RESEARCH ARTICLE 1539
Development 134, 1539-1548 (2007) doi:10.1242/dev.001529
Focal adhesion kinase controls morphogenesis of the
Drosophila optic stalk
Satoshi Murakami1,2, Daiki Umetsu1,2, Yuko Maeyama1, Makoto Sato1,2, Shoko Yoshida1,2 and
Tetsuya Tabata1,2,*
Photoreceptor cell axons (R axons) innervate optic ganglia in the Drosophila brain through the tubular optic stalk. This structure
consists of surface glia (SG) and forms independently of R axon projection. In a screen for genes involved in optic stalk formation,
we identified Fak56D encoding a Drosophila homolog of mammalian focal adhesion kinase (FAK). FAK is a main component of the
focal adhesion signaling that regulates various cellular events, including cell migration and morphology. We show that Fak56D
mutation causes severe disruption of the optic stalk structure. These phenotypes were completely rescued by Fak56D transgene
expression in the SG cells but not in photoreceptor cells. Moreover, Fak56D genetically interacts with myospheroid, which encodes
an integrin ␤ subunit. In addition, we found that CdGAPr is also required for optic stalk formation and genetically interacts with
Fak56D. CdGAPr encodes a GTPase-activating domain that is homologous to that of mammalian CdGAP, which functions in focal
adhesion signaling. Hence the optic stalk is a simple monolayered structure that can serve as an ideal system for studying glial cell
morphogenesis and the developmental role(s) of focal adhesion signaling.
INTRODUCTION
Glial cells play important roles during the formation of the nervous
system. For instance, they provide trophic support to neurons,
modulate axon pathfinding and guide nerve fasciculation (Booth et
al., 2000; 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
1
Laboratory of Morphogenesis, Institute of Molecular and Cellular Biosciences, the
University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan. 2Graduate
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: [email protected])
Accepted 8 February 2007
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
DEVELOPMENT
KEY WORDS: Fak56D, CdGAPr, Optic stalk, Glia, Drosophila
1540 RESEARCH ARTICLE
MATERIALS AND METHODS
Fly stocks
The following mutants and transgenic strains were used: yw flies were used
as wild-type controls. optomotor-blindP1 (ombP1) is a reporter construct that
contains a P-element carrying lacW upstream of the omb (bifid – FlyBase)
gene (Tsai et al., 1997). GMR-Gal4 was described previously (Hay et al.,
1997). UAS-GFP encodes a cytoplasmic form of GFP and UASmCD8::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-␤-galactosidase (␤-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, antirabbit 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
␮g 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⬘-GGTACGCTCAGTTCGTTAGG3⬘) (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
Surface glial cells are organized into a
monolayered tubular structure and form the optic
stalk
During development of the Drosophila adult visual system, R axons
extend toward the brain through the optic stalk (Fig. 1A). In the optic
stalk, two types of glial cells express the glia-specific transcription
factor Repo (Xiong et al., 1994; 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
DEVELOPMENT
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.
Development 134 (8)
Focal adhesion signaling regulates glial cell arrangement
RESEARCH ARTICLE 1541
Fig. 1. SG cells form the optic stalk,
the monolayered tubular structure
that surrounds the R axon bundle.
Unless otherwise noted, all specimens
are late third instar viewed from a
lateral perspective. Gal4 expression
was detected with UAS-mCD8GFP
(green) and glial cells with anti-Repo
(Blue), except where noted.
(A) Developing Drosophila visual
system. Photoreceptor cells extend
their axons (R axons; white) toward
the target region, the lamina (green,
anti-Dachshund), in the optic lobe. R
axons form a bundle in the optic stalk.
(B) R axons (green, visualized with
GMRGal4) are surrounded with glial
cells (white). (C) Processes of WG
(green, visualized using ptc-Gal4)
enwrap R axons (magenta, anti-HRP)
in the optic stalk (arrow). Note that
ptc-Gal4 also labels a subset of
epithelial cells of the eye disc
(arrowhead). (D) SG cells (green,
visualized using NP4702-Gal4) form
the outer sheath (arrows) that
surrounds the R axons (magenta, antiHRP). (E) An optic stalk carrying
NP4702-Gal4 and UAS-GFP. SG cells
(green) are arranged along the AP axis,
and form a sheath by tightly
contacting each other. (E⬘) A single SG cell, which is clonally labeled by mCD8GFP (green), extends two long processes anteroposteriorly. (F) An
optic stalk carrying NP4702-Gal4, viewed from a horizontal perspective. SG cells (green) form a single-cell layer. LamininA (magenta, anti-LamininA)
is detected on the surface of the SG cell layer. (G,H) Quantitative analysis of the optic stalk morphology. (G) An optic stalk stained with anti-Perlecan
(magenta). The green bar indicates the height of the optic stalk, while the white bar indicates the diameter. (H) Quantification of the average length
of height (A/P) and the diameter (D/V) of wild-type optic stalks. The error bars indicate s.d. (I,J) Schematic drawings of the optic stalk. (I) WG cell
processes (gray) loosely enwrap several R axons (magenta). SG cells (green) surround the entire R axon bundle, forming an outer sheath. (J, left) The
optic stalk consists of SG cells (green), which form a monolayered tubular structure, and is surrounded by the BM (gray). (J, right) The optic stalk
viewed from a horizontal perspective (top) or a lateral perspective (bottom). (A,B,C,D,E,G) Anterior is upwards and dorsal is to the right. (F) Lateral
down, dorsal right.
lobe. In addition, virtually no ptclacZ-positive WG cells were seen
in the optic stalk at early third instar stage, except for an occasional
appearance in the basal region of the eye disc (n=12) (Fig. 2F,F⬘).
These data suggest that optic stalk expansion is primarily or wholly
due to SG cell proliferation.
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.
Fak56D is involved in optic stalk morphogenesis
The above results clearly show that SG cells autonomously shape
the optic stalk. We then explored the molecular mechanisms
underlying SG cell development. As the optic stalk is formed before
R axon innervation (Fig. 2), disruption of this structure may affect
some aspects of the projection pattern of R axons, as has been
observed previously in disconnected mutants (Steller et al., 1987).
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
DEVELOPMENT
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 further examined how the optic stalk expands during the early
third instar larval stage. While the size of SG cells did not seem to
change much during this optic stalk expansion (compare Fig. 2C
with D), mitotic cells were observed in the optic stalk glia (Fig.
2E,E⬘). Although glial cells could migrate into the optic stalk during
the expansion, virtually no GFP labeled clones generated on the
optic lobe surface (Fig. 2G-G⬙) entered into the optic stalk.
Therefore a boundary may exist between the optic stalk and optic
1542 RESEARCH ARTICLE
Development 134 (8)
Fig. 2. The optic stalk expands before R axon innervation.
(A-D) Optic stalks at the early larval stages before R axon projection.
(A,B) Optic stalks carrying ombP1 at the late second instar (A,A⬘) and
the early third instar (B,B⬘). lacZ expression is visualized with anti-␤galactosidase (␤-gal) antibody (white). The BM is visualized with antiPerlecan. (A) At the late second instar before R axon innervation, glial
cells (white) form a tubular structure around Bolwig’s nerves (magenta,
anti-HRP). (B) At the early third instar, the optic stalk expands and glial
cells increase in number. One ommatidial column is detected in this
specimen, but R axon innervation is not observed (arrow). (C,D) A late
second instar (C) or an early third instar (D) larval optic stalk carrying
NP4702-Gal4 and UAS-mCD8GFP, viewed from horizontal perspective.
(E,E⬘) Cells in mitosis are visualized with anti-phospho-Histone H3
(magenta) in an early third instar optic stalk. Glial cells (blue, anti-Repo)
in the optic stalk undergo mitosis (arrow). Repo-positive glial cells are
shown alone in E’. (F,F⬘) An early third instar larval optic stalk carrying
NP4702-Gal4, UAS-mCD8GFP and ptc-lacZ (visualized with anti-␤-gal).
The optic stalk consists solely of SG cells (green), while no WG cells
(white) are seen in the optic stalk. (G-G⬙) Clonally labeled glial cells
surrounding the optic lobe (green) never entered into the optic stalk
(white, visualized with ␣-Tubulin). (A-G⬙, except for C and D) Anterior is
to the up, dorsal right. (C,D) Lateral is down, dorsal right. os, optic
stalk.
Fig. 3. The optic stalk is formed independently of R axon
projection. (A-D) Wild-type (A,C) and sine oculis1 (so1) (B,D) optic
stalks at the early pupal stage, viewed from a lateral (A,B) or a
horizontal (C,D) perspective. Glial cells are visualized with anti-Repo
(blue). The BM is visualized with anti-Perlecan (green). In a so1 mutant,
the optic stalk was observed, despite a complete lack of photoreceptor
cells (magenta, anti-HRP). (E) Quantification of the optic stalk growth in
wild-type (white column) and so1 mutant animals (gray column). A
cross-sectional area surrounded by the BM (green in C,D) is measured in
each specimen at larval or early pupal stages. Columns represent the
mean cross-sectional area. The mean cross-sectional area of so1 optic
stalk is not significantly different from that of wild type at the early
pupal stage (P=0.57). The error bars indicate s.d.; n.s. indicates no
significant difference. (F) A late third instar so1 mutant optic stalk
carrying NP4702-Gal4 and UAS-mCD8GFP, viewed from a horizontal
perspective. SG cells (green) form a monolayered tubular structure.
Arrows in B,D and F indicate Bolwig’s nerves. (A,B) Anterior is to the up,
dorsal right. (C,D,F) Lateral is down, dorsal right.
DEVELOPMENT
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.
Expression of a Fak56D transgene in SG cells is
sufficient to rescue the Fak56D optic stalk defect
The above results indicate that Fak56D is required for optic stalk
morphogenesis and R axon bundle formation. The Fak56D mRNA
is expressed in the third instar larvae (Fig. 4F). To address whether
Fak56D acts in SG cells or R axons, we expressed exogenous
Fak56D in Fak56D mutants using the UAS/Gal4 system (Brand and
Perrimon, 1993). 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
RESEARCH ARTICLE 1543
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
Fig. 4. Disruption of optic stalk morphology in the Fak56D mutant. (A,B) R axons in a wild type (A) and a Fak56DCG1 homozygous mutant (B),
visualized with mAb24B10 (white). In a Fak56D mutant, R axons failed to be bundled (arrow). (C) Optic stalks in a wild type (0, left) and in Fak56D
mutants (1–4), visualized with anti-Perlecan (white). Fak56D mutants exhibited defects in optic stalk morphology. Defects were classified into five
groups (0–4), according to the severity of the phenotype. A dashed line indicates the eye disc-optic lobe boundary. Arrows indicate the optic
stalk/optic lobe boundary. (D) Quantification of optic stalk defects in wild type and Fak56D mutants. The percentage of the brain that shows optic
stalk defect is plotted as a function of severity level of the phenotype (0–4 in C). (E) Quantification of optic stalk defects in wild-type animals or
Fak56DCG1 homozygous animals at various developmental stages. Defects are not detected until the early third instar larval stage in Fak56D
mutants, becoming progressively more severe during third instar larval stages. (F) Fak56D expression in the late third instar larvae. Brains and
imaginal discs were stained with the Fak56D sense or the Fak56D antisense RNA probe. Fak56D is ubiquitously expressed. (A-C) Anterior up, dorsal
right. AP, the height of the optic stalk; DV, the diameter of the optic stalk.
DEVELOPMENT
Focal adhesion signaling regulates glial cell arrangement
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
Fig. 5. Rescue of optic stalk defects in Fak56D mutants by SG-cellspecific expression of a Fak56D transgene. (A-C) Expression of Gal4
lines used for rescue experiments, visualized with UAS-mCD8GFP
(green). (D-F) Fak56D optic stalks carrying NP2109-Gal4, UAS-Fak56D
(D) or NP4702-Gal4, UAS-Fak56D (E) or GMR-Gal4, UAS-Fak56D (F).
Arrows indicate optic stalks (white, visualized with anti-Perlecan).
(G) Quantification of optic stalk defects in animals carrying constructs
as described. Expression of exogenous Fak56D in SG cells significantly
rescued optic stalk disruption, while photoreceptor cell expression of
Fak56D did not rescue the phenotype. Note that rescued optic stalks
with NP4702-Gal4 exhibit the thinner morphology than those of wild
type (asterisk). (H) Fak56D overexpression in wild type. Columns
represent mean length of optic stalks carrying NP4702-Gal4 (white
column) or NP4702-Gal4, UAS-Fak56D (gray column). Fak56D
overexpression resulted in thinner optic stalks (***P>0.001), while
length of the optic stalks did not change (P=0.73). The error bars
indicate s.d. (A-F) Anterior up, dorsal right. Genotypes for rescue:
Fak56DCG1/Fak56DCG1; NP2109/+, Fak56DCG1/Fak56DCG1; NP2109/UASFak56D, Fak56DCG1/Fak56DCG1; NP4702/+, Fak56DCG1/Fak56DCG1;
NP4702/UAS-Fak56D, Fak56DCG1/Fak56DCG1; GMR-Gal4/+,
Fak56DCG1/Fak56DCG1; GMR-Gal4/UAS-Fak56D. Genotypes for Fak56D
overexpression: NP4702/+, NP4702/UAS-Fak56D.
Development 134 (8)
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 ␮m; n=46,) than in wild
type (29.4±2.1 ␮m; n=47).
CdGAPr is also involved in optic stalk
morphogenesis
To gain additional insights into the molecular mechanisms
underlying SG cell morphogenesis, we screened 400 Gal4 enhancer
trap lines for SG-cell-specific expression and identified line NP3053
(Fig. 7A), which has an insertion in the first intron of the CdGAPr
gene (Fig. 7B). NP3053-Gal4 is expressed restrictively in SG cells
and WG cells, as well as the glial cells that surround the optic lobe
(Fig. 7A). We found that the optic stalk was severely disrupted in
animals homozygous for CdGAPrNP3053 (Fig. 7C,D). Real-time PCR
demonstrated that CdGAPr transcript level was decreased in
CdGAPrNP3053 homozygotes by approximately six-fold compared
with wild type, thus suggesting that CdGAPrNP3053 is a loss-offunction mutation. CdGAPrNP3053 in trans to another P insertion
allele CdGAPrEY13451, or in trans to the deficiency chromosome
Df(2L)ED1303, which uncovers the CdGAPr locus, also exhibited
an optic stalk defect similar to that of CdGAPrNP3053 homozygotes
(Fig. 7E). Moreover, expression of CdGAPr dsRNA (Billuart et al.,
2001) 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 ␤ subunit. Two genes encode integrin ␤ subunits in
the fly, ␤PS and ␤␷. We found that null mutants for myospheroid,
which encodes ␤PS, 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 ␤PS acts together with Fak56D in optic stalk
morphogenesis.
DISCUSSION
SG cells autonomously form the tubular structure
SG cells are regularly arranged and form the optic stalk, a
monolayered tube that surrounds the entire R axon bundle. During
larval development, the optic stalk becomes larger as SG cells
proliferate, and this seems to adjust the size of the stalk so that it can
wrap around the R axon bundle. The proliferation and
morphogenesis of SG cells could depend on signals from the
incoming R axons, because various cellular events of glial cells, such
as differentiation, migration and proliferation, have been shown to
require interaction with neuronal axons. For example, in the
developing rodent optic nerve, Sonic hedgehog (Shh) from retinal
ganglion cells (RGCs) promotes the proliferation of astrocytes
(Wallace and Raff, 1999; Dakubo et al., 2003). Moreover, initial
formation of the optic stalk during embryogenesis was shown to be
DEVELOPMENT
1544 RESEARCH ARTICLE
Focal adhesion signaling regulates glial cell arrangement
RESEARCH ARTICLE 1545
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.
other, SG cells may migrate along the nearby SG cells that have
extended processes along the AP axis. Such homotypic migration is
reported for neuronal precursors in the adult mammalian
subventricular zone (SVZ). Neuronal precursors migrate to the
olfactory bulb in chains, by sliding along each other without the
assistance of other cell types (Luskin, 1993; Lois and AlvarezBuylla, 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.
Optic stalk morphogenesis
In this study, we have elucidated cellular mechanisms underlying
optic stalk morphogenesis in Drosophila. Optic stalk morphology
and development are precisely regulated, and the optic stalk expands
throughout the larval stages via cell proliferation (Figs 1, 2). We also
found that SG cells were distributed along the AP axis during optic
stalk formation. As cells in a clone are often associated with each
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
DEVELOPMENT
Fig. 6. SG cells fail to be organized into a tubular structure in Fak56D mutants. (A,B) A wild-type (A) and a Fak56D mutant (B) optic stalk at
late third instar larval stage were stained with anti-␣-Tubulin (white). In a wild-type optic stalk, SG cells express a high level of ␣-Tubulin (white, anti␣-Tubulin) (arrow in A). In a Fak56D mutant, SG cells failed to be organized into a tubular structure and instead spread over the surface of the optic
lobe (arrow in B). (C-D⬘) A wild-type (C) or a Fak56D mutant (D) optic stalk of an early third instar larva carrying ombP1 (white, visualized with anti␤-gal). The BM is visualized with anti-Perlecan (green). Anti-HRP (magenta) stained only Bolwig’s nerves. In a Fak56D mutant at the early third instar,
SG cells spread over the surface of the optic lobe (arrow in D). (C⬘,D⬘) Glial cells are shown alone in these panels. The outlines of the optic stalks are
indicated with dashed yellow lines. (E-G) Subsets of SG cells were clonally labeled with UAS-mCD8GFP (green) in a wild type (E) or Fak56D mutants
(F,G). SG cells in the optic stalk were visualized with anti-␣-Tubulin (white). SG cells distributed along the AP axis and made contacts with each other
via their processes (arrowhead in E). In Fak56D mutants, SG cells exhibited normal elongated morphology (arrowheads in F) but occasionally failed
to distribute properly (arrows in G). (H) Quantification of SG cell distribution along the AP axis in wild type (yw) and Fak56D mutants. Positions of
SG cells in clones containing a few cells were classified into four groups.
1546 RESEARCH ARTICLE
Development 134 (8)
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
Fig. 8. A model of Fak56D and CdGAPr control of optic stalk
morphogenesis. SG cells distribute along the AP axis. This might be
important for anteroposteriorly-elongated morphology of the optic stalk.
The mutations of either Fak56D or CdGAPr may cause random
distribution of SG cells, which results in a broadened and shortened optic
stalk. Cells in the same linage are visualized with the same color. Red
arrows indicate directions towards which proliferated cells migrate. Cells
in the optic lobe are shown as orange circles. Anterior up, dorsal right.
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.
Fak56D might also be required for adhesion between SG cells.
siRNA-mediated mammalian FAK knockdown results in loss of Ncadherin-based cell-cell adhesion in Hela cells (Yano et al., 2004).
In addition, it has been shown that overexpression of a kinasedefective 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 ␤PS 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
DEVELOPMENT
Fig. 7. Identification of CdGAPr as a gene involved in optic stalk morphogenesis. (A) Expression of NP3053-Gal4, which is inserted in the
first intron of the CdGAPr, is visualized with UAS-mCD8GFP (green). NP3053-Gal4 exhibits restricted expression in SG cells (arrow) and WG cells
(arrowhead). R axons are visualized with anti-HRP (magenta). (B) CdGAPr gene organization, showing the positions of P-element insertions in the
CdGAPNP3053 and CdGAPr EY13451 lines. (C,D) Optic stalk morphology in a wild type (C) and a CdGAPrNP3053 homozygote (D), visualized with antiPerlecan (magenta). A CdGAPrNP3053 homozygote exhibited optic stalk disruption (D). Arrows indicate optic stalks. (E) Quantification of optic stalk
defects in various allelic combinations of CdGAPr alleles. (F) Quantification of optic stalk defects in animals carrying NP3053-Gal4, UAS-CdGAPrdsRNA. (G) Quantification of the optic stalk defects in trans-heterozygous combinations of Fak56D and CdGAPr alleles, or trans-heterozygous
mutants for Fak56D and myospheroid (mys). (A,C,D) Anterior up, dorsal right.
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 ␤-subunit of integrin, ␤PS. 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 ␤ subunit, ␤␷, compensates for the loss of ␤PS. ␤PS and ␤␷
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.
We thank Adrian Moore for his critical reading of the manuscript. We thank Y.
Watanabe for access to ABI PRISM 7000 and A. Kagami for assistance with
real-time PCR assay. We are grateful to all the members of Tabata laboratory
for helpful discussions during the course of the work. We also thank S.
Baumgartner for providing antibodies, and T. A. Bunch and R. H. Palmer for fly
strains. We thank the Bloomington Stock Center, Exelixis, the National Institute
of Genetics, the Szeged Drosophila Stock Center and the Kyoto Stock Center
for fly stocks. We would also like to thank the Genetic Studies Hybridoma
Bank for antibodies. This work was supported by Grants-in-Aid from the
Ministry of Education, Science and Culture of Japan and by the Toray Science
Foundation to T.T. Support for S.M. was provided by a Predoctoral Fellowship
from the Japan Society for the Promotion of Science for Japanese Junior
Scientists.
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