013).
The data presented in this study suggest a link between the Rac, Hh, and Par-1 signaling pathways.
However, understanding of how these proteins function together to modulate migration
remain a mystery. The results from the suppression screen indicate that overexpression of Hh
can overcome Rac-dependent migration defects. In fact, Hh was the strongest suppressor
obtained from the screen. A simple explanation for this observation is that Ci induces
transcription of one or more as yet unknown downstream target genes required in the migratory
process. However, the entire repertoire of Ci targets has yet to be elucidated and specific
targets (apart from ptc itself) in border cells are unknown. Interestingly, Rac2 was recently uncovered as a potential target of the Hh signaling pathway in the Drosophila embryo. Both Rac1 and Rac2 are essential for border cell migration. Thus, it is intriguing to speculate that upregulation of Rac2 by Hh is a possible
mechanism to overcome the RacN17 migration phenotype. Alternatively, the Hh pathway may
directly or indirectly affect regulation of Rac protein activity either by increasing the amount of active Rac-GTP or by regulating the subcellular localization of activated Rac within the border cell cluster. Hek 293 cells exposed to Shh had increased levels of the active form of the small GTPase RhoA. Similarly, Shh stimulated RhoA and Rac via
phosphoinositide 3-kinase (PI3K) signaling during chemotaxis of fibroblasts. Because border cells do not rely on PI3K activity, another mechanism is likely involved (Geisbrecht, 2013).
What is the connection between Par-1 and the Hh signaling pathway? One possibility is
the emerging requirement for microtubules in Hh signaling. Disruption of microtubules with the drug nocodazole prevents downstream transcriptional responses, possibly due to nuclear
translocation of Gli proteins. Furthermore, Cos2-mediated subcellular
motility and translocation of its cargo Ci requires microtubules in Drosophila. Par-1 is a central player in mediating microtubule polymerization and dynamics. More specifically, phosphorylation of microtubule-associated proteins (MAPs) by
the Par-1 kinase induces detachment of MAPs from microtubules. It is interesting to speculate
that the kinase activity of Par-1 is essential in the Hh pathway to regulate Cos2 or MAP proteins
for Ci mobility. Another possibility is that Par-1 functions through Rac. Overexpression of Par-1 partially suppressed the RacN17 migration defect, similar to overexpression of Hh. Although it is unclear why Par-1 rescued the Rac phenotype, it is possible that Par-1 acts in parallel to Hh signaling to promote Rac-mediated border cell motility. Notably, mammalian MARK2 (Par-1 homolog) promotes microtubule growth downstream of Rac1 at the leading edge of migrating cells. Further studies, however, are needed to determine the precise
molecular relationships amongst the Rac, Hh and Par-1 pathways in collectively migrating
border cells (Geisbrecht, 2013).
Cell shape changes are important for most aspects of morphogenic processes, including cell
contractility and cell migration. Hh signaling induces cell shape changes in the developing
Drosophila eye via regulation of non-muscle myosin II. Thus, the role of Hh in border cells may be to regulate cell shape during migration.
Accumulating evidence points to a non-canonical role for Hh in mediating mammalian cell
migration. Shh can function as a chemoattractant in migrating cells and guidance of axons
independent of Gli-induced gene transcription. In axon
guidance, Shh stimulates phosphorylation and activation of Src kinase and thereby facilitates
axon turning through regulation of the actin cytoskeleton. Specifically, Shh
induced phosphorylation of Src at Tyr-418, an activating site, and polarization of Src family
kinases within the axon. This appears to be consistent with the finding that loss of Hh activity depolarized global p-Tyr distribution. In other migratory cell types, Shh also acts as a chemoattractant that induces cytoskeletal rearrangements and migration independent of the canonical transcriptional respons. However, the mechanism of Hh function in border cell migration is likely to be different
for several reasons. First, Hh is unlikely to function as a long-range chemoattractant, because border cells are the likely source of Hh signal. Second, a role for Src in
border cell migration is unknown at present, so Hh may mediate tyrosine phosphorylation of
other substrates in border cells. Third, these is evidence that Ci is involved in border cell
migration and therefore canonical Hh-induced transcription is predicted to be important.
Nonetheless, the results are consistent with a conserved role for the Hh pathway in regulating cytoskeletal-mediated events in migrating cells, which in border cells likely functions through the Rac GTPase (Geisbrecht, 2013).
Adult stem cells are responsible for
maintaining the balance between cell proliferation and differentiation
within self-renewing tissues. The molecular and cellular mechanisms mediating
such balance are poorly understood. The production of reactive oxygen species
(ROS) has emerged as an important mediator of stem cell homeostasis in
various systems. Recent work demonstrates that Rac1-dependent ROS
production mediates intestinal stem cell (ISC) proliferation in mouse models of
colorectal cancer (CRC). This study used the adult Drosophila midgut and the
mouse small intestine to directly address the role of Rac1 in ISC proliferation and
tissue regeneration in response to damage. The results demonstrate that Rac1 is
necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent
manner. The data point to an evolutionarily conserved role of Rac1 in
intestinal homeostasis and highlight the value of combining work in the mammalian
and Drosophila intestine as paradigms to study stem cell biology (Myant, 2013b).
The epithelium of the posterior adult Drosophila midgut is replenished by ISCs. Each ISC proliferates to give rise to an uncommitted enteroblast (EB), which will differentiate into either an enterocyte (EC) or an enteroendocrine cell (ee). ISCs are the only proliferative cells within the adult fly posterior midgut (Myant, 2013b).
Recent work shows that deletion of Rac1 suppresses intestinal hyperproliferation and ROS production in Apc-deficient mice (Myant, 2013a). It was therefore first asked whether Rac1 is sufficient to drive ROS production within ISCs in the Drosophila midgut. The UAS/Gal4 system was used to specifically overexpress Drosophila Rac1 in ISCs/EBs (progenitor cells) using the temperature-controlled escargot-gal4, UAS-gfp; tubulin-gal80ts driver (esgts > gfp). Overexpression of Rac1 resulted in a dramatic expansion of the esg > gfp cell population and increased ROS production in the midgut. These results suggest that Rac1 overexpression in progenitor cells is sufficient to drive ROS production within the intestinal epithelium (Myant, 2013b).
The epithelium of the adult posterior Drosophila midgut has a remarkable regenerative capacity. Damage induced by agents such as bacterial infection, Bleomycin, or dextran sodium sulfate (DSS) treatment leads to activation of ISC proliferation to regenerate the damaged intestinal epithelium. Previous work demonstrated that ROS production is essential for damaged-induced ISC proliferation in the fly midgut (Buchon, 2009). It was therefore asked whether ROS upregulation was important for the phenotype resulting from Rac1 overexpression in the midgut. Consistent, with the previous report preventing ROS production by NAC impaired ISC proliferation in posterior midguts from flies infected with the pathogenic bacteria Pseudomonas entomophila (Pe). Importantly, NAC treatment strongly suppressed ISC hyperproliferation in Rac1-overexpressing midguts (). These results suggest that ROS production is essential for Rac1-dependent ISC hyperproliferation in the intestine (Myant, 2013b).
It was finally asked whether Rac1 was necessary to drive ISC proliferation in response to damage. This is a question, which also derives from previous work in the mammalian intestine. A genetic approach was used to knockdown Rac1 within progenitors cells of the Drosophila midgut by RNA interference (RNAi) (esgts > Rac1-IR). Knockdown of Rac1 by 2 independent RNAi lines resulted in almost complete suppression of ISC proliferation in regenerating posterior midguts subject to Pe infection. Similar to the Drosophila midgut, the mammalian intestine displays a remarkable regenerative capacity following damage. Therefore the conservation of the requirement for Rac1 during intestinal regeneration across these species was addressed. Rac1 was conditionally deleted from the mouse intestinal epithelium using the vil-Cre-ERT2, and the effect of Rac1 loss on tissue regeneration upon DNA damage was tested. Consistent with the results in the fly midgut, Rac1 deletion significantly suppressed regeneration in the mouse intestinal epithelium (Myant, 2013b).
Altogether, this work results suggest a central conserved role for the small GTPase RAC1 as a driver of ISC proliferation through the production of ROS. These data highlight RAC1 as key player and potential therapeutic target for conditions linked to oxidative stress such as cancer and aging (Myant, 2013b).
Reestablishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how postmitotic diploid cells contribute to repair. Puncture wounds to the adult Drosophila epidermis close initially by forming a melanized scab. Epithelial cells near the wound site fuse to form a giant syncytium, which sends lamellae under the scab to re-epithelialize the damaged site. Other large cells arise more peripherally by initiating endocycles and becoming polyploid, or by cell fusion. Rac GTPase activity is needed for syncytium formation, while the Hippo signaling effector Yorkie modulates both polyploidization and cell fusion. Large cell formation is functionally important because when both polyploidization and fusion are blocked, wounds do not re-epithelialize. These observations indicate that cell mass lost upon wounding can be replaced by polyploidization instead of mitotic proliferation. It is proposed that large cells generated by polyploidization or cell fusion are essential because they are better able than diploid cells to mechanically stabilize wounds, especially those containing permanent acellular structures, such as scar tissue (Losick, 2013).
Puncture wounds of the adult epithelium such as those in this study present multiple challenges. Biosynthetic capacity has been reduced by cell loss, the epithelial barrier has been
breached, and regional mechanical stability has been compromised
by irreversible muscle damage. It was found that the
epithelium employs two novel processes, polyploidization
and cell fusion rather than cell proliferation, to respond to
these challenges (Losick, 2013).
One function of polyploidization is to restore the tissue mass
destroyed during wounding. The number of nuclei induced to
leave quiescence in the adult abdomen and reenter S phase
correlates closely with the wound size. Furthermore,
the levels of epithelial polyploidy observed are sufficient
to generate approximately the same number of new genomes
as were initially lost. More severe cell losses in the abdomen
caused by repeated wounding induce higher levels of endoreplication. This correspondence even holds in the
case of the severe damage to the hindgut pyloric region.
A region of 300 cells (600 genomes)
was reduced to 100 cells following damage, but their ploidy
increased sufficiently to restore a total of 550 genomes, approximately the starting number. Thus, previously
quiescent diploid cells can sense the severity of tissue damage,
reenter the cell cycle, and endoreplicate to levels that
replace the lost cells (Losick, 2013).
During development, animals and their component organs
are able to precisely control their size. Following injury, as
after liver resection, cells proliferate until the normal organ size
is again attained. In many tissues, certain cell types complete
differentiation while undergoing endocycles; hence, mechanisms
to modulate endocycling as well as cell proliferation in
response to tissue size must exist. Indeed, during development, the ploidy
level of cells can increase beyond that normally attained to compensate
for overall reductions in cell number caused by mutation or damage, both in
Drosophila embryos, ovarian follicles, and rectum and in the mammalian liver. This study has extended the known versatility of endocycle regulation by showing that polyploidization can also be induced
in previously differentiated, quiescent diploid cells and then
terminated at an appropriate level as a repair response (Losick, 2013).
Another striking response to adult epidermal wounding is the
generation of a large syncytium by cell fusion. Barrier function
is transiently restored by scab formation, after which a continuous
epithelium must be regenerated. These studies suggest that
syncytium formation at the site of the scab accelerates this
process. When cell fusion was blocked by expressing RacN17,
wound re-epithelialization was significantly slowed. Previous
studies described cell fusion and syncytium formation at the
site of larval epidermal wounds. Larval epidermal cells facilitate
wound closure by sending lamellae under the scab using
actin treadmilling and myosin II-dependent crawling.
The giant syncytia that were studied contain five to ten times as
many cells as in these larval epidermal wounds but may utilize
many of the same processes to speed re-epithelialization.
The giant syncytium may provide several additional advantages.
Concentrating most nuclei at the periphery of the scar
may allow thin cytoplasmic lamellae to rapidly move under the
scab to seal the wound while the cell itself remains firmly fixed
at the wound periphery. Individual diploid cells would have to
migrate under the scab, a zone that is probably not conducive
to organized cellular movement due to the absence of a basement
membrane, polarity signals, and supporting muscle.
Even after the epithelium is restored, the large syncytial cell
may continue to function by stabilizing the scar, a large rigid
structure susceptible to motions that could damage the tissue
and reopen the wound (Losick, 2013).
How does the injured abdomen induce polyploidization and
cell fusion at the appropriate levels and locations? Both
JNK and Hippo signaling are upregulated at the wound
site, suggesting that these pathways regulate the wound
response. Hippo signaling, in conjunction with the TOR and
insulin/IGF pathways, plays an important role in organ size
control in both mammals and insects. Yki, the major
effector of Hippo signaling, is required for the polyploidization
response. Hippo signaling in response to wound damage
may activate cycE, a gene known to be regulated by Yki
in other tissues to stimulate S phase reentry. The modest
2- to 3-fold increase in the Yki-regulated genes expanded
and dIAP may reflect the relative mild nature of these
wounds, which only caused a 25% reduction in cell number
(233 of 913) within the wound region. Thus, Hippo signaling
via Yki may specify how much polyploidization ensues in
response to the magnitude of the wound as well as its spatial location (Losick, 2013).
In order to fulfill this role, Hippo signaling would have to be
activated locally within the tissue in proportion to the magnitude
of the damage. Changes in cell polarity, actin cytoskeletal
dynamics, and cell density can all induce the expression
of Yki-regulated genes. In addition, other signals may
play Hippo-independent roles in controlling abdominal wound
repair. Hemocytes are known to be recruited to the wound
site to help clear debris and microorganisms. Hemocytes are also recruited to adult abdominal wounds
and are present at the 24 hr time point, when cells are both
undergoing cell fusion and reentering S phase.
This suggests that blood cells could liberate factors that
facilitate either of these wound-healing processes. Another
possibility is DNA damage from genotoxic stress, such as
that produced from reactive oxygen species (ROS). ROS are
known to be released after injury to many tissues, and
these products can also induce the endocycle in plant cells. The syncytial and polyploid cells induced by wounding
might themselves send both local and long-range signals
that participate in sensing when organ size and stability have been restored (Losick, 2013).
These experiments provided insight into the distinct and overlapping
roles of polyploidy and cell fusion in the healing process.
Polyploidization appears to be solely responsible for replacing
the lost cells and restoring the tissue back to its initial mass.
However, the 25% reduction in synthetic capacity generated
by the wounds made in this study is probably too small to register
in assays when polyploidization alone is blocked. A 25%
reduction in the time required for re-epithelialization or in the
thickness of the lamella under the scar would not have been
detected. Despite this, a distinct function was detected for
polyploidization by analyzing its role in conjunction with that
of cell fusion (Losick, 2013).
Blocking cell fusion clearly perturbs wound healing, as an
extra day is now required to complete wound closure but healing
is not prevented. In the absence of fusion, polyploidization
still takes place. Indeed, the level of ploidy is
slightly increased under these conditions. However,
when polyploid cells cannot form and cell fusion is also
blocked, wounds usually fail to heal. Thus,
polyploid cells, which are located near the edge of the scab
and extend several cell diameters away, contribute something
critically important to wound repair in addition to restoring
cell mass, but this function is redundant when cell fusion can operate (Losick, 2013).
It is proposed that large cells, whether syncytial or polyploid in
origin, provide a unique mechanical function that helps organize
and control the healing process, and that cannot be provided
by surviving diploid cells. The large size of either type of
cell allows more robust cytoskeletal structures to form and
function than is possible in diploid cells. This is likely the
reason that muscle cells fuse into large syncytia prior to organizing
myofibrils. However, other large cytoplasmic mechanical
structures are present and function in many other types
of polyploid cells that are less familiar. For example, megakaryocytes,
which extrude segments of their cytoplasm as platelets,
contain long branching β1-tubulin-based processes that
are required for platelet release. Polyploid jump reflex neurons
in Drosophila produce exceptionally long and thick axons
that allow signals to be transmitted with great speed.
Trophoblast giant cells accumulate stress fibers and specialized
podosomes, which may structurally support placenta development (Losick, 2013).
Mechanical tension is already known to play a critical role in
mammalian epithelial wound healing. Cells tend to migrate
toward regions of higher ECM rigidity ('durotaxis'). Large
cells may be necessary at the wound site to generate an
appropriate mechanical environment for migrating cells to
complete their movements under the scar and close the
wound. In the abdomen, the transverse muscle bands that
normally span the abdomen did not undergo repair. Large cells
may also be particularly advantageous for dealing with mechanical
stability issues that require balancing forces over a
substantial area due to the size of the damaged region and
the presence of altered structures such as scar tissue. The
central syncytium may be advantageous not only in rapidly
closing the wound but also because a large cell can better
stabilize the scar and prevents it from breaking loose. The
enlarged peripheral cells, whether polyploid or syncytial,
may more easily generate stabilizing forces to protect the
wounded region during the normal flexure and stress on the
abdomen (Losick, 2013).
These same considerations would apply equally to mammalian
as well as Drosophila tissue. In many damaged mammalian
tissues, extracellular matrix deposits of fibrin and collagen
initially form a fibrin clot/scab to hold edges of the wound
together, but collagen protein deposits can persist in a lasting
mark at the injury site in the form of a scar. In tissues where
cell proliferation is limited, such as the heart, scar formation is
necessary to maintain tissue integrity but also leads to stiffness
and reduced heart function. Polyploidization may
frequently take place in response to mammalian tissue damage
that repairs imperfectly and leaves scar tissue, such as
in the heart, but this has received little attention. Cardiomyocytes
reenter the cell cycle after injury, leading to a low level
of cell division as well as polyploidy and multinucleation at
the scar periphery. Consequently, the establishment
of a model system for studying the control of polyploidization
and syncytium formation in response to wounding is likely to
provide insight to questions of wide significance (Losick, 2013).
Actin and microtubule cytoskeletons have overlapping, but distinct roles in the
morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin
and microtubule cytoskeletons appears to be required for the growth of wing hairs, since treatment of
cultured pupal wings with either cytochalasin D or vinblastine is able to slow prehair extension. At
higher doses, a complete blockage of hair development is seen. The microtubule cytoskeleton is also
required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule
cytoskeleton results in the development of multiple prehairs along the apical cell periphery. The
multiple hair cells are a phenocopy of mutations in the inturned group of tissue polarity genes, which
are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton
also plays a role in maintaining prehair integrity during prehair development, since treatment of pupal wings
with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy
of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode
products that interact with the actin cytoskeleton. Several other mutant genotypes produce branched or split bristles or hairs. For example, mutations in singed, chickadee and capping protein produce bristles and/or hairs that are split, bent or stunted in ways that partially resemble cytochalasin D treatment. However, the phenotypes associated with these mutations do not resemble those seen with CD treatment as closely as the phenotype associated with crinkled (e.g. there is not hair splitting in sn mutants). The recent finding that mutations in the small G-protein rho result in an inturned-like phenotype and that the expression of a dominant negative form of rac also results in multiple hair cell phenotype is interesting with regard to the interaction of the actin and microtubule cytoskeletons. Small G-proteins of the rho and rac families are thought to interact with the actin cytoskeleton, yet they produce a wing hair phenotype that is similar to what is seen with the disruption of the microtubule cytoskeleton. This could be due to both the small G-proteins and the micotubule cytoskeleton being required for localizing a common component or activity to the vicinity of the distal vertex, or to the small G-proteins affecting the structure of the microtubule cytoskeleton, or to the microtubular cytoskeleton functioning in the localization of the small G-proteins or, alternatively, these two classes of proteins could be functioning in parallel pathways that function independently to restrict prehair initiation to the distal region of the cell. The observation that the expression of a dominant negative form of rac1 causes a disruption of the microtubule array suggests the possibliity that the phenotypes associate with G-protein loss could be due to their disrupting the structure/function of the microtubule cytoskeleton and not to their being part of the frizzled signaling/signal transduction pathway (Turner, 1998).
Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the
two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial
planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling.
Dishevelled is required in both signaling pathways. Genetic and overexpression assays have been used to
show that Dishevelled activates JNK cascades. In contrast to the action of wingless-pathway components, mutations in rhoA, hemipterous, basket, and jun as well as deficiencies removing the Rac1 and Rac2 genes show a strong dominant suppression of a Dishevelled overexpression phenotype in the compound eye. In an in vitro assay, expression of Dsh has been shown to induce phosphorylation of Jun, indicating that Dsh is a potent activator of the JNK pathway. Whereas the PDZ domain of Dsh, known to be required in the transduction of the wingless signal, is dispensable for signal-independent induction of Jun phosphorylation, the C-terminal DEP domain of Dsh is found to be essential. The planar polarity-specific dsh1 allele is found to be mutated
in the DEP domain. These results indicate that different Wnt/Fz signals activate distinct intracellular
pathways, and Dishevelled discriminates among them by distinct domain interactions (Boutros, 1998).
How can Fz/Dsh signaling be linked to small GTPase and JNK/MAPK pathways? Recent studies provided evidence
that links G protein-coupled receptors, which share structural features with Fz proteins, to MAPK signaling through
heterotrimeric G proteins and PI-3 kinases. It is intriguing to speculate that a subset of Fz proteins might signal through a
similar pathway. It was also shown recently that XWnt5A and rFz2, in a heterologous assay, increase intracellular
calcium via G proteins and phosphoinositol signaling. A mutation in the beta-subunit of a heterotrimeric G protein in C. elegans prevents correct spindle orientation, a process that is believed to be dependent on a Wnt and a Fz receptor, but not on Arm. Further studies regarding a possible involvement of PI-3K and G proteins in planar
polarity signaling may provide additional insight to the diversity of Fz-related signaling pathways (Boutros, 1998 and references).
Cdc42 and Rac1 contribute differently to the organization of epithelial cells in the Drosophila wing imaginal
disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin
assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the
other hand, is required for processes that involve polarized cell shape changes during both pupal and
larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation.
Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar,
cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped.
Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates
elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These
observations suggest that a common Cdc42-based mechanism may govern polarized cell shape changes
in a wide variety of cell types (Eaton, 1995).
The wing of Drosophila is covered by an array of distally pointing hairs. A hair begins as
a single membrane outgrowth from each wing epithelial cell, and its distal orientation is determined by
the restriction of outgrowth to a single distal site on the cell circumference. The roles of Cdc42 and Rac1 were examined in the formation of
wing hairs. Cdc42 is required for localized actin polymerization in the extending hair.
Interfering with Cdc42 activity by expression of a dominant negative protein abolishes both localized
actin polymerization and hair outgrowth. In contrast, Rac1 is important for restricting the site at which
hairs grow out. Cells expressing the dominant negative Rac1N17 fail to restrict outgrowth to a single
site and give rise to multiple wing hairs. This polarity defect is associated with disturbances in the
organization of junctional actin and also with disruption of an intricate microtubule network that is
intimately associated with the junctional region. Apical junctions and microtubules are also
involved in structural aspects of hair outgrowth. During hair formation, the apical microtubules that
point distally elongate and fill the emerging wing hair. As the hair elongates, junctional proteins are
reorganized on the proximal and distal edges of each cell (Eaton, 1996).
The Drosophila myoblast city (mbc) locus was previously identified on the basis of a defect in myoblast
fusion. The mbc transcript and its encoded protein are expressed in a broad range of tissues,
including somatic myoblasts, cardial cells, and visceral mesoderm. The gene is also expressed in the pole cells
and in ectodermally derived tissues, including the epidermis. Consistent with this latter expression, mbc
mutant embryos exhibit defects in dorsal closure and cytoskeletal organization in the migrating
epidermis. Both the mesodermal and ectodermal defects are reminiscent of those induced by altered
forms of Drac1 and suggest that mbc may function in the same pathway. MBC bears striking
homology to human DOCK180, which interacts with the SH2-SH3 adapter protein Crk and may play a
role in signal transduction from focal adhesions. Taken together, these results suggest the possibility
that MBC is an intermediate in a signal transduction pathway from the rho/rac family of GTPases to
events in the cytoskeleton and that this pathway may be used during myoblast fusion and dorsal
closure (Erickson, 1997).
mbc mutants have been identified as suppressors of a rac1 overexpression phenotype. To determine whether Rac and Cdc42 GTPases can disrupt eye
development, transgenic flies were generated in which wild-type Rac1,
Rac2, and Cdc42 GTPases were expressed in the developing eye under the
control of the synthetic glass multimer reporter
(GMR). Flies
harboring a single copy of the rac1 transgene exhibit an
externally rough eye, and retinal
sections reveal a loss of pigment cells and a disruption of the
normal ommatidial morphology, with occasional loss of photoreceptors. With two copies of the GMR-rac1 transgene,
a complete disruption of normal eye structure is observed; a similar
phenotype is observed in GMR-rac2 transgenic flies.
The GMR-cdc42 transgenic flies exhibit externally rough eyes
distinct from those seen in the rac1 and rac2
transgenics. Retinal sectioning reveals missing
photoreceptors and a disruption of ommatidial morphology.
Although the cdc42-induced eye phenotype somewhat resembles
the GMR-rho1 phenotype, the
cdc42 transgenics also exhibit an abnormal rhabdomere morphology. The postmitotic elongation event that
establishes the depth of the retina was also examined. Overexpression of either rho1 or cdc42, but not
rac1, disrupts the normal elongation of all retinal cells. Thus, each of these members of the Rho GTPase family, when
overexpressed, induces distinct alterations of normal eye development (Nolan, 1998).
To identify specific components of a Rac1 signaling pathway in
Drosophila, rac1 transgenic flies were used to screen
for dominant mutations that specifically suppress the
rac1-induced rough eye phenotype but not that caused by
GMR-cdc42 or GMR-rho1. Chromosomal deficiences that cover either rac1 or
rac2 each suppress the GMR-rac1 eye defect,
confirming that the phenotype is sensitive to the levels of endogenous
Rac activity and that Rac1 and Rac2 are normally expressed during eye
development. To identify rac1-suppressing
mutations, mutagenized wild-type males were mated with
GMR-rac1 females and the resulting F1 progeny were
examined for suppression of the rough eye phenotype. A total of 23,000 F1 flies were screened, and 36 dominantly suppressing mutations were identified. Three complementation groups were
established on the basis of lethality, and a single complementation
group of 11 alleles has been termed Suppressor of rac1
[Su(rac)1]. Each of the Su(rac)1 alleles
dominantly suppresses the GMR-rac1-induced rough eye surface as well as the underlying retinal morphology, rescuing the percentage of normal appearing ommatidia from 3% in GMR-rac flies to 97% in
GMR-rac1/Su(rac)1 flies. Each of these alleles
also suppresses the GMR-rac2-mediated defect, although none suppresses a GMR-rho1 phenotype. These data suggest that
Su(rac)1 encodes a specific component of a Rac-mediated
signaling pathway (Nolan, 1998).
Because a specific requirement for Rac activity, but not that of
Cdc42, has been demonstrated in the fusion of myoblasts during muscle
development (Luo, 1994), the musculature of
Su(rac)1 mutants was examined. Myoblast fusion is normally completed by stage 15; however, in
stage 15 Su(rac)1 mutants, myoblasts are largely unfused. Meiotic mapping localized Su(rac)1 alleles to a
chromosomal region similar to that of a previously reported gene,
mbc, that is also associated with a loss-of-function myoblast
fusion defect. Null alleles of mbc fail to complement the
lethality and myoblast fusion phenotype of several alleles of
Su(rac)1. Moreover, mbc alleles also suppress the
GMR-rac1 phenotype. Together, these results
indicate that Su(rac)1 is allelic to mbc.
Although the role of Rac in myoblast fusion is unknown, these results
suggest the Mbc mediates the activity of Rac in this morphogenetic
process in which actin rearrangements have been implicated previously (Nolan, 1998).
Other phenotypes were examined that would be consistent with
aberrant Rac signaling. Drosophila Rac1 has been implicated in
axonal outgrowth (Luo, 1994), and mbc
mutants exhibit a low penetrance defect in the fasciculation of axons of the ventral nerve cord neurons. Specifically, some
mbc mutant embryos exhibit improper spacing between
commissures and, in extreme cases, a lack of fasciculation of the
longitudinal connectives, possibly because of abnormal migration of the
central nervous system (CNS) neurons across the ventral surface. In
support of a role for Mbc in cell migration is the recent observation that mutations in ced-5, the C. elegans homolog of
mbc, result in defective migration of the distal tip cells of
the gonad. Additionally, mutations in
mig-2, a C. elegans gene encoding a Rac-like GTPase,
also affect distal tip cell migration and axon outgrowth. Moreover, the mammalian Rac GTPase appears to regulate the
motility of cultured fibroblasts. It is possible
that a pathway mediated by both Rac and Mbc regulates neuronal
migration and axon growth, and may explain the CNS defects observed in
mbc mutant embryos (Nolan, 1998 and references).
Cell transfection and in vitro nucleotide exchange assays with each DH domain of human Trio have suggested that GEF1 preferentially activates Rac, whereas GEF2 activates Rho (Debant, 1996; Bellanger, 1998a). To explore the relationship between trio and Rac in Drosophila, the compound eye was used as an established system to test for genetic interactions in GTPase signaling pathways. Rac overexpression under the control of the eye-specific promoter GMR creates a mispatterened 'rough' eye in which individual ommatidia are misshapen. However, removal of a single copy of trio causes a dramatic suppression of this Rac gain-of-function phenotype. This is true for additional trio alleles. In contrast, overexpression of Rho also generates a rough eye, but this phenotype is not significantly altered by reduction in trio activity. This suggests that in the Drosophila retina, trio functions to activate one or more of the Drosophila Rac-like genes but not Rho (Bateman, 2000).
In the embryo, previous studies have shown that the same nerve branches affected by trio mutations are also most sensitive to Rac perturbation. Although occasional ISNb stop short phenotypes are observed, the predominant ISNb and SNa bypass phenotypes induced by Drac1N17 overexpression are distinct from phenotypes caused by loss of trio function. This difference likely reflects Drac1N17 interference with multiple neural activators of Rac GTPases. However, it was reasoned that if trio is involved in Rac activation in the embryonic motor nervous system, the penetrance of the Drac1N17 phenotype should be sensitive to changes in the genetic dose of trio. Consistent with this hypothesis, removal of a single copy of trio in embryos expressing Drac1N17 causes a distinct increase in the penetrance of ISNb bypass. This was true for all alleles of trio tested. Moreover, coexpression of Drac1N17 and a wild-type trio transgene results in a dramatic suppression of the ISNb bypass phenotype, consistent with the model that trio is an activator of Rac GTPases in the embryonic motor nervous system (Bateman, 2000).
Despite phenotypic differences between Drac1N17 and trio in the motor nervous system, analysis of the CNS in embryos lacking Rac function reveals defects identical to those observed in trio mutants. Specifically, expression of the dominant-negative Drac1N17, under the control of the neural-specific GAL4 driver C155, causes a failure of the lateralmost Fas II-positive longitudinal pathway to properly connect at stage 17 (13.7%). In contrast, neural expression of either Dcdc42N17 or DRho1N19 does not cause defects in longitudinal pathfinding, indicating that the CNS phenotype is specific to interference with Rac-like GTPase function. Thus, in the CNS, mutant phenotypes of Drac1N17 and trio are consistent with disruption of a common pathway (Bateman, 2000).
Rhodopsin is essential for photoreceptor morphogenesis; photoreceptors lacking rhodopsin degenerate in humans, mice, and Drosophila. Transgenic expression of a dominant-active Drosophila Rho guanosine triphosphatase, Rac1, rescues photoreceptor morphogenesis in rhodopsin-null mutants; expression of dominant-negative Rac1 results in a phenotype similar to that seen in rhodopsin-null mutants. Rac1 is localized in a specialization of the photoreceptor cortical actin cytoskeleton, which is lost in rhodopsin-null mutants. Thus, rhodopsin appears to organize the actin cytoskeleton through Rac1, contributing a structural support essential for photoreceptor morphogenesis (Chang, 2000).
Sensory neurons present a challenge for morphogenesis: to harness the generic mechanisms of the cytoskeleton to shape a cell to the needs of its specific sensory protein. For photoreceptors, it is clear that morphogenesis and maintenance of the photosensitive organelle (in Drosophila, the rhabdomeres; in vetrebrates, the outer segments rods and cones) depends on the organelle's sensory protein: rhodopsin. Rhabdomeres and outer segments are orderly stacks of photosensitive plasma membrane organized from enormously expanded apical cell surfaces. The forces that constrain this expansion and organize it into a dense stack are incompletely understood, but the cortical actin cytoskeleton and its associated proteins are substantial contributors. It has been suggested that in addition to its sensory role, Drosophila rhodopsin organizes the cortical actin cytoskeleton into an essential morphogenetic constraint: the rhabdomere terminal web (RTW). The RTW defines the regular, curving base of the rhabdomere that partitions the rhabdomere from the photoreceptor cytoplasm. In rhodopsin-null mutants, the rhabdomere base fails to organize correctly, and the rhabdomere collapses deep into the photoreceptor cytoplasm in convoluted sheets of apposed membrane (Chang, 2000 and references therein).
A chimeric protein that decorates F-actin with green fluorescent protein (GFP) reveals the RTW as bundled microfilaments extending from the rhabdomere base deep into the photoreceptor. Before rhodopsin expression, the RTW of developing photoreceptors shows less microfilament bundling, resembling a house painter's brush. At about 90% of pupal development (pd), after the onset of rhodopsin expression at 75% pd, RTW microfilaments elongate commensurate with the increasing microvillar length and gathered into bundles (Chang, 2000).
RTW maturation and rhabdomere morphogenesis fail in photoreceptors lacking rhodopsin. Paralleling the normal initiation of microvillar organization observed in rhodopsin-null mutants, the RTW of mutant photoreceptors appears normal before the time when rhodopsin expression would normally commence. The RTW growth and bundling that normally follow rhodopsin expression fail in rhodopsin-null photoreceptors. Unlike wild-type rhabdomeres, the smaller, flattened rhabdomeres formed in the rhodopsin-null mutant collapse into the photoreceptor cytoplasm in convoluted sheets of apposed membrane during the first day after eclosion. The actin cytoskeleton becomes thoroughly disorganized in the absence of rhodopsin (Chang, 2000).
Although rhodopsin contributes about 50% of rhabdomere membrane protein, it is unlikely to support morphogenesis by a simple mass effect. Smaller, but ultrastructurally normal rhabdomeres form in mutants in which Rh1 is reduced by over 99%. Furthermore, a pulse of rhodopsin expression restricted to a narrow window of development is sufficient to rescue rhabdomere morphogenesis in photoreceptors otherwise lacking rhodopsin. It is proposed that an additional role for rhodopsin is to contribute an activity required to organize the RTW into an effective subapical barrier (Chang, 2000).
Drosophila Rac1, localizes to the rhabdomere base beginning with the onset of microvillar organization during midpupal development; it remaines subapical in adult eyes. To explore potential Rac1 functions in rhabdomere morphogenesis, dominant-negative N17Rac1 was expressed at defined stages of eye development. N17Rac1 expression during rhabdomere morphogenesis leads to reduced, disordered rhabdomeres. Fewer microvilli are seen in cross section, and a well-defined rhabdomere base is not formed; apposed sheets of rhabdomere membrane involute into the photoreceptor cytoplasm. Although these defects are reminiscent of those seen in rhodopsin-null mutants, the phenotype is not a consequence of a failure of rhodopsin delivery to the rhabdomeres. The actin cytoskeleton, however, appears diffuse and disordered as a result of transgene expression (Chang, 2000).
The resemblance of the rhabdomere base defects caused by N17Rac1 to those seen in rhodopsin-null mutants suggests that rhodopsin might exert its structural effect through Rac1. If so, it was reasoned that expression of constitutively active V12Rac1 might rescue rhabdomere morphogenesis in photoreceptors lacking rhodopsin. To test this idea, V12Rac1 was expressed during rhabdomere morphogenesis in ninaEI17 mutants that lack rhodopsin in photoreceptors R1 to R6. Substantial rescue of rhabdomere morphogenesis was observed. Occasional loops of rhabdomere membrane intrude into the photoreceptor, but most terminate at a well-defined base. The RTW is more tightly organized in V12Rac1-expressing animals. Similar to rhodopsin-null rhabdomeres rescued by a pulse of rhodopsin expression, V12Rac1-expressing animals show substantial rescue 5 days after eclosion. Thus, V12Rac1 appears to supply a durable organizing activity lost in rhodopsin-null mutants (Chang, 2000).
Similar to the requirement of small amounts of rhodopsin for normal morphogenesis, rescue appears quite sensitive to Rac1V12. About 18% of R1 to R6 rhabdomeres are rescued in non-heat-shocked hsGAL/SM1;UAS-Rac1V12 ninaEI17 eyes, rising to 90% in animals heat-shocked at 80% pd. Substantial rhabdomeres and tighter organization of the RTW are evident. To examine rescue specificity among Rho small GTPases, constitutively active V12Cdc42 and V14Rho were overexpressed in ninaE-null mutants. V12Cdc42 rescues rhodopsin-null morphogenesis, but V14Rho does not. Neither Rho nor Cdc42 immunolocalizes to the RTW of normal flies, but Cdc42 has been found to activate Rac in other systems and may do so here (Chang, 2000).
The observations reported here suggest that Rac1 links rhodopsin to photoreceptor morphogenesis: Targeted delivery of rhodopsin to the developing rhabdomere promotes localized Rac1 activity that, in turn, orchestrates assembly of the RTW. In rhodopsin-null photoreceptors, failure to correctly organize the RTW, likely including a failure of microfilament cross linking, would allow sheets of self-adhesive rhabdomere membrane to intrude unopposed into the photoreceptor cytoplasm. How rhodopsin contributes to Rac1 activity, as well as its downstream effectors, remains to be determined. Two attractive effector candidates are nonmuscle myosin II and moesin, which localize to the base of the developing rhabdomere and which, in other systems, lie downstream of small GTPases (Chang, 2000).
An actin barrier may also shape vertebrate photoreceptors, constraining newly added photosensitive membrane to the outer segment. Actin and actin-associated proteins localize to the site of outer segment disc membrane evagination, and nascent outer segment disc membrane intrudes into the cytoplasm of rabbit photoreceptors exposed to cytochalasin D. Given the several parallels between vertebrate and Drosophila retinal development and the highly conserved mechanisms of the cytoskeleton, it is interesting to speculate that vertebrate rhodopsin may also regulate the photoreceptor cytoskeleton. It is possible that some mutant rhodopsins, including those causing human retinitis pigmentosa, may result in photoreceptor degeneration because of an inability to correctly organize the actin cytoskeleton (Chang, 2000).
Putative constitutively active and dominant-negative Drac1 proteins were expressed in PNS tissues. In wild-type embryos, each segment contains three highly stereotyped clusters of PNS neurons connected by axon bundles. When expressed in neurons, Drac1 dominant negative mutant proteins cause axon outgrowth defects in
peripheral neurons without affecting dendrites. Loss of axons between the dorsal and lateral clusters were observed. In addition to defects in axonal initiation Rac1 mutation also causes defects in axon elongation. Dominant negative Rac1 also causes abnormal neuronal accumulation of filamentous actin. When expressed in muscle precursors, altered Rac proteins cause
complete failure of, or abnormality in, myoblast fusion. Expressions of analogous mutant Dcdc42
proteins cause qualitatively distinct morphological defects, suggesting that similar GTPases in the same
subfamily have unique roles in morphogenesis (Luo, 1994).
Previous genetic studies of intersegmental nerve b (ISNb) development have identified several
cell-surface proteins required for correct axon guidance to appropriate target muscles. Of all the proteins currently known to control ISNb guidance, Drac1 and Dlar are most likely to mediate target entry as opposed to defasciculation, because both display a parallel bypass phenotype. The small GTPase Drac1 plays a key role in this guidance process. Neuronal expression of the dominant negative mutation Drac1(N17) causes axons
to bypass and extend beyond normal synaptic partners. GTPase mutations were placed under the control of the yeast transcriptional activator GAL4. Combination of a neuronal GAL4 'driver' with a GTPase cDNA 'reporter' under the control of the GAL4 upstream activator sequence (UAS) results in specific cDNA expression. Neuronal-specific expression of either Drac1(V12) or Dcdc42(V12) causes ISNb motor growth cones to arrest outgrowth just as axons begin to explore the periphery. This highly penetrant motor phenotype is analogous to the growth cone arrest observed in sensory neurons when they express the same GTPase mutations. SNa axons also require Rac to reach correct targets. The growth cone arrest seen in the V12 backgrounds suggests that hyperactivation of different GTPase pathways disrupts leading edge motility. This phenotype is consistently
reproduced by pharmacological blockade of actin assembly, carried out by cytochalasin D treatment. Genetic interactions between
Drac1(N17) and the receptor-tyrosine phosphatase Dlar suggest that ISNb
guidance requires the integration of multiple, convergent signals. Double mutant Drac1(N17-Dlar compound mutants display a penetrance two- to three-fold higher than would be expected if the defects were simply additive. Thus, Rac function in ISNb axons is quite sensitive to the dosage of Dlar protein. This synergistic genetic interaction suggests that Rac and Dlar function together, though not in a simple linear pathway. The existence of multiple inputs during target entry may explain why null mutations in Dlar (and other choice point genes) are not completely penetrant on their own (Kaufmann, 1998)
Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. The guanine nucleotide exchange factor (GEF) Trio has been identified as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. It is proposed that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension (Newsome, 2000).
The small GTPases Rac and Rho act as cellular switches in many important biological
processes. In the fruit fly Drosophila, RhoA participates in the establishment of planar polarity, a process mediated by the receptor Frizzled (Fz). Thus far, analysis of Rac in this process has not been possible because of the absence of mutant Rac alleles. The roles of Rac and Rho in establishing the polarity of ommatidia in the Drosophila eye have been investigated. By expressing a dominant negative or a constitutively activated form of Rac1, Rac signaling is interfered with specifically and ommatidial polarity is disrupted. The resulting defects are similar to the loss/gain-of-function phenotypes typical of tissue-polarity genes. Through genetic interaction and rescue experiments involving a polarity-specific, loss-of-function dishevelled (dsh) allele, Rac1 has been found to act downstream of Dsh in the Fz signaling pathway, but upstream of, or in parallel to, RhoA. Rac signals to the nucleus through the Jun N-terminal kinase (JNK) cascade in this process. By generating point mutations in the effector loop of RhoA, it has been found that RhoA also signals to the nucleus during the establishment of ommatidial polarity. Nevertheless, Rac and RhoA activate transcription of distinct target genes. Thus Rac is specifically required downstream of Dsh in the Fz pathway. It functions upstream or
in parallel to RhoA and both signal to the nucleus, through distinct effectors, to establish planar polarity in the Drosophila eye (Fanto, 2000).
Random mutagenesis of activated mammalian RhoV14 has led to the identification of mutations in the effector loop (a portion of the GTPase responsible for interaction with several effectors) that block either its action on cytoskeletal dynamics or on transcriptional activation of SRF. The F39V mutation impedes the formation of actin stress fibers but does not interfere with the activation of SRF-mediated transcription, separating the two effects of RhoV14. The mutation E40L interferes with both SRF activation and the formation of stress fibers (Fanto, 2000).
The relevant mutations were recapitulated in the activated Drosophila RhoV14 protein and they were expressed under the control of sev-gal4 in the eye disc (sev;RhoV14 F39V and sev;RhoV14 E40L). The sev;RhoV14 F39V flies display a phenotype that is indistinguishable from that of sev;rhoV14 alone, with loss of photoreceptors and misorientation of otherwise wild-type clusters. This was evident even when the transgene was expressed at lower levels. Increasing the expression levels of sev;RhoV14 F39V (two copies) led to an enhancement of both the polarity and the photoreceptor recruitment phenotypes. In contrast, sev;RhoV14 E40L flies never displayed polarity defects, both when the transgene was expressed at low and at very high levels. Nevertheless, this mutant maintained the ability of sev;RhoV14 to cause photoreceptor loss: although a large number of ommatidia had lost several photoreceptors, all the remaining ommatidia with wild-type complement had the correct polarity. This indicates that removing the function required for nuclear signaling (equivalent to SRF activation in cell culture) eliminated the ability of sev;RhoV14 to induce polarity defects, suggesting that nuclear signaling by RhoA is critical for ommatidial polarity determination (Fanto, 2000).
To better characterize nuclear signaling by Rac and RhoA, the expression of puckered (puc) and Delta (Dl) were studied. Dl is the only known transcriptional target of Fz signaling in R3, and puc-lacZ expression serves as a measure of JNK activity in vivo. The puc gene is a transcriptional target of JNK signaling in Drosophila, and encodes a dual specificity protein phosphatase that acts as a negative regulator of JNK itself in a feedback loop. In the wild type, very weak beta-galactosidase expression from the puc enhancer trap line is detectable in all photoreceptor precursors. Expression of sev;racV12 lead to strong upregulation of puc-lacz in one or, more frequently, two cells of the cluster, identified as R3/R4 precursor cells, consistent with the expression pattern of sev;RacV12. These data resemble the upregulation of puc-lacz when the JNK pathway has been activated in the same cells (Fanto, 2000).
In contrast, RhoV14 affects puc-lacz expression differently. Although in sev;RhoV14 eye discs puc-lacz expression is upregulated in some cells at a later stage, these were not identifiable as the R3/R4 pair, but were often found in the position of the R2/R5/R8 precursors (where sev is not expressed). This suggests that the effect seen is not a direct consequence of Rho activation, but more likely a secondary effect (RhoAV14 E40L fails to induce significant puc-lacz expression). Thus, the direct transcriptional activation of puc-lacz in R3/R4 correlates with the genetic interactions with the JNK module, suggesting a difference in the action of Rac and RhoA (Fanto, 2000).
An important aspect of R3/R4 cell fate and ommatidial polarity determination is the upregulation of Dl expression in the R3 precursor by Fz. Dl then signals to Notch on the R4 precursor, resulting in the choice of the R4 cell fate. In addition to Fz, other components of the Fz/planar-polarity pathway have also been found to upregulate Dl transcription. Thus, whether Rac and RhoA also regulate Dl transcription was investigated by monitoring Dl-lacZ expression in sev;RacV12 and sev;RhoV14 eye discs (Fanto, 2000).
In the wild type, Dl is expressed dynamically in photoreceptor precursors behind the furrow. Within the R3/R4 pair, it is expressed in R3 from rows 4 to 8, whereas it remains at lower levels in R4. In contrast to the difference in puc expression, both sev;RacV12 and sev;RhoV14 upregulated Dl-lacz expression in both R3/R4 precursors. The RhoAV14 E40L isoform that is impaired in nuclear signaling does not affect Dl expression, confirming the importance of nuclear signaling by RhoA. These effects are very similar to those of sev;Fz, supporting the idea that Rac and RhoA act downstream of Fz in the regulation of the R3/R4 cell fate. Their different effects on puc-lacz indicate that their downstream effectors in nuclear signaling are distinct (Fanto, 2000).
Recent studies have shown the small GTPases, Rac1,
Rho, and CDC42, to have a role in axon guidance. To
assess their participation in synapse assembly and function
various forms of Drac1 have been expressed in the
giant fiber system of Drosophila. Overexpression of wild-type
Drac1 in the giant fiber (GF) leads to a disruption in
axonal morphology; axons often terminate prematurely in
a large swelling in the target area but lack the normal
lateral bend where the synapse with the jump motor neuron
would normally be found. Electrophysiological assays
reveal longer latencies and lowering following frequencies
indicating defects in the synapse between the GF and
the tergotrochanteral motor neuron (TTMn). Thickened
abnormal GF dendrites are also observed in the brain.
Overexpression of the dominant-negative form of Drac1,
(N17), results in axons that produced extra branches in
the second thoracic neuromere (T2); however, the synaptic
connection to the TTMn is present and functions
normally. Conversely, expression of the constitutively active
form, Drac1(V12), results in a complete lack of neurite
outgrowth and this was also seen with overexpression
of Dcdc42(V12). In the absence of a GF, these flies show
no response in the jump (TTM) or flight (DLM) muscles
upon brain stimulation. Taken together these results
show that the balance of actin polymerization and depolymerization
determines local process outgrowth and
thereby synapse structure and function (Allen, 2001).
Growth, guidance and branching of axons are all essential
processes for the precise wiring of the nervous system. Rho
family GTPases transduce extracellular signals to regulate the
actin cytoskeleton. In particular, Rac has been implicated in
axon growth and guidance. Loss-of-function phenotypes of three Rac GTPases have been analyzed in Drosophila mushroom body neurons. Progressive loss of combined
Rac1, Rac2 and Mtl activity leads first to defects in axon branching, then guidance, and finally growth. Expression of a Rac1
effector domain mutant that does not bind Pak rescues growth,
partially rescues guidance, but does not rescue branching defects
of Rac mutant neurons. Mosaic analysis reveals both cell autonomous
and non-autonomous functions for Rac GTPases, the
latter manifesting as a strong community effect in axon
guidance and branching. These results demonstrate the central
role of Rac GTPases in multiple aspects of axon development in
vivo, and suggest that axon growth, guidance and branching could be controlled by differential activation of Rac signaling pathways (Ng, 2002).
The Drosophila genome has two Rac genes that share 92%
amino acid sequence identity and have overlapping expression
patterns. A highly related Mig-2-like (Mtl) gene, the ortholog
of Caenorhabditis elegans mig-2, is present on the same
chromosome. To isolate loss-of-function mutants of Rac1 and
Rac2, small deficiencies were generated by means of imprecise excision
of nearby P-elements. The Rac2Delta excision disrupts
only the Rac2 open reading frame (ORF), and hence is a
Rac2-specific null mutation, but is homozygous viable. The
Df(3)Rac1 excision disrupts the Rac1 ORF and two adjacent
genes. Rac1 point mutations were then recovered from
an ethylmethane sulphonate (EMS) screen for mutations that were
lethal over Df(3)Rac1 in a Rac2Delta
homozygous background, but viable in a Rac2Delta background. Sequence analysis and transgenic rescue have established that these mutants are recessive, loss-of-function Rac1 alleles (Ng, 2002).
Three Rac1 missense mutations were recovered, each altering an
amino acid conserved within Rho GTPases. The strongest
allele, Rac1J11, changes glycine 60 to glutamate (Gly60Glu). Structural, biochemical and genetic criteria all indicate that this is a null
allele. Structurally, Gly 60 forms a hydrogen bond with the
gamma-phosphate of GTP, and is invariant in all members of the GTPase
superfamily. Introduction of a glutamate is predicted to disrupt
this interaction. Indeed an analogous Gly60Asp mutation in H-Ras
disrupts the activity of both wild-type and constitutively active
proteins. Biochemically, the Rac1J11
mutation markedly impairs
binding of GTP to Rac1 in vitro, reducing it to less than 10%
compared with wild type. Genetically, homozygous Rac1J11
phenotypes are indistinguishable from Rac1J11/Df(3)Rac1 in both the mushroom bodies and visual system (Ng, 2002).
Mushroom body (MB) neurons of the Drosophila brain
were used to examine the role of Rac in axon development. Adult MB neurons
derive from four neuroblasts per brain hemisphere. Each neuroblast
sequentially generates three classes of neurons with distinct patterns
of axon projection. Each MB neuron sends a single primary
neurite that gives rise to both dendritic branches and an axon. MB
axons fasciculate tightly in the anteriorly projected axon peduncle.
In the anterior brain, each early born class of gamma-neuron has one
principal medial branch, whereas each neuron of the later-born alpha'/
ß' -or alpha/ß-classes has bifurcated axons with one dorsal (alpha' or alpha)
and one medial (ß' or ß) projection. These axonal lobes can be distinguished using the fasciclin II (FasII) marker, which stains alpha/ß' -axons strongly, gamma-axons weakly, but does not stain alpha'/ß'-axons, cell bodies or dendrites of MB neurons (Ng, 2002).
Three sets of experiments were carried out to analyze Rac function in MB
axon development. In the first set, by examining viable adults, gross
axon defects were found in animals in which the whole brain was isogenic for
particular Rac mutant combinations. In the second and third
sets, the MARCM system was used to generate neuroblast and
single-cell homozygous mutant clones that are positively labelled by
MB Gal4-OK107 driven marker expression. These mosaic
brains are unlikely to contain unlabelled clones outside the MB
lineage, since clones were induced in newly hatched larvae, when
proliferation is largely confined to the MB neuroblasts. Progressive defects were found in MB axon branching, guidance and growth as wild-type copies of Rac1, Rac2 and Mtl, referred to hereafter as Rac GTPases, were removed.
Although all three Rac GTPases contribute to the fidelity of MB
axon development, there is a differential dependence, with loss of
Rac1 having the largest effect and loss of Rac2 the smallest effect (Ng, 2002).
Axon branching is most sensitive to loss of Rac GTPase activity.
Defective branching included the generation of alpha- but not ß-axonal
branches and vice versa. This was revealed either by FasII
immunostaining or expression of a murine (m)CD8-green fluorescent
protein (GFP) fusion protein in the whole MB or in neuroblast clones. Rac1J11 heterozygotes exhibit significant branching defects. The percentage of branching defects increases as additional wild-type copies of Rac
genes are removed. The absence of a specific axonal
lobe might be caused either by a failure of individual axons to
bifurcate, or by misguidance of the bifurcated branches. To distinguish
between these possibilities, single-cell clones
were generated in the later-born alpha/ß'-neurons. In all cases where FasII staining reveals a branching defect in the MB as a whole, only a
single unbranched alpha-or ß-axon was detected for every alpha/ß'-neuron
labelled. These axons extend normally and follow appropriate trajectories in the remaining lobe. These observations indicate that the lack of one particular axonal lobe, as observed in isogenic or largely isogenic mutant brains or
neuroblast clones, is caused by a failure in axon branching (Ng, 2002).
Axon guidance displays an intermediate sensitivity to loss of Rac
GTPases. Whereas Rac1J11 heterozygotes exhibit mainly branching
defects, in Rac1J11 homozygotes, most MB axons are misguided. In mosaic animals, 21% of neuroblast clones homozygous for Rac1J11
exhibited defective guidance. This percentage
increased to 55% if the neuroblast clone was additionally homozygous
for Rac2Delta, and to 73% if the whole brain was also heterozygous for MtlDelta. The predominant guidance defects were
caused by accumulation of most or all axons in a ball-like structure
at the beginning of the axon peduncle that was intensely stained for
FasII. By labelling single mutant gamma-neurons, it was found
that individual axons tended to wind around the FasII-positive axon
'balls', similar to those seen in the neuroblast clones.
Therefore, these axon balls, whether they are observed in isogenic
mutant brains, neuroblast clones, or single neuron clones, are
caused by misguidance of MB axons. Axon growth is least sensitive to loss of Rac GTPases. Defects in axon growth were found mainly in MB neurons homozygous for
Rac1J11;Rac2Delta;MtlDelta. Ball-like axon accumulations were only rarely observed in this genotype. Instead, these axons terminated prematurely or failed to enter FasII-positive regions
altogether. Most of the remaining axons exhibited severe guidance defects, extending their axons in a non-stereotypical fashion. To confirm this interpretation of a growth defect, single-cell clones homozygous for Rac1J11;Rac2Delta;MtlDelta were examined in brain hemispheres in which most of the FasII-positive gamma-axons
(representing non-clonal tissue) were correctly patterned as in wild
type. It was found that 55% of homozygous Rac1J11;Rac2Delta;MtlDelta
single-cell clones exhibited axon-stalling defects, mostly at the peduncle, as compared with less than 5% in Rac1J11;Rac2Delta
single-cell clones. These data indicate that Rac1, Rac2
and Mtl collaborate to control axon growth in a cell-autonomous manner (Ng, 2002).
The dendritic region of MB neuroblast clones homozygous for
Rac1J11;Rac2Delta;MtlDelta
still possessed mCD8-GFP-positive neurites,
which were presumably contributed by the initial neurite outgrowth
from the cell body and elaboration of MB dendrites.
Quantitative analysis of single-cell Rac1J11;Rac2Delta;MtlDelta
clones revealed significant reduction in both total dendritic length and number of dendritic segments per neuron, indicating that Rac GTPases are also required for dendritic growth and branching (Ng, 2002).
The differential sensitivity of axon growth, guidance and branching
to loss of Rac function could in principle reflect the fact that
axons must grow in order to be assayed for guidance and branching
defects, and may need correct guidance to reach appropriate
branching points. However, such a simple hierarchical model
cannot explain the data. If Rac were equally required for growth,
guidance and branching, and these processes were simply epistatic
to one another as proposed by this heirarchical model, then growth
defects should always be more frequent than guidance defects,
which in turn should always be more frequent than branching
defects. For example, if growth, guidance and branching were
equally reduced by 20%, then in a population of 100 axons, 20
would show a growth defect, 16 would show a guidance defect (20%
of the 80 axons that grow), and 13 would show a branching defect
(20% of the 64 axons that grow and navigate correctly). However,
this is not what was observed; instead, a shift was seen from branching
to guidance to growth defects as the combined level of wild type Rac
was progressively reduced. That axon branching can be
selectively perturbed is best exemplified in genotypes involving a
hypomorphic allele Rac1J10. Furthermore, axon branching
defects could be disrupted without any growth or guidance
defects, whereas guidance could be disrupted without
obvious growth defects. These data strongly suggest that
axon growth, guidance and branching are separable events requiring
increasing amounts of combined Rac GTPase activity in vivo (Ng, 2002).
One model that could account for these observations is that
growth, guidance and branching use different Rac effector pathways.
To test this idea, use was made of Rac effector domain mutants.
The Rac Phe37Ala mutation (RacF37A) abolishes the ability of
activated Rac to induce lamellipodia formation without affecting
the Pak/JNK pathway. However, the Rac Tyr40Cys mutation
(RacY40C) blocks Rac binding to effectors containing the 'CRIB'
motif, including Pak, but does not affect lamellipodia formation. Overexpression of analogous Rac1F37A and
Rac1Y40C mutants, or wild-type Rac1, did not disrupt MB axon
patterning in a wild-type background. Therefore the MARCM system could be used to express these effector mutants specifically
in Rac mutant clones to determine which effector pathways are
required for MB axon growth, guidance and branching (Ng, 2002).
MB axon growth defects in single-cell Rac1J11;Rac2Delta;MtlDelta
clones were largely rescued by transgenic expression of wild-type Rac1 or
Rac1Y40C, but not Rac1F37A, indicating that direct
binding of CRIB effector proteins is not required for Rac function
in axon growth. To assess the effector pathways involved in guidance
and branching, these same transgenes were expressed in Rac1J11;Rac2Delta
neuroblast clones. In this background, 81% of axons show mutant
phenotypes, predominantly guidance (55%) and branching (24%)
defects. Expression of wild-type Rac1 markedly rescues these
defects, reducing the fraction of abnormal axon phenotypes to
53%. The remaining branching and guidance defects were
probably caused by influences of nearby non-clonal MB neurons heterozygous for Rac1J11;Rac2Delta, which exhibit a
similar degree of branching and guidance defects. Neither
Rac1Y40C nor Rac1F37A expression reduces the percentage of total
axonal defects (78% and 88%, respectively). However, expression of
Rac1Y40C (but not Rac1F37A) results in a marked shift in the
distribution of axonal defects, with most showing branching (45%)
rather than guidance (31%) defects. Thus, compared with wild-type Rac1, expression of Rac1Y40C in a Rac mutant background is able to rescue growth, partially rescue guidance, but is unable to rescue branching defects (Ng, 2002).
These results suggest that different downstream effector pathways
mediate axon growth, guidance and branching. In particular, Rac
binding of CRIB-domain effectors such as Pak is not required for
axon growth, but may contribute to axon guidance and branching.
This is consistent with genetic analyses indicating a requirement for
Drosophila Pak in axon guidance but not growth.
The mosaic analysis revealed an unexpected degree of cell non-autonomous
effects in axon guidance and branching caused by
defective Rac activity. If every MB axon were to choose its pathway
independently, then in a brain hemisphere containing one homozygous
Rac1J11;Rac2Delta
neuroblast and three heterozygous neuroblasts,
one would expect to observe a mixture of wild-type and
mutant trajectories. Remarkably, all FasII-positive axons in the
same hemisphere as the mutant neuroblast clone invariably exhibited
the same guidance defect. This cannot
be explained simply by the guidance defect caused by Rac1J11;Rac2Delta
heterozygous neurons, since only 8% of such hemispheres exhibited
this defect. Such a cell non-autonomous effect is also
observed in axon branching. If every branching-defective alpha/ß-neuron were to make an independent decision to form either a
single dorsal or medial branch, then in neuroblast clones where
hundreds of axons are examined together, one would expect to see alpha
thinning of both axonal lobes rather than the absence of a single
lobe. Instead, in Rac1J11;Rac2Delta
neuroblast clones that exhibited
branching defects, all MB axons in the neuroblast clone projected
either dorsally (one-third) or medially (two-thirds). Most non-clonal
axons (as revealed by FasII staining) would always make the
same choice as the mutant clone. Whereas homozygous
mutant axons could induce their heterozygous neighbours to make
the same errors, it is possible that heterozygous axons could reduce
the error rate of nearby homozygous mutant axons. This could be
one explanation for why the phenotypes of homozygous mutant
animals are of higher penetrance than those of homozygous mutant
neuroblast clones (Ng, 2002).
These observations suggest a marked community effect in MB
axon guidance and branching: axons of mixed genotypes make their
choices together. In any given animal, the collective choice of a
normal versus mutant projection is likely to be influenced by the
severity of the genotype, and the relative number of homozygous
versus heterozygous axons. Such collective decision making is
probably a result of tight fasciculation among MB axons, which is
not disrupted in Rac mutants. It will be interesting to test whether
this community effect reflects a general feature of axon development
in a complex central nervous system environment (Ng, 2002).
This analysis of Rac GTPases in MB axon development suggests a
mechanistic link between axon growth, guidance and branching.
Although there is evidence that axon growth and guidance have
different cytoskeletal requirements, their connections are not
well understood. Little is known about intracellular signaling
mechanisms that regulate axon branching. This study shows that
axon growth, guidance and branching require increasing amounts
of combined Rac GTPase activity in vivo. The requirement of Rac GTPases for axon outgrowth and guidance in C. elegans has also been reported recently. It is proposed that axon branching, guidance and growth specified by extracellular
cues require different amount of Rac GTPase activation in the
growth cone, which in turn engage different downstream pathways
to specify distinct cytoskeletal changes (Ng, 2002).
Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance
receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively
active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and
ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout
enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho
suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by
heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase
(ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho
or ctRho. These data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin
activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho (Fritz, 2002).
Thus, when
expressed alone, only ctDrac and ctDcdc42 cause midline
crossing errors. However, the mutant GTPases interact
genetically with mutations in robo, Sos, and chic and with
overexpression of ctMLCK. The interactions are surprisingly
specific. Midline crossing errors caused by expression
of ctDrac or ctDcdc42 are suppressed by heterozygous loss
of Profilin and enhanced by expression of ctMLCK. These
results indicate that Drac1 and Dcdc42 encourage axons to
cross the midline by regulating actin polymerization and/or
myosin activity. CtRho and dnRho interact strongly with
expression of ctMLCK or heterozygous loss of Robo, which
suggests that regulation of myosin activity by Rho is crucial
for midline repulsion. This work demonstrates that Rho,
Drac1, and Dcdc42 are involved in dictating which axon
may cross the midline, presumably by aiding in the transduction
of attractive and/or repulsive cues operating at the
midline. By using mutations in signaling molecules known
to prevent pCC/MP2 axons from crossing the midline, this
analysis concentrates on how Rho, Drac1, and Dcdc42 may
regulate cytoskeletal dynamics in response to midline repulsive
cues (Fritz, 2002).
The Rho family of GTPases was first studied in fibroblasts
where activation of Cdc42, Rac, or Rho results in
production of filopodia, lamellapodia, and stress fibers,
respectively. In wound-healing assays,
Rac appears to control actin polymerization to provide
the protrusive force needed for movement, while Cdc42
determines cell polarity to localize Rac activity to the
leading edge of the cell. Rho seems to play a role in adhesion
and spreading during cell migration.
These same processes are involved in growth cone motility,
which makes the Rho GTPases candidates for regulation of
cytoskeletal dynamics during axon guidance. Experiments
in neurons, both in vitro and in vivo, indicate that activation
of Rac and/or Cdc42 increases axon outgrowth and this
is opposed by activation of Rho, which leads to growth cone
collapse or retraction. This is consistent with
findings that expression of ctDcdc42 or ctDrac allow
axons to ignore repulsive signals at the midline and continue
extending across the midline (Fritz, 2002).
Rho family GTPases activate a number of effectors that
may affect axon outgrowth by regulating adhesion, myosin
force generation, and/or actin polymerization. The ctDrac- and
ctDcdc42-induced midline crossing errors are suppressed
by heterozygous loss of Profilin, an actin-binding
protein, which stimulates actin polymerization. Since reducing
actin polymerization partially rescues the ctDrac
and ctDcdc42 phenotypes as well as errors caused by
heterozygous loss of Robo, it is likely that the midline
crossing errors are caused by excessive actin polymerization.
Increased actin polymerization may produce more
filopodia to explore the midline, which leads to midline
crossing. There are several pathways through which Drac1
and Dcdc42 might affect actin polymerization. The Cdc42/
Rac effector p21-activated kinase (PAK) activates LIM kinase
to phosphorylate cofilin, an actin-depolymerizing factor
required for neurite outgrowth. Cdc42 also activates actin polymerization through WASP,
which stimulates polymerization by binding to the Arp2/3
complex. The activation of WASP by Cdc42 is enhanced by
Profilin, which may explain why the suppression of the
ctDcdc42 phenotype is stronger than that of the ctDrac-induced
errors. However, actin polymerization
may not be the only process regulated by Rho family
GTPases to increase outgrowth (Fritz, 2002).
The interactions between the Drac1 and Dcdc42 and
ctMLCK indicate that misregulation of myosin activity
may contribute to ctDrac- and ctDcdc42-induced axon
guidance errors. Coexpression of ctMLCK with ctDrac or
ctDcdc42 results in a strong enhancement of midline
crossing errors, while expression of dnDrac or dnDcdc42
suppresses the defects caused by increased myosin activity.
This suggests that Drac1 and/or Dcdc42 activate myosin
activity in the growth cone to increase outgrowth. One
mechanism may be through activation of PAK, which leads
to phosphorylation of myosin regulatory light chains (MLC)
to increase myosin activity. However, it has been shown that PAK also phosphorylates
and inactivates MLCK, resulting in less myosin
activity. In vitro, PAK phosphorylates
MLCK at serine 439, which is present in ctMLCK, and
serine 991, which has been removed from ctMLCK, so the
impact of this pathway on the truncated ctMLCK protein is
uncertain. Alternatively, it is possible that the interaction of Drac1 or
Dcdc42 and ctMLCK is a secondary effect to increased actin
polymerization. If increased actin polymerization is causing
more filopodial exploration of the midline, increasing myosin activity through ctMLCK expression could cause axons
to cross the midline before they can retract filopodia encountering
repulsive signals. Separating the relative contributions
of Drac1 and Dcdc42 to actin polymerization and
myosin activity will require more specific experiments
involving the effectors of Drac1 and Dcdc42 (Fritz, 2002).
The data suggest that Drac1 and Dcdc42 activation must
be prevented or reduced for axons to respond to repulsive
signals at the midline. The midline crossing errors seen in
Drac1 mutants are strongly enhanced by a partial loss of
Robo, which suggests that midline repulsion requires a
down-regulation of Drac1 activity. Down-regulation of Rac
activity occurs in response to other repulsive signals, such
as Ephrin and Semaphorin. A mechanism for this is suggested by experiments
showing that Plexin-B, the receptor for Semaphorin,
binds specifically to activated Rac, most likely to prevent it
from activating effectors. Experiments in cell culture systems have confirmed
that Robo-mediated signaling involves down-regulation of
Cdc42. Activation of Robo by Slit recruits srGAP1 to the
CC3 domain of Robos cytoplasmic tail, where it interacts
with and inactivates Cdc42. Although
srGAP1 does not affect the activity of Rac, srGAP2 and
srGAP3 also bind to Robo, and one of these may regulate
Rac activity. Down-regulation of Cdc42
and Rac by Robo-dependent repulsive signals is consistent
with recent experiments showing that activation of DCC
by chemoattractive Netrins stimulates neurite outgrowth
and results in activation of Cdc42 and Rac1. Together, these data and the
literature led to the hypothesis that Robo prevents axons
from crossing the midline by decreasing Drac and Dcdc42
activity so that actin polymerization and myosin force
generation are reduced (Fritz, 2002).
Down-regulation of Dcdc42 and Drac1 by Robo may also
repel axons by preventing coupling of the actin cytoskeleton
to the substrate. Rac is required for localization of
E-Cadherin to cell-cell contacts and recruiting actin to
Cadherin binding sites. Cdc42 and Rac
promote Cadherin-mediated adhesion by preventing IQGAP,
a CaM-binding Ras GAP, from interfering with the
interaction of ß-catenin with alpha-catenin. Integrin-mediated adhesion also involves signaling
through Rho family GTPases. By reducing actin and myosin dynamics
and decoupling the cytoskeleton from the substrate, downregulation
of Drac and Dcdc42 by repulsive guidance receptors
would prevent axons from extending across the midline (Fritz, 2002).
The role of Rho in midline repulsion is more difficult to
determine since both dnRho and ctRho enhance the
midline crossing phenotype of heterozygous robo mutants.
This is consistent with the data in which both
dnRho and ctRho enhance the ctMLCK phenotype. Similar
complexities are seen in the literature; expression of a
Rho GEF, which is expected to increase Rho activity,
leads to increased attraction to the midline, even though
activation of Rho usually leads to growth cone collapse or
retraction. The complexity of the Rho interactions is understandable
when the dual role of myosin activity during
axon guidance is considered. The most documented connection
between myosin activity and Rho is through the
effector Rho Kinase (RhoK). RhoK phosphorylates MLC
and also inactivates myosin phosphatase by phosphorylating
its myosin binding subunit, leading to increased
phosphorylation of MLC and therefore increased myosin
activity. Myosin
activation is needed both for the retrograde flow of actin
that retracts filopodia and for the force that propels the
growth cone forward. Repulsive guidance signals are expected to increase
retrograde flow while preventing forward movement (Fritz, 2002).
Clearly, regulation of Rho family GTPase activity is
necessary to prevent axons from crossing the midline inappropriately.
Midline repulsive signaling involves regulation
of all three GTPases; Drac1 and Dcdc42 are likely downregulated,
while Rho seems to be activated downstream of
repulsive signals. The Rho family GTPases influence actin
polymerization and/or myosin force generation to regulate
the processes of growth cone motility that are required for
proper response to axon guidance signals (Fritz, 2002).
Rho family small GTPases are thought to be key molecules in the regulation of cytoskeletal organization, especially for actin filaments. In order to examine the functions of Rac1 and Cdc42 in axon guidance at the midline of the central nervous system in Drosophila embryos, Rac1 and Cdc42 were either activated or inactivated in all postmitotic neurons. The phenotypes of Cdc42 activation and Rac1 inactivation were similar to those of roundabout mutants, in that many extra axons crossed the midline. Rac1 inactivation is dominant over Roundabout receptor activation. These observations indicate that Rac1 and Cdc42 have distinct functions in downstream signalling events triggered by Roundabout receptors. In order to further examine the functional difference between Rac1 and Cdc42 in the growth cone morphogenesis, primary embryonic cultures were used to closely observe neurite formation. Activation of Rac1 and Cdc42 is shown to have distinct effects on neurite formation, particularly on growth cone morphology and the actin filaments within. Both Rac1 and Cdc42 activation induced large growth cones and long filopodia, but Cdc42 did so more efficiently than Rac1. Only Rac1 activation, however, induced thick actin bundles in the filopodia. A clear difference was found between Rac1 and Cdc42 in terms of the response to an inhibitor of actin polymerization. These results suggest that Cdc42 is specifically involved in the regulation of actin filaments in growth cones, whereas Rac1 is involved in additional functions (Matsuura, 2004).
In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. The gene kette (correctly termed Hem-protein, or simply Hem) is required for the normal projection of the VUM axons and interference with kette function disrupts glial migration. In spite of the fact that glial migration is disrupted in kette mutants, both the axon guidance and glial migration phenotypes have their origin in midline neuron expression and not in midline glial expression. Axonal projection defects are found for many moto- and interneurons in kette mutants. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The Hem/Kette protein is homologous to the transmembrane protein HEM-2/NAP1 (Nck-associated protein) evolutionary conserved from worms to vertebrates. In the
CNS, the membrane protein Kette could be participating directly in the
neuron-glial interaction at the midline, where it could act as a signal
to direct glial migration. Alternatively, Kette could serve as a
receptor of possibly glial-derived signals during VUM growth cone
guidance. The experimental data suggest that Kette transduces
information to the neuronal cytoskeleton, which is in agreement with a
receptor function (Hummel, 2000).
The vertebrate homolog of KETTE has been shown to interact with the
first SH3 domain of the Nck adapter protein (Kitamura, 1996). The
Drosophila homolog of Nck is encoded by dreadlocks (Garrity, 1996). dock was identified in a screen for
mutations affecting axonal pathfinding and targeting of the adult
photoreceptor neurons. In wild-type third instar
larvae, the different photoreceptor cells stop their axonal growth in two distinct layers of the optic lobe, the lamina and the medulla. In contrast, dock mutant photoreceptor
cells fail to establish this specific targeting, leading to a
disruption of the lamina neuropile organization. In ~70%
of the third instar larvae homozygous for the hypomorphic
ketteDelta2-6 allele (n = 25),
a weak disorganization was found of the lamina plexus and an abnormal
bundling of R-cell axons in the medulla. The remaining larvae
showed a stronger disorganization of the R-cell axons (20%) or were
indistinguishable from wild type (10%). Further reduction of the
kette gene function results in an enhancement of this axonal
phenotype in 50% of the analyzed transheterozygous mutant
kette larva. If one
copy of dock is removed in the background of a hypomorphic kette mutation, a considerable enhancement of the larval projection phenotype is observed in 60% of the individuals. In addition, a significant enhancement of the
homozygous dock phenotype is observed when removing one copy of
kette in a dock mutant background (Hummel, 2000).
A reduction
in the size of the longitudinal connectives in the embryonic CNS is observed in dock mutants. This phenotype resembles a hypomorphic kette connective
phenotype. In mutant dockP2 embryos, commissure
separation is also affected, comparable with the hypomorphic phenotype
seen in ketteJ1-70 embryos. In correlation
with the commissural phenotype, the VUM axons do not project properly
in mutant dock embryos (Hummel, 2000).
In summary, these data show that both kette and dock
mutants genetically interact and share a number of phenotypic traits. This suggests that these genes might be acting in the same genetic pathway during axonal pathfinding (Hummel, 2000).
The Rho family of small GTPases constitutes important regulatory factors also interacting with Nck. To analyze the functional interaction of KETTE with members of
the Rho family, both the activated as well as the dominant-negative
versions of Cdc42 and Rac1 were expressed in the midline cells of wild-type and kette mutant embryos. Expression of both of these mutant proteins in all
midline cells using the simGAL4 driver line results in similar
axonal defects. The projection of the VUM neurons resembles
the phenotype observed in kette mutant embryos. In addition,
the cell bodies of the VUM neurons appear sometimes displaced. In stage 16 embryos, the segmental commissures appear fused,
which again indicates the importance of the midline neurons for the
migration of the midline glia. Only a weak
commissural disorganization is observed when the different Cdc42 or
Rac1 proteins are expressed in the midline glial cells only. In all
experiments, the expression of Rac1 appears to have more pronounced
effects on the axonal morphology (Hummel, 2000).
To further test the interaction of kette and Rac1, activated Rac1 was expressed in all midline cells of mutant
ketteJ4-48 embryos. The commissures appeared
separated, indicating that the midline glial cells are able to migrate
between anterior and posterior commissures. Concomitantly, the
connectives are further distant from the midline),
indicating that expression of activated Rac1 can partially
rescue the kette phenotype (Hummel, 2000).
Among others, the Nck adapter protein transduces signals via CDC42
and Rac1 to the Actin cytoskeleton. A GFP-moesin
transgene was used to analyze the Actin cytoskeleton
of mutant kette embryos. This protein binds to the F-actin
fibers and thus allows a determination of their subcellular distribution using confocal microscopy. In wild-type embryos, F actin is found in
axonal processes that are arranged in the typical ladder-like pattern. Prominent expression is also detected in
the epidermis and the somatic musculature. In similar focal planes, kette embryos appear very different. Within the CNS, the typical fused commissure phenotype of mutant kette
embryos is evident. Frequent intense granular
staining is observed in the CNS and in the lateral body wall. Furthermore, the regular appearance of the cytoskeleton is disrupted in
both mesoderm and ectoderm. In a tangential section of
the dorsal epidermis, individual cells can be seen in wild-type
embryos. Some cells form hairs, characterized by thin F-actin bundles. In mutant ketteC3-20 embryos, pronounced
F-actin bundles are found, which often have a wavy appearance. In addition, the cortical actin cytoskeleton appears to
stain weaker compared with wild-type embryos (Hummel, 2000).
Thus, mutations in kette affect the organization of the
cytoskeleton. kette is expressed in neurons and is needed for
correct axonal pathfinding. The KETTE protein seems to interact with
the SH2-SH3 adapter Dock and at least part of the kette
function might be mediated via small GTPases such as Rac1 (Hummel, 2000).
In addition to Kette function in axonal pathfinding, defects are observed in the morphology of trichomes and bristles in flies
homozygous for the weak hypomorphic ketteDelta2-6 allele. Around 10% of the
bristles appear wavy or do bend sharply and wing trichomes are enlarged
and sometimes split. These phenotypes resemble those
observed for mutations affecting the organization of the F-actin
bundles or following expression of mutated Cdc42 or Rac1. Similarly, elevated levels
of GTPase function in the developing eye cause late developmental
defects as observed in hypomorphic kette mutations. cdc42
mutations have been isolated, but, presumably due
to maternal contribution, loss of cdc42 function does not lead
to an embryonic CNS phenotype. Both Cdc42 and Rac1 are
important regulators of the actin cytoskeleton. The
transduction of extracellular signaling to small GTPases is believed to
involve Nck-type adapter proteins. Several
phenotypic traits of kette are shared by mutations in the
Drosophila gene dock, which encodes a Nck homolog.
Furthermore, dock and kette genetically interact. The
genetic data in combination with the kette loss-of-function
and kette overexpression phenotypes led to the proposal of a model
relating Dhem2/NAP1 function to cytoskeleton organization (Hummel, 2000).
The vertebrate KETTE homolog is HEM-2/NAP1 with 86%
amino acid identity over the entire ORF, indicating that presumed
protein-protein interactions are also conserved. To date, no
hem-2 mutation has been described in vertebrates. The first
SH3 domain of Nck was used to isolate Nck-associated proteins (NAP) and
led to the identification of HEM-2/NAP1. Binding of
HEM-2/NAP1 to Nck appears to be mediated by a 140-kD
protein. Interestingly, in a screen for proteins
interacting with activated Rac1, a complex consisting of
HEM-2/NAP1 and a 140-kD protein was isolated. Thus, the 140-kD protein might be a novel adapter linking
HEM-2/NAP1 signaling along two routes to the small
GTPases. It will be of interest to identify Drosophila genes
interacting with kette (Hummel, 2000 and references therein).
The Drosophila Nck homolog is encoded by dock. dock function appears highly
specialized for growth-cone guidance since no mutant phenotypes have been
reported in the mesoderm or the ectoderm. Because kette shows more pleiotropic defects, other
adapter proteins may interact with the Kette protein (Hummel, 2000).
During axonal pathfinding, coordinated cytoskeletal remodeling
occurs at the tip of the extending neurites, the growth cone. The Rho family of
GTPases mediates the regulation of the reorganization of the actin
cytoskeleton induced by extracellular signals: Cdc42, Rac1, and RhoA. In fibroblast cells, the different
GTPases induce different cellular responses. Similarly, different
functions appear to be associated with the different Drosophila GTPases. Rho as well as Cdc42 function is needed
for cell shape changes during gastrulation, dorsal closure, bristle, and hair formation. Bristle and hair formation are similarly affected by kette (Hummel, 2000).
These data suggest that Kette provides a novel
mechanism linking extracellular signals to the neuronal cytoskeleton.
Central relay proteins are SH2-SH3 adapter proteins that control the
organization of the actin cytoskeleton via a number of downstream
proteins. Biochemical data suggest that additional proteins (p140 kD)
may bypass the function of SH2-SH3 adapter proteins, but a detailed analysis awaits its isolation. The Kette
protein might interact with extracellular signals, which, in the CNS,
might possibly be presented by glial cells. To gain further insight in
the neuron-glial interaction at the midline, future work will be
directed toward the identification of these components (Hummel, 2000).
The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. This study demonstrates that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. Phenotypic similarities and genetic interactions were observed among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. It is proposed that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner (Abe, 2014).
Initially acquired memory dissipates rapidly if not consolidated. Such memory decay is thought to result either from the inherently labile nature of newly acquired memories or from interference by subsequently attained information. This study reports that a small G protein Rac-dependent forgetting mechanism contributes to both passive memory decay and interference-induced forgetting in Drosophila. Inhibition of Rac activity leads to slower decay of early memory, extending it from a few hours to more than one day, and to blockade of interference-induced forgetting. Conversely, elevated Rac activity in mushroom body neurons accelerates memory decay. This forgetting mechanism does not affect memory acquisition and is independent of Rutabaga adenylyl cyclase-mediated memory formation mechanisms. Endogenous Rac activation is evoked on different time scales during gradual memory loss in passive decay and during acute memory removal in reversal learning. It is suggested that Rac's role in actin cytoskeleton remodeling may contribute to memory erasure (Shuai, 2010).
When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys) is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and it has been shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. This study teste the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. Rac1 was shown to be required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results it is concluded that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. This study shows that Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila larvae are often raise at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes (Xavier, 2011; full text of article).
Nutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (RasACT) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with RasACT in tumorigenesis, a genome-wide screen was carried out for genes that when overexpressed throughout the developing Drosophila eye enhance RasACT-driven hyperplasia. RasACT-cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wild-type cells, only Rac1, an activated allele of Rho1 (Rho1ACT), RhoGEF2, and pbl cooperated with RasACT, resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or >Rac1 with RasACT upregulated Jun kinase (JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system,
upregulation of JNK alone was not sufficient for cooperation with RasACT, while in the clonal setting, JNK upregulation was sufficient for RasACT-mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of RasV12-expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2+ human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg
and aPKC, with RasACT in the whole-tissue context. Collectively this analysis reveals the importance of the RhoGEF/Rho-family/JNK pathway in cooperative tumorigenesis with RasACT (Brumby, 2011).
One goal of cell biology is to understand how cells adopt different shapes in response to varying environmental and cellular conditions. Achieving a comprehensive understanding of the relationship between cell shape and environment requires a systems-level understanding of the signalling networks that respond to external cues and regulate the cytoskeleton. Classical biochemical and genetic approaches have identified thousands of individual components that contribute to cell shape, but it remains difficult to predict how cell shape is generated by the activity of these components using bottom-up approaches because of the complex nature of their interactions in space and time. This study describes the regulation, in cultured Drosophila neural cells, of cellular shape by signalling systems using a top-down approach. The shape diversity generated by systematic RNAi screening was exploited, and the shape space a migratory cell explores was comprehensively defined. A simple Boolean model, involving the activation of Rac and Rho GTPases in two compartments is suggested to explain the basis for all cell shapes in the dataset (see Boolean model - Figure 4). Critically, a probabilistic graphical model was generated to show how cells explore this space in a deterministic, rather than a stochastic, fashion. The predictions made by the model were evaluated using live-cell imaging. This work explains how cross-talk between Rho and Rac can generate different cell shapes, and thus morphological heterogeneity, in genetically identical populations (Sailem, 2014 24451547).
The Drosophila melanogaster homolog of the ced-1 gene from Caenorhabditis elegans is draper, which encodes a cell surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, a genetic screen of chromosomal deficiencies was performed to identify loci that dominantly modify the phenotype of over-expression of Draper isoform II, which suppresses differentiation of the posterior crossvein in the wing. The existence of 43 genetic modifiers of Draper II was deduced. 24 of the 37 suppressor loci and 3 of the 6 enhancer loci have been identified. A further 5 suppressors and 2 enhancers were identified from mutations in functionally related genes. These studies indicated positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott-Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in the engulfment process downstream of Draper activation. The relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador-Warts-Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are further investigated and discussed (Fullard, 2014).
Some memories last longer than others, with some lasting a lifetime. Using several approaches memory phases have been identified. How are these different phases encoded, and do these different phases have similar temporal properties across learning situations? Place memory in Drosophila using the heat-box provides an excellent opportunity to examine the commonalities of genetically-defined memory phases across learning contexts. This study determines optimal conditions to test place memories that last up to three hours. An aversive temperature of 41°C was identified as critical for establishing a long-lasting place memory. Interestingly, adding an intermittent-training protocol only slightly increased place memory when intermediate aversive temperatures were used, and slightly extended the stability of a memory. Genetic analysis of this memory identified four genes as critical for place memory within minutes of training. The role of the rutabaga type I adenylyl cyclase was confirmed, and the latheo Orc3 origin of recognition complex component, the novel gene encoded by pastrel, and the small GTPase rac were all identified as essential for normal place memory. Examination of the dopamine and ecdysone receptor (DopEcR) did not reveal a function for this gene in place memory. When compared to the role of these genes in other memory types, these results suggest that there are genes that have both common and specific roles in memory formation across learning contexts. Importantly, contrasting the timing for the function of these four genes, plus a previously described role of the radish gene, in place memory with the temporal requirement of these genes in classical olfactory conditioning reveals variability in the timing of genetically-defined memory phases depending on the type of learning (Ostrowski, 2014).
Temperature as an aversive reinforcer interacts with training conditions to induce place memories of different stabilities. Previous work showed that intermittent training for Drosophila in space and place memory increases memory performance up to two hours after training. Shown in this study is that temperatures at or above 41°C are needed for induction of this longer lasting memory. That is, 37°C and below can act as an aversive reinforcer and condition flies to avoid a part of the training chamber, but continued avoidance decays within minutes of training. It is only with a temperature of 41°C that an hours-long memory is induced with massed and intermittent training. This abrupt difference in the length of the memory after training with the higher temperature may reflect a threshold of some sort, the steepness of which is currently unknown. This could arise from a differential input to the reinforcing circuit from separate sensory systems, like the Trp family of receptors, or from altered output from one of these sensory systems. Future studies on different temperature responsive proteins may differentiate between these possibilities (Ostrowski, 2014).
Genetic analysis challenges the use of time as a critical factor in determining a memory phase. Memory phases in the fly were initially examined after classical olfactory conditioning where an odorant is typically paired with an aversive electric shock or a rewarding sugar. Four different memory phases have been classified based roughly on time after training and genetic/pharmacological manipulations. Short-term memory after olfactory learning is measured within minutes of training; long-term memory and anesthesia resistant memory start to be active within hours and are increasingly important for memories at the 24 h range and longer. An intermediate memory is thought to be important in the interval between short-term and long-term memories. That time alone is a critical factor in determining these phases loses support when comparing flies with different mutations in aversive and rewarded olfactory memory. For example, the long-known mutant radish was originally shown to be important in the hours-long range after aversive olfactory training and genetically classified the anesthesia-resistant memory. Interestingly, this gene is important within minutes of training in rewarded olfactory memory (Ostrowski, 2014).
Several genes that are important for early to late phases of classical olfactory conditioning are critical on a finer time scale in place memory. Mutation of both the rut and lat genes leads to reduced aversive olfactory memory tested immediately after training, as well as longer time points. Although it is currently unclear when during the life-cycle these genes are important for place memory, mutation of rut and lat reduces memory directly after training. Furthermore, both the rut and lat products have been implicated in synaptic plasticity at the neuromuscular junction (NMJ), which suggests a role for these genes in early stages of learning and memory. It is pretty straight-forward that the rut-encoded type I adenylyl cyclase is also acting early on in associative processes in place learning. The lat gene encoding a subunit of the origin of replication (orc3) is also localized to the pre-synaptic specializations at the NMJs). The lat-orc3 also acts early-on in associative processes for place learning. How the lat-orc3 product is related to regulation of cAMP levels is, however, not as clear. The rut and lat results add to our understanding of an apparently common set of short-term changes in memory between olfactory and place memory, which include a common function of the S6 kinase II, an atypical tribbles kinase, and the arouser EPS8L3. And, the recently identified role of the foxp transcription factor specifically in operant learning, as tested in a flight simulator, suggests another set of genes that could be important for operant place memory in the minutes range (Ostrowski, 2014).
Late memory phases in classical olfactory conditioning depend on a set of genes that are important for place memory within minutes. The first challenge to a common timing of a memory phase came from the radish gene. In contrast to a role in the hours range after olfactory learning, radish mutant flies have a deficit in operant place memory within minutes of training. Furthermore, the pst gene (CG8588), encoding a novel product, has been previously shown to have a specific defect in aversive olfactory memory 24 h after spaced training. That is, the pst mutant flies have a normal short-term olfactory memory but a defective memory 1 day later. Interestingly, in the heat-box pst mutant flies already show a significant decrement in place memory immediately after training. This place memory defect seems to get worse within the first hour after training, reduced to ~50% of normal after 60 min. Thus, this 'long-term memory gene' is also involved in a memory within minutes of training in a second learning situation (Ostrowski, 2014).
Using the classical aversive olfactory learning paradigm the rac small GTPase has been identified as a key regulator in memory retention. Inhibition of Rac activity slows early olfactory memory decay, leading to elevated memory levels one hour after training, but becoming increasingly important 2 h after training. There does not appear to be an effect of Rac inhibition in olfactory memory in the minutes range after training. Transgenic flies with inhibited Rac function also have an increase in memory retention after place memory training. However, the first evidence of an increase in memory performance is within 10 min. Impressively, significant place memory was still evident up to 5 h after training, far beyond the range that can be typically measured in wild-type flies. Thus, while rac has a more general role in stabilizing memories, the timing of this function depends again on the type of memory trace that is formed (Ostrowski, 2014).
Not all memory genes first identified in other contexts, however, play a significant role in place memory. The DopEcR gene has been implicated in several behaviors, including a 30 min memory after courtship conditioning. This G-protein linked receptor is responsive to both dopamine and the steroid hormone ecdysone. Remarkably, DopEcR has been shown to interact with the cAMP cascade through double mutant and pharmacological tests. Using conditions that induce a robust and lasting place memory, the DopEcR mutant flies do not show a defect in memory directly after training or at 1 h post-training. This is despite the fact that the rut and cAMP-phosphodiesterase genes (dunce) are critical for place memory. It may be that DopEcR is not required for this type of learning and would be consistent with the independence of place memory from dopamine signaling. Alternatively, other redundant pathways may compensate for the reduction in DopEcR function caused by the DopEcRPB1 allele. One might further speculate that other types of behavioral plasticity, such as reversal learning or memory enhancement after unpredicted high temperature exposures in the heat-box might be more sensitive to DopEcR changes. Future experiments will determine if this is the case (Ostrowski, 2014).
Memory stability across learning contexts in Drosophila has some common genetic mechanisms, but the timing for gene action depends on the type of learning. That this study has added several genes here, including lat, pst, and rac as regulators of memory stability in operant place memory suggests that there are at least some common molecular processes in memory stability across different learning types. However, the timing of these genetically-defined phases depends on what is learnt. It is speculated that an ideal system to regulate memory stability would be one that activates its own decline. That is, a given memory type should activate the process of decreasing memory expression. This might work with the recruitment of a reinforcing pathway, like the dopaminergic signal that is important for both the acquisition of an associative olfactory memory and the active process of forgetting that association. In this case an odor associated with shock gives rise to a memory trace in mushroom body neurons that depends on a set of dopamine neurons that is important for both memory acquisition and decline. Whether this type of aminergic-based system applies to other forms of memory is not yet known. However, if an aminergic-based signal is common in memory decline, as appears to be the case with the Rac-based mechanism, differences in the types of aminergic neurons or innervation targets could give rise to the altered stabilities of behaviorally expressed memories (Ostrowski, 2014).
The role of Rac in dorsal closure
Rac1 Effects of mutation part 2/2
Rac1:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| References
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