|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 14 March 2007
doi: 10.1242/dev.001529
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Laboratory of Morphogenesis, Institute of Molecular and Cellular Biosciences,
the University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan.
2 Graduate Program in Biophysics and Biochemistry, Graduate School of Science,
the University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan.
* Author for correspondence (e-mail: ttabata{at}iam.u-tokyo.ac.jp)
Accepted 8 February 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Fak56D, CdGAPr, Optic stalk, Glia, Drosophila
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Leiserson et al., 2000;
Lemke, 2001;
Gilmour et al., 2002;
Chotard and Salecker, 2004). At
present the molecular and cellular mechanisms that regulate glial cell
behavior, such as migration and morphogenesis, are largely unknown. The main
classes of central nervous system glia in Drosophila exhibit many
morphological and functional similarities to their mammalian counterparts
(Freeman and Doherty, 2006);
therefore they represent a good model for studying general mechanisms
underlying glial development and behavior.
The Drosophila visual system is one of the most extensively
studied nervous systems (Huang and Kunes,
1996; Kunes, 2000;
Clandinin and Zipursky, 2002;
Chotard and Salecker, 2004). It
consists of the compound eyes and the optic ganglia. The compound eye consists
of approximately 750 ommatidial units, each of which contains eight
photoreceptor cells. During the development of the visual system,
photoreceptor cells send their axons (R axons) from the eye primordium (eye
disc) to their targets in the optic lobe of the brain through a structure
called the optic stalk. All R axons from an eye disc come together and make a
single thick bundle in the optic stalk
(Fig. 1A). The optic stalk also
contains two kinds of glial cells, the wrapping glial (WG) cells and surface
glial (SG) cells (Meinertzhagen and
Hanson, 1993; Perez and
Steller, 1996; Hummel et al.,
2002). The WG cells are intermingled with R axons and extend long
processes that wrap around several R axons, so that the entire R axon bundle
is subdivided into groups of smaller bundles. The WG processes form an inner
sheath to segregate these bundles from each other during the pupal stage. By
contrast, SG cells form an outer sheath that surrounds the entire bundle of R
axons. It is clear that the optic stalk glia play roles in R axon innervation
and ensheathment (Rangarajan et al.,
1999; Hummel et al.,
2002); however, the precise structure and development of the optic
stalk remain largely unknown. Hence, we have focused our efforts to elucidate
SG cell development and optic stalk morphogenesis.
We found that SG cells in the optic stalk have characteristic bipolar
morphology and form a single-cell monolayer covered by a basement membrane
(BM). Optic stalk expansion occurs before R axon innervation. Moreover, even
in those mutants with no R axon innervation, SG cells form a single-cell-layer
tube that develops normally. Therefore, we conclude that SG cells autonomously
form the optic stalk. We then sought to elucidate the molecular mechanisms
underlying optic stalk formation. In screening for mutants that exhibit
defects in R axon innervation or for genes that are specifically expressed in
the optic stalk, we identified the Fak56D and CdGAPr genes
as encoding important components required for optic stalk formation.
Fak56D (Fox et al.,
1999; Fujimoto et al.,
1999; Palmer et al.,
1999) is a Drosophila homolog of mammalian focal adhesion
kinase (FAK; also known as Ptk2), which is known to be a main regulatory
component of focal contacts (Parsons,
2003; Mitra et al.,
2005). Focal contacts are large integrin complexes that anchor the
cytoskeleton of cells to the extracellular matrix
(Critchley, 2000). While focal
contacts generate traction forces during migration, their disassembly is also
crucial to the control of cell migration
(Lauffenburger and Horwitz,
1996; Webb et al.,
2002; Webb et al.,
2004). FAK is involved in cell migration through disassembly of
focal contacts (Ilic et al.,
1995; Gilmore and Romer,
1996; Parsons et al.,
2000), or through regulation of cytoskeletal rearrangement via Rho
GTPases (Hildebrand et al.,
1996; Zhai et al.,
2003). In Drosophila, several studies indicate that
Fak56D also acts in focal contacts in a similar way to that of mammalian FAK.
Fak56D is tyrosine-phosphorylated and localized to focal contacts upon the
plating of embryonic cells onto extracellular matrix components
(Palmer et al., 1999).
However, as no Fak56D loss-of-function phenotype has been reported,
the in vivo function of Fak56D remains elusive.
In Fak56D mutant animals, SG cells were not arranged into a
tubular structure, although cell proliferation and differentiation were
normal. Clone labeling analysis suggested that SG cell distribution along the
anteroposterior (AP) axis was defective in Fak56D mutants. We also
identified CdGAPr as a possible functional partner of
Fak56D. CdGAPr has a GTPase activating domain that is homologous to
that of mammalian CdGAP (Sagnier et al.,
2000). It has been shown that mammalian CdGAP regulates cell
cytoskeletal rearrangement through Rac or Cdc42
(Lamarche-Vane and Hall,
1998); however, in vivo function of neither CdGAP nor CdGAPr has
been described. We found that loss-of-function alleles of CdGAPr
exhibited a Fak56D-like phenotype. Moreover, a strong genetic
interaction was observed between Fak56D and CdGAPr,
indicating that these genes act together to regulate SG cell behavior.
Recently, mammalian CdGAP was shown to localize to focal contacts, and to be
required for regulation of cell morphology and motility
(LaLonde et al., 2006). Our
results provide strong evidence that optic stalk shape is controlled by
mechanisms acting autonomously in SG cells, in which Fak56D and
CdGAPr play crucial roles.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
GMR-Gal4 was described previously (Hay et
al., 1997). UAS-GFP encodes a cytoplasmic form of GFP and
UAS-mCD8::GFP encodes a membrane targeted form of GFP
(Lee and Luo, 2001).
Act>CD2>Gal4 was described previously
(Pignoni and Zipursky, 1997).
Df(2R)BSC26 (Parks et al.,
2004), EY13451 (Bellen et al.,
2004), ED(2L)1303 (Ryder et
al., 2004) and CdGAPr-6.1
(Billuart et al., 2001) were
obtained from the Bloomington Stock Center. Fak56DCG1 was
described previously (Grabbe et al.,
2004), as was mys1 and
mysXG43 (Bunch et al.,
1992), UAS-Fak56D
(Palmer et al., 1999),
sine oculis1 (Kunes et
al., 1993) and ptcGal4 (G559.1)
(Shyamala and Bhat, 2002).
NP4702-Gal4, NP2109-Gal4 and NP3053-Gal4 were isolated from a screening of
enhancer trap lines (NP lines) (Hayashi et
al., 2002) for SG cell-specific expression.
Immunohistochemistry
Immunohistochemistry was performed as described previously
(Huang and Kunes, 1996).
Rabbit anti-Perlecan (also known as Trol - FlyBase)
(Friedrich et al., 2000) and
rabbit anti-LamininA (Gutzeit et al.,
1991) were used at a dilution of 1:3000. Mouse anti-Repo, mouse
anti-Dac and mouse anti-Caoptin (referred to as mAb24B10 throughout the text)
were obtained from the Developmental Studies Hybridoma Bank and were used at a
1:10 dilution. Rabbit anti-phospho-Histone H3 (Upstate Biotechnology) was used
at a 1:200 dilution. Mouse anti-
-Tubulin (Sigma) was used at a dilution
of 1:500. Rabbit anti-ß-galactosidase (ß-gal) (Cappel) was used at a
dilution of 1:1000. Goat Cy3 anti-HRP (Accurate Chemical and Scientific) was
used at a dilution of 1:200. Following secondary antibodies (Jackson) were
used at 1:200 dilutions: anti-mouse Cy3, anti-mouse Cy5, anti-mouse FITC,
anti-rabbit Cy3, anti-rabbit Cy5. Specimens were viewed on a Zeiss LSM510
confocal microscope.
Quantitative analysis of the optic stalk expansion
For analysis of the optic stalk growth, the number of pixels within an area
of the optic stalk cross section encircled by the BM, which is visualized with
anti-Perlecan, was calculated with Adobe Photoshop. Statistical analysis was
performed using Microsoft Excel. Statistical differences were compared by
Student's t-test.
In situ hybridization
In situ hybridization was performed as described previously
(Nagaso et al., 2001). Sense
and antisense RNA probes were synthesized using the DIG RNA labeling kit
(Roche).
Analysis of mRNA levels by real-time PCR
Total RNA was isolated from late third instar larval brains of yw
or CdGAPrNP3053 homozygotes. Reverse transcription was
performed with 3 µg total RNA and Superscript II reverse transcriptase
(Invitrogen). The resulting cDNA was amplified by SYBR Premix Ex Taq (Takara),
and products were detected on an ABI PRISM 7000 (Applied Biosystems). The fold
decrease of CdGAPr transcripts was calculated relative to Actin
5C transcripts.
The primers used for real-time PCR were: CdGAPr (5'-AATCGCCCACTTTCAGTGTC-3' and 5'-GGTACGCTCAGTTCGTTAGG-3') (123 bp); Actin 5C (5'-AAGTGCGAGTGGTGGAAGT-3' and 5'-CATGCGCCCAAAACGATGA-3') (125 bp).
Labeling a subset of SG cells with mCD8GFP
For labeling a subset of SG cells, males with the genotype of
Actin>CD2>Gal4 were mated to
hsflp122; UAS-CD8::GFP females. For labeling a subset of
SG cells in Fak56D mutants, males with the genotype of
Actin>CD2>Gal4;Fak56DCG1/CyoAct
were mated to hsflp122;
Fak56DCG1/CyoAct; UAS-CD8::GFP females. The progeny at
60-84 hours after egg laying (AEL) were heat shocked (37°C for 5 minutes).
Dissections were carried out at late third instar larval stage.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Halter et al., 1995)
(Fig. 1B). These cells are WG
and SG, as classified according to their position, morphology and marker
expression (Fig. 1I). We found
that ptcGal4 and NP4702-Gal4 specifically labeled WG and SG,
respectively, at late third instar larval stage. Using green fluorescent
protein (GFP), we closely observed the morphology of WG and SG cells. As
described previously (Hummel et al.,
2002), WG cell bodies are located in the eye disc or inside the
lumen of the optic stalk and are intermingled with R axons. They extend long
processes along R axons and establish axonal ensheathment
(Fig. 1C). Each WG cell process
wraps several ommatidial R axons into a bundle within the optic stalk.
However, NP4702-expressing SG cell bodies are located in the optic stalk
closely associated with the R axon bundle, but never intermingled with R axons
(Fig. 1D)
(Hummel et al., 2002). We
found that SG cells have long processes extending along the AP axis
(Fig. 1E,E'). These SG
cells form a monolayered tube that ensheathes the entire R axon bundle
(Fig. 1F). We measured the size
of the optic stalk and found that it was invariable, suggesting that size of
the optic stalk structure is precisely regulated
(Fig. 1G,H). We also found that
the optic stalk is covered by the BM. Major components of the BM, LamininA and
Perlecan, were localized on the surface of the SG cells
(Fig. 1F,G and
Fig. 2). Together, SG cells
form a monolayered tubular structure that is covered by the BM
(Fig. 1J), resulting in the
formation of the outer sheath of R axons. This SG cell tubular structure
corresponds to the optic stalk (Steller et
al., 1987; Kunes and Steller,
1993).
Optic stalk growth is regulated independently of R axon innervation
The Drosophila larva has a visual system much simpler than that of
the adult. This structure is known as Bolwig's organ, consists of 12
photoreceptors and develops in the embryonic stage. In disconnected
mutant larvae, Bolwig's nerves fail to project properly, which leads to
failure of optic stalk formation and R axon projection
(Steller et al., 1987). These
results indicate that the optic stalk is formed along Bolwig's nerves and
later serves as the R axon pathway. However, development of the optic stalk at
a later stage is largely unknown. We examined optic stalk development and
asked if R axons are required for development of the optic stalk. At second
instar larval stage before photoreceptor cell differentiation, glial cells
form a tubular structure in which Bolwig's nerves extend
(Fig. 2A,A',C). The SG
cell marker (NP4702-Gal4) is expressed in these glial cells
(Fig. 2C). At early third
instar stage the R axons have yet to extend; however, we found that the optic
stalk expands and glial cells increase in number
(Fig. 2B,D). The mean
cross-sectional area of the early third instar optic stalk was about five
times larger than that of the late second instar
(Fig. 3E). In addition, on
average 38 glial cells were observed (n=25) in the early third instar
optic stalk, while only 15 glial cells on average were observed in late second
instar larvae (n=13). These data indicate that the optic stalk
undergoes expansion at larval stages before R axon innervation.
|
We next examined if optic stalk formation is affected in the absence of R
axons. In sine oculis (so) mutants eye formation is
defective, and thus no R axons project
(Kunes et al., 1993;
Perez and Steller, 1996).
However, we found that in these mutants the optic stalk grew as normally as
the wild type, although it failed to maintain a round-shaped cross section
(Fig. 3A-F). These data
indicate that the proliferation and arrangement of SG cells are autonomously
regulated and independent of R axons.
|
|
). We
then searched mutants for abnormal R axon projection and identified
Fak56D. A null mutant allele for Fak56DCG1
(Grabbe et al., 2004) showed a
defect in R axon bundle formation at the optic stalk
(Fig. 4A,B). We found that the
optic stalk was also severely disrupted in the Fak56D mutant
(Fig. 4C). As the severity of
the optic stalk phenotype was variable, we classified the phenotype into five
groups (Fig. 4C). Class 0
represents the wild-type phenotype, in which the length is at least two times
longer than the diameter. In class 1, length is shortened or the diameter is
broadened but length is still longer than diameter. In class 2, length is
equal to diameter. In class 3, length is shorter than diameter. In class 4,
length is much shorter than diameter and the optic stalk is only barely seen.
In order to avoid ambiguity, we considered classes 2-4 to be optic stalk
defective. According to this classification, about 80% of
Fak56DCG1 animals exhibited significant defects in optic
stalk morphogenesis (Fig. 4D).
The phenotype of Fak56DCG1 in trans to a
deficient chromosome that uncovers the Fak56D locus was
indistinguishable from that of Fak56DCG1 homozygotes
(Fig. 4D). We never observed a
shortened optic stalk with a diameter larger than its length in late second
instar Fak56D mutants, although slightly truncated optic stalks were
occasionally observed (Fig.
4E). Defects in optic stalk morphology became clearer and more
severe as development proceeded to the third instar stage
(Fig. 4E). Therefore, these
data indicate that Fak56D is not required for the initial
establishment of the optic stalk but is required during optic stalk
expansion.
|
). We expressed Fak56D using NP2109-Gal4, which is
specifically expressed in SG cells and WG cells but not in photoreceptor cells
(Fig. 5A). Alternatively we
used NP4702-Gal4, which is expressed in SG cells but not in photoreceptor
cells (Fig. 5B). In both cases
the optic stalk defect was significantly rescued
(Fig. 5D,E,G). In contrast,
expression of Fak56D only in photoreceptor cells using GMR-Gal4
(Fig. 5C) did not rescue the
optic stalk defect (Fig. 5F,G).
These data provide strong evidence that Fak56D is autonomously
required in SG cells for optic stalk formation but is not required in R axons.
We also found that overexpressed Fak56D in a wild-type background resulted in
a thinner optic stalk (Fig.
5H), further supporting a crucial role for Fak56D in
controlling optic stalk morphology.
SG cell arrangement is disrupted in Fak56D mutants
We next investigated which process of SG cell development is affected in
Fak56D mutants. Mammalian FAK is reported to function in the
regulation of cell proliferation (Gilmore
and Romer, 1996). However, during the early larval stages in which
SG cells proliferate, no significant difference in SG cell numbers was
observed between wild type and Fak56D mutants. Therefore, it is
unlikely that Fak56D plays a role in regulation of SG cell
proliferation during optic stalk expansion. We then examined the morphogenesis
of the optic stalk. In wild type, strong expression of -Tubulin was
observed in SG cells within the optic stalk
(Fig. 6A). In Fak56D
mutants, the intensity of -Tubulin was preserved. However, in contrast
to wild type, the SG cells failed to form a tubular structure but were instead
spread over the optic lobe surface (Fig.
6B). This abnormal distribution of SG cells was also confirmed
using ombP1 as an alternative SG cell marker (data not
shown). We also found that this defect in SG cell localization occurs before R
axon innervation (Fig.
6C-D'), thus supporting the hypothesis that the primary
defect of the Fak56D mutant is in SG cells.
To gain more insight into the Fak56D phenotype, we examined how SG cells are distributed during optic stalk formation. We found that clonally labeled SG cells with GFP are always distributed along the AP axis in a chain-like shape (Fig. 6E). We almost never observed clones that spread along the dorsoventral axis (n=58). This suggests that SG cells are actively arranged during optic stalk expansion rather than simply distributed as a result of cell division. In Fak56D mutants, SG cells exhibited the same elongated morphology as wild type (Fig. 6F). However, we occasionally observed SG cells that failed to correctly distribute along the AP axis (Fig. 6G). In order to precisely quantify the SG cell distribution, we generated clones consisting of a few cells in wild type or Fak56D mutants and examined the positions of SG cells. We found that SG cells in Fak56D mutants dispersed along the AP axis to a lesser extent than wild-type cells examined under the same conditions (Fig. 6H). Mean distance between SG cells within clones was shorter (P<0.001) in Fak56D mutants (18.6±1.8 µm; n=46,) than in wild type (29.4±2.1 µm; n=47).
|
) with NP3053-Gal4 resulted in this similar optic stalk defect
(Fig. 7F). Therefore, these
genetic data suggest that CdGAPr is required for optic stalk
formation. We also found that CdGAPr and Fak56D genetically interact. Trans-heterozygous mutants for CdGAPr and Fak56D exhibited the same defect as Fak56D homozygotes in optic stalk formation (Fig. 7G). In comparison, heterozygous mutations in either gene alone exhibited only slight defects in optic stalk formation (Fig. 7G). These data suggest that CdGAPr acts together with Fak56D within a common genetic pathway.
Mammalian FAK and CdGAP are known to act in integrin signaling. We next
tested whether integrins and Fak56D act within the same signaling cascade.
Integrins function as heterodimers of one and one ß subunit. Two
genes encode integrin ß subunits in the fly, ßPS and ß
. We
found that null mutants for myospheroid, which encodes ßPS,
showed significant genetic interaction with Fak56D and animals
trans-heterozygous for myospheroid and Fak56D exhibited a
similar phenotype to Fak56D homozygotes
(Fig. 7G). This suggests that
ßPS acts together with Fak56D in optic stalk
morphogenesis.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Dakubo et al.,
2003). Moreover, initial formation of the optic stalk during
embryogenesis was shown to be dependent on the axons from larval photoreceptor
cells (Steller et al., 1987).
However, our data demonstrate that SG cells in Drosophila form the
optic stalk independently of R axons. First, SG cells proliferate before R
axon innervation, which leads to the expansion of the optic stalk. Second, in
so1 mutants the optic stalk develops normally despite
complete lack of R axon innervation. In addition, specific expression of a
Fak56D transgene in SG but not photoreceptor cells rescued a defect in optic
stalk morphology, hence indicating the presence of an intrinsic mechanism in
SG cells to regulate optic stalk morphogenesis. Some kinds of glial cells are
reported to develop independently of axon innervation in a similar way to SG
cells. For instance, during Drosophila visual system development,
retinal basal glia (RBG) are derived from the optic stalk and migrate into the
eye disc. This process was shown to be independent of R axons
(Rangarajan et al., 1999). In
these cases, glial cells must behave by unknown mechanisms independent of axon
cues. As our results demonstrated that SG cell development is highly
independent of R axons, this system can provide an excellent model to
elucidate mechanisms underlying axon-independent glial development and
behavior.
|
;
Lois and Alvarez-Buylla, 1994;
Wichterle et al., 1997). Such
directed distribution of SG cells is likely to be necessary for keeping the
optic stalk thin and long. The mechanism regulating tube morphogenesis is
largely unknown. Our finding that SG cells undergo cell arrangement during
optic stalk expansion provides an interesting insight into the morphogenesis
of a tubular structure. It is known that the optic stalk disappears during the pupal stage and that the ommatidia are set much closer to the optic lobe. We found that SG cells begin to locate at the surface of the optic lobe during early pupal stages (data not shown). This is similar to what we observed in Fak56D mutants during larval stages. It seems that the optic stalk is degenerating during the pupal stage through relocation of SG cells to the optic lobe, and this process might be regulated by FAK activity.
The role of Fak56D in optic stalk morphogenesis
In an attempt to identify the molecular mechanisms underlying optic stalk
formation, we found that the optic stalk was disrupted in Fak56D
mutants. Expression of a Fak56D transgene in SG cells, but not
photoreceptor cells, rescued the defect. This indicates that Fak56D
is autonomously required in SG cells. Fak56D is apparently required
during the expansion of the optic stalk. As we did not detect any differences
in number of SG cells between wild type and Fak56D mutants, it is
unlikely that Fak56D regulates the proliferation of SG cells. We
found that SG cells were mis-localized on the surface of the optic lobe in
Fak56D mutants instead of forming a tubular structure. Visualization
of newly divided cells by clonal labeling suggests that SG cells tend to
migrate along the AP axis, and this could lead to formation of a longitudinal
tube structure (Fig. 8). In
Fak56D mutants SG cells partially lose tendency to migrate along the
AP axis; this could lead to enlargement of the diameter as opposed to the
length. Ectopic localization of SG cells on the optic lobe observed in
Fak56D mutants is likely to be the result of the optic stalk
widening. Mammalian FAK plays a central role in cell migration
(Mitra et al., 2005); hence in
Drosophila Fak56D may regulate optic stalk formation via regulation
of cell migration.
|
|
). In addition, it has been shown that overexpression of
a kinase-defective mutant of FAK in cultured cells blocks the
hyperosmolarity-induced E-cadherin accumulation at the cell periphery
(Quadri et al., 2003). As SG
cells are closely attached to each other, adhesion between SG cells may be
important for keeping optic stalk tubular structure. SG cells are also
attached to the BM throughout optic stalk formation. Because Fak56D was
implied to regulate integrin adhesion
(Grabbe et al., 2004), it is
possible that Fak56D is required for SG cells to form proper adhesion to the
BM.
CdGAPr and ßPS integrin act together with Fak56D
In addition to Fak56D, we also identified CdGAPr as a
regulator of optic stalk morphology. Fak56D and CdGAPr
exhibited a strong genetic interaction. As both mammalian FAK and CdGAPr are
known to act at focal contacts, this raised the possibility that focal
contacts are important for optic stalk morphogenesis. Main components of focal
contacts, such as integrins or Paxillin, are conserved between vertebrates and
invertebrates (Wilcox et al.,
1989; Bokel and Brown,
2002; Chen et al.,
2005). In Drosophila, Fak56D is implicated in
integrin-involving molecular mechanisms
(Palmer et al., 1999;
Grabbe et al., 2004). We found
a genetic interaction between Fak56D and myospheroid
(mys), which encodes a ß-subunit of integrin, ßPS. This
suggests that Fak56D and CdGAPr act together with integrins in focal contacts
to regulate SG cell behavior. Because the interaction between
Fak56DCG1 and mys1 (a null allele) is
much weaker than the interaction between Fak56DCG1 and
CdGAPr hypomorphic alleles, it is possible that another ß
subunit, ß, compensates for the loss of ßPS. ßPS and
ß were shown to act together to regulate midgut cell migration
(Devenport and Brown, 2004).
It is also possible that Fak56D function is regulated via other receptors,
such as G-protein coupled receptors (GPCRs). Mammalian Pyk2, another FAK
family member, is known to be activated via GPCRs
(Dikic et al., 1996;
Yu et al., 1996).
CdGAPr encodes a GAP domain that regulates the activity of
Rho-family GTPases. The mammalian CdGAP
(Lamarche-Vane and Hall, 1998;
LaLonde et al., 2006) was
shown to regulate Rac and Cdc42, which are known regulators of the actin
cytoskeleton (Nobes and Hall,
1995), and was reported to be required for cell morphology and
motility (Etienne-Manneville and Hall,
2002). This raises the possibility that FAK regulates cytoskeletal
rearrangement via activation of CdGAP, although exact functional interaction
between the two proteins remains elusive. In fact, the mammalian FAK interacts
directly with guanine nucleotide exchange factors or GTPase-activating
proteins in the regulation of cytoskeleton
(Hildebrand et al., 1996;
Liu et al., 2002;
Medley et al., 2003;
Zhai et al., 2003). More
biochemical studies are required for elucidating the molecular mechanisms that
underlie cell behaviors regulated by focal adhesion signaling.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W.,
Tsang, G., Evans-Holm, M., Hiesinger, P. R., Schulze, K. L., Rubin, G. M. et
al. (2004). The BDGP gene disruption project: single
transposon insertions associated with 40% of Drosophila genes.
Genetics 167,761
-781.
Billuart, P., Winter, C. G., Maresh, A., Zhao, X. and Luo, L. (2001). Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway. Cell 107,195 -207.[CrossRef][Medline]
Bokel, C. and Brown, N. H. (2002). Integrins in development: moving on, responding to, and sticking to the extracellular matrix. Dev. Cell 3,311 -321.[CrossRef][Medline]
Booth, G. E., Kinrade, E. F. and Hidalgo, A. (2000). Glia maintain follower neuron survival during Drosophila CNS development. Development 127,237 -244.[Abstract]
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]
Bunch, T. A., Salatino, R., Engelsgjerd, M. C., Mukai, L., West, R. F. and Brower, D. L. (1992). Characterization of mutant alleles of myospheroid, the gene encoding the beta subunit of the Drosophila PS integrins. Genetics 132,519 -528.[Abstract]
Chen, G. C., Turano, B., Ruest, P. J., Hagel, M., Settleman, J.
and Thomas, S. M. (2005). Regulation of Rho and Rac signaling
to the actin cytoskeleton by paxillin during Drosophila development.
Mol. Cell. Biol. 25,979
-987.
Chotard, C. and Salecker, I. (2004). Neurons and glia: team players in axon guidance. Trends Neurosci. 27,655 -661.[CrossRef][Medline]
Clandinin, T. R. and Zipursky, S. L. (2002). Making connections in the fly visual system. Neuron 35,827 -841.[CrossRef][Medline]
Critchley, D. R. (2000). Focal adhesions-the cytoskeletal connection. Curr. Opin. Cell Biol. 12,133 -139.[CrossRef][Medline]
Dakubo, G. D., Wang, Y. P., Mazerolle, C., Campsall, K.,
McMahon, A. P. and Wallace, V. A. (2003). Retinal ganglion
cell-derived sonic hedgehog signaling is required for optic disc and stalk
neuroepithelial cell development. Development
130,2967
-2980.
Devenport, D. and Brown, N. H. (2004).
Morphogenesis in the absence of integrins: mutation of both Drosophila beta
subunits prevents midgut migration. Development
131,5405
-5415.
Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S. A. and Schlessinger, J. (1996). A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature 383,547 -550.[CrossRef][Medline]
Etienne-Manneville, S. and Hall, A. (2002). Rho GTPases in cell biology. Nature 420,629 -635.[CrossRef][Medline]
Fox, G. L., Rebay, I. and Hynes, R. O. (1999).
Expression of DFak56, a Drosophila homolog of vertebrate focal adhesion
kinase, supports a role in cell migration in vivo. Proc. Natl.
Acad. Sci. USA 96,14978
-14983.
Freeman, M. R. and Doherty, J. (2006). Glial cell biology in Drosophila and vertebrates. Trends Neurosci. 29,82 -90.[CrossRef][Medline]
Friedrich, M. V., Schneider, M., Timpl, R. and Baumgartner, S. (2000). Perlecan domain V of Drosophila melanogaster. Sequence, recombinant analysis and tissue expression. Eur. J. Biochem. 267,3149 -3159.[Medline]
Fujimoto, J., Sawamoto, K., Okabe, M., Takagi, Y., Tezuka, T.,
Yoshikawa, S., Ryo, H., Okano, H. and Yamamoto, T. (1999).
Cloning and characterization of Dfak56, a homolog of focal adhesion kinase, in
Drosophila melanogaster. J. Biol. Chem.
274,29196
-29201.
Gilmore, A. P. and Romer, L. H. (1996). Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol. Biol. Cell 7,1209 -1224.[Abstract]
Gilmour, D. T., Maischein, H. M. and Nusslein-Volhard, C. (2002). Migration and function of a glial subtype in the vertebrate peripheral nervous system. Neuron 34,577 -588.[CrossRef][Medline]
Grabbe, C., Zervas, C. G., Hunter, T., Brown, N. H. and Palmer,
R. H. (2004). Focal adhesion kinase is not required for
integrin function or viability in Drosophila.
Development 131,5795
-5805.
Gutzeit, H. O., Eberhardt, W. and Gratwohl, E.
(1991). Laminin and basement membrane-associated microfilaments
in wild-type and mutant Drosophila ovarian follicles. J. Cell
Sci. 100,781
-788.
Halter, D. A., Urban, J., Rickert, C., Ner, S. S., Ito, K., Travers, A. A. and Technau, G. M. (1995). The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster. Development 121,317 -332.[Abstract]
Hay, B. A., Maile, R. and Rubin, G. M. (1997).
P element insertion-dependent gene activation in the Drosophila eye.
Proc. Natl. Acad. Sci. USA
94,5195
-5200.
Hayashi, S., Ito, K., Sado, Y., Taniguchi, M., Akimoto, A., Takeuchi, H., Aigaki, T., Matsuzaki, F., Nakagoshi, H., Tanimura, T. et al. (2002). GETDB, a database compiling expression patterns and molecular locations of a collection of Gal4 enhancer traps. Genesis 34,58 -61.[CrossRef][Medline]
Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (1996). An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase. Mol. Cell. Biol. 16,3169 -3178.[Abstract]
Huang, Z. and Kunes, S. (1996). Hedgehog, transmitted along retinal axons, triggers neurogenesis in the developing visual centers of the Drosophila brain. Cell 86,411 -422.[CrossRef][Medline]
Hummel, T., Attix, S., Gunning, D. and Zipursky, S. L. (2002). Temporal control of glial cell migration in the Drosophila eye requires gilgamesh, hedgehog, and eye specification genes. Neuron 33,193 -203.[CrossRef][Medline]
Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M. and Yamamoto, T. (1995). Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377,539 -544.[CrossRef][Medline]
Kunes, S. (2000). Axonal signals in the assembly of neural circuitry. Curr. Opin. Neurobiol. 10, 58-62.[CrossRef][Medline]
Kunes, S. and Steller, H. (1993). Topography in the Drosophila visual system. Curr. Opin. Neurobiol. 3, 53-59.[CrossRef][Medline]
Kunes, S., Wilson, C. and Steller, H. (1993). Independent guidance of retinal axons in the developing visual system of Drosophila. J. Neurosci. 13,752 -767.[Abstract]
LaLonde, D. P., Grubinger, M., Lamarche-Vane, N. and Turner, C. E. (2006). CdGAP associates with actopaxin to regulate integrin-dependent changes in cell morphology and motility. Curr. Biol. 16,1375 -1385.[CrossRef][Medline]
Lamarche-Vane, N. and Hall, A. (1998). CdGAP, a
novel proline-rich GTPase-activating protein for Cdc42 and Rac. J.
Biol. Chem. 273,29172
-29177.
Lauffenburger, D. A. and Horwitz, A. F. (1996). Cell migration: a physically integrated molecular process. Cell 84,359 -369.[CrossRef][Medline]
Lee, T. and Luo, L. (2001). Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci. 24,251 -254.[CrossRef][Medline]
Leiserson, W. M., Harkins, E. W. and Keshishian, H. (2000). Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment. Neuron 28,793 -806.[CrossRef][Medline]
Lemke, G. (2001). Glial control of neuronal development. Annu. Rev. Neurosci. 24, 87-105.[CrossRef][Medline]
Liu, Y., Loijens, J. C., Martin, K. H., Karginov, A. V. and
Parsons, J. T. (2002). The association of ASAP1, an ADP
ribosylation factor-GTPase activating protein, with focal adhesion kinase
contributes to the process of focal adhesion assembly. Mol. Biol.
Cell 13,2147
-2156.
Lois, C. and Alvarez-Buylla, A. (1994).
Long-distance neuronal migration in the adult mammalian brain.
Science 264,1145
-1148.
Luskin, M. B. (1993). Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11,173 -189.[CrossRef][Medline]
Medley, Q. G., Buchbinder, E. G., Tachibana, K., Ngo, H.,
Serra-Pages, C. and Streuli, M. (2003). Signaling between
focal adhesion kinase and trio. J. Biol. Chem.
278,13265
-13270.
Meinertzhagen, I. A. and Hanson, T. E. (1993). The development of the optic love. In The Development of Drosophla melanogaster. Vol. II (ed. M. Bate and A. Martinez Arias), pp. 1363-1491. Plainview, NY: Cold Spring Harbor Laboratory Press.
Mitra, S. K., Hanson, D. A. and Schlaepfer, D. D. (2005). Focal adhesion kinase: in command and control of cell motility. Nat. Rev. Mol. Cell Biol. 6, 56-68.[CrossRef][Medline]
Nagaso, H., Murata, T., Day, N. and Yokoyama, K. K.
(2001). Simultaneous detection of RNA and protein by in situ
hybridization and immunological staining. J. Histochem.
Cytochem. 49,1177
-1182.
Nobes, C. D. and Hall, A. (1995). Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81,53 -62.[CrossRef][Medline]
Palmer, R. H., Fessler, L. I., Edeen, P. T., Madigan, S. J.,
McKeown, M. and Hunter, T. (1999). DFak56 is a novel
Drosophila melanogaster focal adhesion kinase. J. Biol.
Chem. 274,35621
-35629.
Parks, A. L., Cook, K. R., Belvin, M., Dompe, N. A., Fawcett, R., Huppert, K., Tan, L. R., Winter, C. G., Bogart, K. P., Deal, J. E. et al. (2004). Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat. Genet. 36,288 -292.[CrossRef][Medline]
Parsons, J. T. (2003). Focal adhesion kinase:
the first ten years. J. Cell Sci.
116,1409
-1416.
Parsons, J. T., Martin, K. H., Slack, J. K., Taylor, J. M. and Weed, S. A. (2000). Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement. Oncogene 19,5606 -5613.[CrossRef][Medline]
Perez, S. E. and Steller, H. (1996). Migration of glial cells into retinal axon target field in Drosophila melanogaster. J. Neurobiol. 30,359 -373.[CrossRef][Medline]
Pignoni, F. and Zipursky, S. L. (1997). Induction of Drosophila eye development by decapentaplegic. Development 124,271 -278.[Abstract]
Quadri, S. K., Bhattacharjee, M., Parthasarathi, K., Tanita, T.
and Bhattacharya, J. (2003). Endothelial barrier
strengthening by activation of focal adhesion kinase. J. Biol.
Chem. 278,13342
-13349.
Rangarajan, R., Gong, Q. and Gaul, U. (1999). Migration and function of glia in the developing Drosophila eye. Development 126,3285 -3292.[Abstract]
Ryder, E., Blows, F., Ashburner, M., Bautista-Llacer, R.,
Coulson, D., Drummond, J., Webster, J., Gubb, D., Gunton, N., Johnson, G. et
al. (2004). The DrosDel collection: a set of P-element
insertions for generating custom chromosomal aberrations in Drosophila
melanogaster. Genetics
167,797
-813.
Sagnier, T., Grienenberger, A., Mariol, M., Berenger, H., Pradel, J. and Graba, Y. (2000). Dynamic expression of d-CdGAPr, a novel Drosophila melanogaster gene encoding a GTPase activating protein. Mech. Dev. 94,267 -270.[CrossRef][Medline]
Shyamala, B. V. and Bhat, K. M. (2002). A positive role for patched-smoothened signaling in promoting cell proliferation during normal head development in Drosophila. Development 129,1839 -1847.
Steller, H., Fischbach, K. F. and Rubin, G. M. (1987). Disconnected: a locus required for neuronal pathway formation in the visual system of Drosophila. Cell 50,1139 -1153.[CrossRef][Medline]
Tsai, S. F., Jang, C. C., Prikhod'ko, G. G., Bessarab, D. A.,
Tang, C. Y., Pflugfelder, G. O. and Sun, Y. H. (1997). Gypsy
retrotransposon as a tool for the in vivo analysis of the regulatory region of
the optomotor-blind gene in Drosophila. Proc. Natl. Acad. Sci.
USA 94,3837
-3841.
Wallace, V. A. and Raff, M. C. (1999). A role for Sonic hedgehog in axon-to-astrocyte signalling in the rodent optic nerve. Development 126,2901 -2909.[Abstract]
Webb, D. J., Parsons, J. T. and Horwitz, A. F. (2002). Adhesion assembly, disassembly and turnover in migrating cells - over and over and over again. Nat. Cell Biol. 4,E97 -E100.[CrossRef][Medline]
Webb, D. J., Donais, K., Whitmore, L. A., Thomas, S. M., Turner, C. E., Parsons, J. T. and Horwitz, A. F. (2004). FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 6,154 -161.[CrossRef][Medline]
Wichterle, H., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1997). Direct evidence for homotypic, glia-independent neuronal migration. Neuron 18,779 -791.[CrossRef][Medline]
Wilcox, M., DiAntonio, A. and Leptin, M.
(1989). The function of PS integrins in Drosophila wing
morphogenesis. Development
107,891
-897.
Xiong, W. C., Okano, H., Patel, N. H., Blendy, J. A. and
Montell, C. (1994). repo encodes a glial-specific homeo
domain protein required in the Drosophila nervous system. Genes
Dev. 8,981
-994.
Yano, H., Mazaki, Y., Kurokawa, K., Hanks, S. K., Matsuda, M.
and Sabe, H. (2004). Roles played by a subset of integrin
signaling molecules in cadherin-based cell-cell adhesion. J. Cell
Biol. 166,283
-295.
Yu, H., Li, X., Marchetto, G. S., Dy, R., Hunter, D., Calvo, B.,
Dawson, T. L., Wilm, M., Anderegg, R. J., Graves, L. M. et al.
(1996). Activation of a novel calcium-dependent protein-tyrosine
kinase. Correlation with c-Jun, N-terminal kinase but not mitogen-activated
protein kinase activation. J. Biol. Chem.
271,29993
-29998.
Zhai, J., Lin, H., Nie, Z., Wu, J., Canete-Soler, R.,
Schlaepfer, W. W. and Schlaepfer, D. D. (2003). Direct
interaction of focal adhesion kinase with p190RhoGEF. J. Biol.
Chem. 278,24865
-24873.
