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First published online May 30, 2007
doi: 10.1242/10.1242/dev.004242
1 Center for Basic Neuroscience, Department of Pharmacology, NA4.301/5323 Harry
Hines Blvd, The University of Texas Southwestern Medical Center, Dallas, TX
75390, USA.
2 Solomon H. Snyder Department of Neuroscience, Howard Hughes Medical Institute,
The Johns Hopkins University School of Medicine, 725 North Wolfe Street,
Baltimore, MD 21205, USA.
Authors for correspondence (e-mails:
kolodkin{at}jhmi.edu;
jonathan.terman{at}utsouthwestern.edu)
Accepted 2 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: p130Cas, Axon guidance, Drosophila, CasL
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Tessier-Lavigne and Goodman,
1996). These guidance cues include both membrane-bound and
secreted proteins that instruct the navigation of axons and dendrites through
both attraction and repulsion. Cell surface receptors for these guidance cues
initiate the signal transduction pathways essential for steering neuronal
processes (Dickson, 2002).
However, the signaling pathways initiated by guidance cue receptors, and the
molecular mechanisms through which these signaling cascades alter axonal and
dendritic cytoskeletal dynamics to direct attractive or repulsive steering
events, are still poorly defined.
Some of the mechanisms that direct growing axons are similar to those that
promote cell migration (Ridley et al.,
2003). Proteins originally characterized as important for cell
migration through their effects on cytoskeletal dynamics have more recently
been shown to play roles in axon guidance during development. Integrins are a
large family of receptors crucial for promoting cell migration, supporting
cellular adhesion to extracellular matrix (ECM) components, and regulating
intracellular actin filament dynamics
(Hynes, 2002;
Vicente-Manzanares et al.,
2005). Integrins also participate in directing growing axons to
their targets (Clegg et al.,
2003; Nakamoto et al.,
2004). Genetic analyses in C. elegans, Drosophila and
mice reveal that axon extension in integrin mutants in vivo is not compromised
during development; however, defects in axon guidance result from loss of
integrin function (Baum and Garriga,
1997; Billuart et al.,
2001; Hoang and Chiba,
1998; Pietri et al.,
2004; Stevens and Jacobs,
2002). In combination with in vitro and in vivo observations
addressing the effects integrins and their ligands exert on growing axons
(Adams et al., 2005;
Bonner and O'Connor, 2001;
Garcia-Alonso et al., 1996;
Gomez et al., 1996;
Hopker et al., 1999;
Kuhn et al., 1995), these
studies define functions for integrin signaling in guiding growing axons,
strongly suggesting that integrins are essential for axon guidance.
The molecular mechanisms by which integrins guide neuronal processes during
development remain to be determined. Integrin receptors are heterodimers
composed of a ligand-binding 
-subunit and a ß-subunit that
together signal through their cytoplasmic domains
(Hynes, 2002). Numerous
molecules function downstream of integrin receptor activation to mediate cell
migration, including tyrosine kinases such as focal adhesion kinase (FAK) and
Src, Rho GTPases, focal adhesion proteins including Paxillin, and proteins in
the Crk-associated substrate (Cas) family
(Wiesner et al., 2005). Cas
proteins are intriguing integrin signaling components because they are Src
homology 3 (SH3)-domain-containing proteins that physically link integrins to
kinases, phosphatases, guanine nucleotide exchange factors and proteins that
nucleate actin filaments (Chodniewicz and
Klemke, 2004; O'Neill et al.,
2000). Furthermore, Cas proteins transduce intracellular signals
that stimulate actin filament assembly
(Bouton et al., 2001) and act
as force sensors during cell motility
(Sawada et al., 2006). Do Cas
proteins provide a crucial link between integrin activation and actin dynamics
essential for guiding growing axons to their targets in vivo? We show here
that Cas proteins are highly expressed in the nervous system and together with
integrins direct discrete axonal steering events during development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In situ hybridization
Standard RNA in situ analysis of wild-type Drosophila embryos was
carried out using sense and antisense DCas cRNA probes
(Terman et al., 2002). No
specific staining was observed in sense controls. In situ analyses on specific
genotypes were conducted with the aid of a `blue balancer' chromosome and
staining embryos with a ß-galactosidase antibody (1:3000, Cappell).
|
). Monoclonal antibodies against Fasciclin II (Fas2) (1D4),
BP102, Myc (9E10), muscle myosin (FMM5), Even-skipped (3C10), ß1 integrin
betaPS (CF.6G11), 1 integrin alphaPS1 (DK.1A4), 2 integrin
alphaPS2 (CF.2C7) were obtained from Developmental Studies Hybridoma Bank,
University of Iowa. Embryos heterozygous for both DCas and ß1,
1 or 2 were genotyped using a `blue balancer' and the respective
integrin antibodies to identify integrin heterozygotes. Cas antibodies
[Phospho-p130Cas (Tyr410); #4011, Lot #1, Cell Signaling Technologies
(Fonseca et al., 2004)] were
used for western analysis (1:500) and Drosophila immunostaining
(1:300).
Genetics and phenotypic characterization
Flies were obtained from Bloomington [Df(3L)ED201,
DCasDf(3L)Exel6083 and integrin mutants ß1
(mys1), 1 (mewM6) and 2
(ifk27E)] and the Drosophila Genetic Resource
Center [DCasP1 (NP4466)]. Drosophila genetics and
scoring of axon guidance defects were performed using standard approaches
(Terman et al., 2002). All
images were captured using a Zeiss Axioimager upright microscope with an
Axiocam HRc camera and Axiovision software. Brightness, contrast and color
balance of images were adjusted using Adobe Photoshop.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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13 kb of genomic sequence and includes at least four exons
(Fig. 1A). The longest
DCas cDNA encodes a protein of 793 amino acids
(Fig. 1B). In addition, we
found a smaller DCas EST that when translated is missing the DCas SH3
domain (data not shown; accession number AAF47336).
The Cas family includes the mammalian p130Cas (Cas, Bcar1), Cas-L (Hef1,
Nedd9) and Efs (Sin) proteins, all of which contain, along with several
additional conserved motifs, a highly conserved SH3 domain that is important
for Cas-dependent cell migration (Fig.
1B) (Cary et al.,
1996; Cary et al.,
1998; Garton and Tonks,
1999). DCas, like vertebrate Cas proteins, includes this
N-terminal SH3 domain and is 70% identical at the amino acid level to the SH3
domain of human CAS (Fig.
1B,C). Cas proteins are also characterized by their `substrate'
and `serine-rich (Ser)' regions, which constitute a major portion of the Cas
protein and contain several conserved tyrosine, serine and threonine
phosphorylation sites (Fig.
1B,C). Many of these amino acid residues are conserved between
DCas and human CAS, suggesting they are important for Cas function
(Fig. 1C). Among these
conserved tyrosine residues are five YxxP motifs that, when phosphorylated,
serve as binding sites for proteins with Src homology 2 (SH2) or
phosphotyrosine-binding (PTB) domains. These proteins include the adaptor
proteins Crk and Nck (Dock) and also the phosphatase Ship2 (Inppl1)
(Fig. 1C)
(Bouton et al., 2001). DCas
also contains a stretch of conserved amino acids found in all vertebrate Cas
proteins, YDYV, that serves as an Src tyrosine kinase-binding site
(Fig. 1C)
(Bouton et al., 2001). At its
C-terminus, DCas includes residues, conserved in all mammalian Cas proteins,
that are important for dimerization, suggesting that DCas might also exist as
a dimer (Fig. 1B,C)
(Law et al., 1999). Human CAS
and EFS, but not CAS-L, also contain a proline-rich (PxxP) region immediately
C-terminal to their SH3 domains. However, although DCas contains conserved
proline residues both within this region and C-terminal to the serine-rich
region, it is most similar to CAS-L (Fig.
1B,C). Therefore, Drosophila, unlike mammals, has only
one Cas family member, eliminating issues of functional redundancy in
assessing Cas contributions to neural development.
|
We next assessed the distribution of endogenous DCas protein. Our attempts
to generate a DCas antibody were unsuccessful. However, we tested several
different antibodies generated against Cas proteins and found that an antibody
against an epitope highly conserved between mammalian and Drosophila
Cas (Fonseca et al., 2004)
recognizes DCas (see Fig. S1 in the supplementary material). Western analysis
using this polyclonal antibody revealed that this vertebrate Cas antibody
recognizes bands at 105 kDa and 140 kDa in Drosophila
embryos, and that these bands are absent in embryos harboring a deletion of
the DCas gene (Fig.
2E). In Drosophila embryos, DCas immunostaining was
observed during neural development in a pattern consistent with that of
DCas mRNA and with the expression of a MycDCas
transgene under the control of a GAL4 enhancer trap insertion in
DCas (DCasP1-GAL4;
Fig. 1A; see Fig. S1D,E in the
supplementary material). DCas protein first appeared in motor and CNS
projections during initial stages of axon outgrowth. At later embryonic
stages, DCas immunostaining was present in neuronal cell bodies and in axons
that contribute to all motor axon pathways
(Fig. 2F,G; data not shown). We
also observed DCas immunostaining in the chordotonal sensory organs (not
shown) and in muscle attachment sites (Fig.
2G). Importantly, no Cas immunostaining was observed in embryos
harboring a small deficiency that includes the DCas locus
(Fig. 2H), showing that this
Cas antibody generated against vertebrate Cas specifically recognizes
DCas.
To further address the role of Cas proteins in neurons, we asked whether
any of the mammalian Cas proteins is also expressed in axons during
development. p130Cas and Cas-L transcripts are found in the
developing and postnatal brain (Huang et
al., 2006; Merrill et al.,
2004), and p130Cas localizes to axons in early development and
into adulthood in the spinal cord, cerebellum and cerebral cortex
(Huang et al., 2006;
Liu et al., 2007;
Nishio and Suzuki, 2002). We
used several antibodies that specifically recognize Cas proteins and examined
their patterns of immunostaining in the developing rat spinal cord. We
observed that mammalian Cas family members are indeed highly expressed in
neurons during development (see Fig. S2 in the supplementary material).
DCas is required for axon pathfinding during development
To determine whether Cas proteins serve axon guidance functions during
development, we analyzed DCas mutant Drosophila embryos. A
search of public Drosophila stock collections identified two
potential DCas loss-of-function (LOF) mutants as well as a large
deficiency that removes DCas (Df(3L)ED201). One of these
potential DCas LOF mutants, DCasP1, is the
GAL4-containing P-element transposon situated in the intron
downstream from the start of DCas translation
(Fig. 1A). Another potential
DCas LOF mutant is a small deficiency (Df(3L)Exel6083) that
removes DCas and five putative adjacent genes. No DCas mRNA
transcript or immunostaining were observed in embryos homozygous for the
DCas deficiencies (Fig.
2D,H), and very little DCas transcript or immunostaining
was present in homozygous DCasP1 embryos
(Fig. 2C, data not shown).
These expression data, in combination with genetic experiments described
below, show that Df(3L)Exel6083 is a DCas-null allele and
that DCasP1 is a DCas hypomorphic LOF allele.
We assessed DCas function in the development of the stereotypic
Drosophila embryonic neuromuscular connectivity pattern as this is an
excellent system to study axon guidance events, including motor axon
fasciculation and defasciculation and target recognition
(Araujo and Tear, 2003). All
motor axon trajectories can be easily observed in late stage 16 to early stage
17 embryos using the mAb 1D4 (anti-Fasciclin II), and individual motor axons
can be followed to their targets (Van
Vactor et al., 1993). All five motor axon pathways, and also the
transverse nerve, were found to be defective in DCas mutant embryos
(Figs 3 and
4;
Table 1; data not shown). The
ISNb pathway in wild-type embryos, and also in DCas heterozygotes, is
formed correctly by motor axons defasciculating from the ISN and extending
dorsally through the ventral musculature to innervate muscles 6, 7, 12 and 13
(Fig. 3A,J;
Table 1). In DCas
mutants, ISNb axons exhibited highly penetrant axon guidance phenotypes, often
failing to innervate muscles 6 and 7 and muscles 12 and 13
(Fig. 3B-E,J;
Table 1). These guidance
phenotypes include the failure of ISNb axons to defasciculate from the ISN
(Fig. 3B), ISNb axons stalling
within the ISNb following defasciculation from the ISN
(Fig. 3C), ISNb axons bypassing
their target muscles (Fig. 3D),
and abnormal ISNb axonal trajectories to their targets
(Fig. 3E). Characterization and
quantification of these phenotypes reveal that these defects are indicative of
increased motor axon fasciculation (Table
1), as has been observed in the absence of several different
guidance-cue signaling cascades that regulate fasciculation of developing
Drosophila motor projections
(Araujo and Tear, 2003;
Van Vactor, 1998). In
DCas mutant embryos, SNa motor axons
(Fig. 4;
Table 1) and CNS axons (see
Fig. S2 in the supplementary material;
Table 1) also exhibit highly
penetrant guidance defects consistent with increased axonal fasciculation.
Phenotypes characteristic of decreased fasciculation are not observed to any
appreciable extent in DCas LOF mutants
(Table 1). Importantly, we
observed no defects in muscle integrity or neuronal cell fate determination in
DCas LOF mutants (see Fig. S1 in the supplementary material).
|
|
). We
utilized the DCasP1 allele to express MycDCas
under control of what is likely to be the endogenous DCas promoter,
as DCasP1 is a GAL4-containing P-element
insertion located downstream of the DCas ATG
(Fig. 1A) and drives gene
expression in a pattern similar to that detected with a Cas antibody (see Fig.
S1D,E in the supplementary material). Indeed, expression of the
MycDCas transgene in a DCas mutant background
that includes the DCasP1 allele significantly rescued the
embryonic ISNb (Fig. 3H), SNa
(Fig. 4I) and CNS (see Fig. S2
in the supplementary material) axon guidance phenotypes
(Table 1). To confirm that this
rescue results from DCas expression in neurons, we placed
UAS-MycDCas under the transcriptional control of the
neuron-specific driver ELAV-GAL4, which drives robust expression of
MycDCas in motor and CNS neurons (see Fig. S1C in the supplementary
material). Neuronal MycDCas expression rescued ISNb
(Fig. 3I), SNa
(Fig. 4J) and CNS (see Fig. S2
in the supplementary material) DCas embryonic axon guidance defects
(Table 1). Therefore, axon
guidance phenotypes observed in DCas mutant embryos result from a
lack of neuronal DCas, demonstrating that DCas plays a key role in the
regulation of motor axon defasciculation.
Overexpression of neuronal DCas results in motor and CNS axon guidance defects
To further characterize DCas neuronal functions, we asked whether neuronal
overexpression of DCas affects axon pathfinding. Overexpression of
MycDCas in all neurons in a wild-type background led to
highly penetrant motor axon guidance phenotypes
(Fig. 5;
Table 1). Interestingly, most
of these DCas gain-of-function (GOF) axon guidance defects are
similar to those we observed in DCas LOF mutants: reductions in motor
axon defasciculation at characteristic choice points
(Fig. 5;
Table 1). For example, when one
copy of the MycDCas transgene was expressed in all neurons
with one copy of the ELAV-GAL4 driver in a wild-type background
(designated `+'), ISNb axons often failed to innervate their muscle targets
and instead remained fasciculated in the ISNb nerve
(Fig. 5A;
Table 1). Increasing the levels
of neuronal DCas (++) resulted in more-severe motor axon pathfinding defects,
including the inability of ISNb axons to defasciculate from the ISN nerve
(Fig. 5B), and extensive
stalling and aberrant bundling of ISNb axons along their trajectory
(Fig. 5C). Increasing neuronal
DCas levels still further (+++) produced dramatic motor axon stalling within
their nerve roots, just after their exit from the ventral nerve cord,
resulting in noticeably fewer motor axons extending into the periphery
(Fig. 5G). SNa motor axon
guidance phenotypes in DCas neuronal GOF mutants were also similar to
those we observed in DCas LOF mutants and exhibited a similar
dosage-dependent increase in the severity of abnormal fasciculation and
stalling defects (Fig. 5D-G).
We also found that DCas GOF dramatically affects CNS axonal
trajectories, producing phenotypes reminiscent of those observed in the
absence of the Slit receptor Roundabout (Robo) or by increased Netrin
attractive signaling (see Fig. S2 in the supplementary material)
(Bashaw and Goodman, 1999;
Seeger et al., 1993). We also
observed increased fasciculation and stalling of CNS axons (see Fig. S2 in the
supplementary material). As was the case for DCas LOF mutants, we
observed no defects in neuronal cell fate in these neuronal DCas GOF
mutants (see Fig. S1 in the supplementary material).
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Cas signals integrin-dependent axon guidance during development
We next sought to better define the signaling pathway through which Cas
regulates axon fasciculation. In mammals, Cas proteins function downstream of
several different receptors in non-neuronal cells, including growth factor
receptors, G-protein-coupled receptors, T-cell receptors, B-cell receptors and
integrins (Defilippi et al.,
2006). Interestingly, integrin receptor subunit mutations in
Drosophila give rise to CNS and motor axon guidance defects that are
strikingly similar to those we observe in DCas mutants
(Hoang and Chiba, 1998;
Stevens and Jacobs, 2002),
suggesting that Cas might function together with integrin receptors to guide
axons.
Drosophila integrins, like vertebrate integrins, are composed of
an -subunit and a ß-subunit. In Drosophila, there is one
gene encoding a typical laminin-binding-type -subunit (1, called
mew), one encoding an RGD-binding-type -subunit (2,
called if), and a single ß-subunit gene (ß1, called
mys) very similar to the prototype vertebrate ß1 receptor
(Bokel and Brown, 2002;
Brower, 2003). To investigate
the connection between integrin and Cas signaling, we revisited the role of
integrin receptor function in embryonic motor axon pathfinding and found that
integrin-null mutant embryos exhibit defects that are qualitatively and
quantitatively similar to DCas mutants (Figs
3,
4, and see Fig. S2 in the
supplementary material; Table
1) (see also Hoang and Chiba,
1998). Embryos harboring null alleles for either 1
(mewM6) or 2 (ifK27E)
integrin genes exhibited ISNb and SNa axon guidance defects very
similar to those observed in DCas mutants, including increased
fasciculation resulting in the absence of muscle innervation
(Fig. 3F,G;
Fig. 4G,H;
Table 1) (see also
Hoang and Chiba, 1998). We
also observed CNS axon guidance defects in both 1 and
2 integrin mutants that were similar to those we observed in
DCas mutants (see Fig. S2 in the supplementary material;
Table 1) (see also
Hoang and Chiba, 1998;
Stevens and Jacobs, 2002).
To further address DCas involvement in integrin-mediated axon guidance, we
looked for dominant genetic interactions between DCas and integrin
subunit LOF mutations. Such transheterozygous interactions provide genetic
support for two proteins functioning together in the same signaling pathway.
We asked whether removal of a single copy of DCas dominantly enhances
heterozygosity at the 1, 2 or ß1
integrin loci. We found that in 1, 2,
ß1 or DCas heterozygotes, motor axon trajectories were
not significantly different from wild type
(Fig. 3A,
Fig. 6A,E;
Table 1). However, removal of a
single copy of DCas together with a single copy of 1,
2 or ß1 integrin resulted in highly penetrant
axon guidance defects, suggesting that these three genes function in the same
signaling pathway (Fig. 6B-E;
Table 1). Importantly, the
phenotypes resulting from dominant enhancement by DCas are indicative
of increased fasciculation, similar to those observed in DCas,
1 or 2 integrin LOF embryos
(Table 1).
|
;
Yee and Hynes, 1993). If
integrins are indeed necessary for activating DCas signaling, removing a
single copy of ß1 integrin should suppress DCas GOF
phenotypes. When low levels of DCas were expressed in all neurons in an
otherwise wild-type background, moderate guidance defects were observed
involving axons of the ISNb, SNa and CNS third longitudinal
(Fig. 5A,D,
Fig. 6F,H and see Fig. S2 in
the supplementary material; Table
1). Removing a single copy of the ß1 integrin gene
in this same neuronal DCas GOF genetic background significantly
rescued axon guidance phenotypes resulting from DCas GOF
(Fig. 6G,H;
Table 1). Importantly, these
results also show that neuronal overexpression of DCas does not simply
function in a dominant-negative fashion to block integrin, or other, signaling
pathways.
Taken together, our analysis of integrin LOF mutations and our observation
of robust genetic interactions between DCas and 1,
2 and ß1integrin mutants strongly support DCas
serving a crucial role in transducing in vivo integrin-mediated signaling
events essential for directing axon guidance. Furthermore, our results show
that integrin/Cas signaling is necessary for axonal defasciculation events
during neural development.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and Cas proteins have also been implicated as mediators of
axon outgrowth and turning (Huang et al.,
2006; Liu et al.,
2007; Yang et al.,
2002). Integrin effects on axon outgrowth have been studied
extensively in vitro, but certain integrin functions suggested by these
experiments have thus far not been reflected in the integrin mutant phenotypes
observed in vivo (reviewed by Clegg et
al., 2003). Examination of integrin mutant mice, flies and worms
does not reveal a decrease in axon outgrowth during development
(Baum and Garriga, 1997;
Billuart et al., 2001;
Hoang and Chiba, 1998;
Pietri et al., 2004;
Poinat et al., 2002;
Stevens and Jacobs, 2002)
(this study). Indeed, our in vivo functional analysis shows that one role of
integrin/Cas signaling in neurons during development is to regulate the degree
of fasciculation, or adhesion, among axons. Drosophila embryonic
motor axons have proven useful for modeling these types of fasciculation
defects and identifying the genes that govern them
(Araujo and Tear, 2003;
Van Vactor, 1998). Loss of
DCas or integrin receptors from Drosophila motor neurons results in
axons remaining abnormally fasciculated, growing past their normal
defasciculation choice points, or sometimes stalling at these choice points.
DCas neuronal overexpression produces similar hyperfasciculation defects,
dependent upon integrins, including abnormal bundling and stalling of motor
axons. Although other interpretations are possible, these results suggest a
model in which integrin/Cas signaling normally serves to slow growing axons at
choice points in vivo: experimentally decreasing integrin/Cas signaling
attenuates adhesive functions that serve to slow growing axons, whereas
increasing integrin/Cas signaling results in excessive adhesion among axons,
or between axons and other substrates upon which they extend
(Fig. 7).
|
;
Bozyczko and Horwitz, 1986;
Hammarback et al., 1988;
Letourneau, 1975a;
Raper et al., 1983). How might
adhesive interactions influence axonal guidance decisions in vivo? Neuronal
growth cones tend to form extensive lamellae, which are indicative of strong
adhesive interactions, when cultured on highly adhesive substrata containing
integrin ligands (Hammarback and
Letourneau, 1986; Letourneau,
1975b; Letourneau,
1979). These adhesive interactions stabilize elongating nerve
fibers by promoting filopodial extension and expansion of growth cone surfaces
(Burden-Gulley et al., 1995;
Gomez et al., 1996;
Hammarback et al., 1988;
Letourneau, 1979). Disruption
of axon-substrate attachment in vitro with integrin function-blocking
antibodies encourages axon-axon adhesive interactions (fasciculation) in place
of axon-substrate adhesion (Bozyczko and
Horwitz, 1986). Furthermore, contact with integrin ligands can
slow axon elongation, as axons encountering an increasing gradient of laminin
peptide exhibit reduced velocity, but growth cone velocity returns to previous
rates when axons turn down the gradient
(Adams et al., 2005). This in
vitro observation resembles in vivo situations in which growth cones slow at a
choice point, exhibit increased morphological complexity and then extend along
distinct pathways (Godement et al.,
1994; Gomez and Spitzer,
1999; Tosney and Landmesser,
1985). Drosophila motor axon growth cones also exhibit
similar changes in morphological complexity upon contacting different
substrates in vivo, suggesting that similar processes function to generate
motor axon trajectories (Broadie et al.,
1993; Johansen et al.,
1989; Sink and Whitington,
1991). Different combinations of integrin ligands might be
responsible for these effects. When vertebrate growth cones in vitro contact
either the 1 or 2 integrin ligands, laminin and fibronectin
respectively, they decelerate, pause and exhibit short-term growth arrest
(Gomez et al., 1996;
Kuhn et al., 1995).
Interestingly, our in vivo observations show that DCas functions with both
1 (laminin-binding) and 2 [RGD (e.g. Tiggrin)-binding] integrins
to mediate correct axon navigation by regulating motor axon fasciculation at
choice points, suggesting that integrin/Cas-mediated spatial regulation of
growth cone extension underlies correct navigation at these choice points.
The molecular mechanisms underlying integrin-mediated axon guidance remain
to be completely defined. However, results derived from analysis of
integrin/Cas signaling on cell migration shed light on how Cas and integrins
might specify axonal defasciculation events in vivo. During cell migration,
Cas proteins serve to establish linkage between migrating cells and the ECM
(Defilippi et al., 2006). Cas
plays an important role in regulating cytoskeletal organization, cell adhesion
and force sensing, and fibroblasts isolated from p130Cas-null mutant
mouse embryos exhibit disorganized and short actin filaments and decreased
cell migration (Cho and Klemke,
2000; Honda et al.,
1999; Honda et al.,
1998). In non-neuronal cells, Cas becomes phosphorylated in
response to integrin engagement by many ECM components, including fibronectin
and laminin (Defilippi et al.,
2006). FAK and Src family kinases have been implicated in
integrin-dependent phosphorylation of Cas. Interestingly, recent in vitro
observations reveal that FAK signaling at sites of integrin-mediated adhesion
controls axon pathfinding (Ivankovic-Dikic
et al., 2000; Rico et al.,
2004; Robles and Gomez,
2006). Furthermore, pharmacological inhibitors of Src family
kinases decrease the level of neuronal phosphorylated Cas in vitro
(Huang et al., 2006;
Liu et al., 2007), supporting
a role for Src kinases in regulating Cas proteins in neurons. Finally, the
activity of Rho-family small GTPases is also regulated by Cas interactions
with the guanine nucleotide exchange factor Dock180 (Dock1)
(Defilippi et al., 2006).
Taken together, these links between Cas signaling components and cytoskeletal
reorganization suggest that some of these signaling proteins might also
influence axon guidance in vivo during development.
Our results demonstrate that integrin/Cas-mediated signaling is necessary
but not sufficient for axonal defasciculation, revealing that
integrin/Cas-mediated axon guidance must be integrated with other axon
guidance signaling cascades to regulate axon defasciculation events during
development (Fig. 7). The
identity of these other axon guidance pathways is not known. The
attractive/permissive guidance cue Netrin binds to integrins, and functions
with integrins in non-neuronal cells
(Yebra et al., 2003). The
Netrin receptor Deleted in colorectal cancer (DCC) has been found to utilize
the integrin effector FAK and recently p130Cas, to mediate Netrin-dependent
attractive growth cone steering (Li et
al., 2004; Liu et al.,
2004; Liu et al.,
2007; Ren et al.,
2004). Ephrins, best known for their role as repulsive axon
guidance cues, also induce cell adhesion and actin cytoskeletal changes in
fibroblasts in a p130Cas-dependent manner
(Carter et al., 2002).
Repulsive axon guidance cues may also regulate integrin/Cas-dependent axon
guidance during development. The axonal repellent Slit genetically interacts
with integrins and their ligands to guide commissural axons in
Drosophila (Stevens and Jacobs,
2002). Semaphorin and Ephrin-mediated repulsive effects on
non-neuronal cells also appear to involve inhibition of integrin signaling
events (Miao et al., 2000;
Serini et al., 2003).
Interestingly, a crucial component of semaphorin-dependent repulsive axon
guidance, a member of the molecule interacting with Cas-L (MICAL) family,
physically associates with Cas-L (Suzuki
et al., 2002; Terman et al.,
2002) and our preliminary data suggest that these interactions are
functionally important for axon guidance (A.L.K. and J.R.T., unpublished). Our
observation that Cas functions with integrins to mediate axon guidance during
development suggests new directions to better understand how integrin/Cas
signaling modulates neuronal guidance through interactions with distinct axon
guidance signaling pathways.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/12/2337/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Present address: Harvard Medical School, Boston, MA 02115, USA
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adams, D. N., Kao, E. Y., Hypolite, C. L., Distefano, M. D., Hu, W. S. and Letourneau, P. C. (2005). Growth cones turn and migrate up an immobilized gradient of the laminin IKVAV peptide. J. Neurobiol. 62,134 -147.[CrossRef][Medline]
Araujo, S. J. and Tear, G. (2003). Axon guidance mechanisms and molecules: lessons from invertebrates. Nat. Rev. Neurosci. 4,910 -922.[CrossRef][Medline]
Bashaw, G. J. and Goodman, C. S. (1999). Chimeric axon guidance receptors: the cytoplasmic domains of slit and netrin receptors specify attraction versus repulsion. Cell 97,917 -926.[CrossRef][Medline]
Baum, P. D. and Garriga, G. (1997). Neuronal migrations and axon fasciculation are disrupted in ina-1 integrin mutants. Neuron 19,51 -62.[CrossRef][Medline]
Bentley, D. and Caudy, M. (1983). Navigational substrates for peripheral pioneer growth cones: limb-axis polarity cues, limb-segment boundaries, and guidepost neurons. Cold Spring Harb. Symp. Quant. Biol. 48,573 -585.[Medline]
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]
Bonner, J. and O'Connor, T. P. (2001). The
permissive cue laminin is essential for growth cone turning in vivo.
J. Neurosci. 21,9782
-9791.
Bouton, A. H., Riggins, R. B. and Bruce-Staskal, P. J. (2001). Functions of the adapter protein Cas: signal convergence and the determination of cellular responses. Oncogene 20,6448 -6458.[CrossRef][Medline]
Bozyczko, D. and Horwitz, A. F. (1986). The participation of a putative cell surface receptor for laminin and fibronectin in peripheral neurite extension. J. Neurosci. 6,1241 -1251.[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]
Broadie, K., Sink, H., Van Vactor, D., Fambrough, D., Whitington, P. M., Bate, M. and Goodman, C. S. (1993). From growth cone to synapse: the life history of the RP3 motoneuron. Dev. Suppl. 1993,227 -238.
Brower, D. L. (2003). Platelets with wings: the maturation of Drosophila integrin biology. Curr. Opin. Cell Biol. 15,607 -613.[CrossRef][Medline]
Burden-Gulley, S. M., Payne, H. R. and Lemmon, V. (1995). Growth cones are actively influenced by substrate-bound adhesion molecules. J. Neurosci. 15,4370 -4381.[Abstract]
Carter, N., Nakamoto, T., Hirai, H. and Hunter, T. (2002). EphrinA1-induced cytoskeletal re-organization requires FAK and p130(cas). Nat. Cell Biol. 4, 565-573.[Medline]
Cary, L. A., Chang, J. F. and Guan, J. L. (1996). Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J. Cell Sci. 109,1787 -1794.[Abstract]
Cary, L. A., Han, D. C., Polte, T. R., Hanks, S. K. and Guan, J.
L. (1998). Identification of p130Cas as a mediator of focal
adhesion kinase-promoted cell migration. J. Cell Biol.
140,211
-221.
Cho, S. Y. and Klemke, R. L. (2000).
Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell
migration and suppress apoptosis during invasion of the extracellular matrix.
J. Cell Biol. 149,223
-236.
Chodniewicz, D. and Klemke, R. L. (2004). Regulation of integrin-mediated cellular responses through assembly of a CAS/Crk scaffold. Biochim. Biophys. Acta 1692,63 -76.[Medline]
Clegg, D. O., Wingerd, K. L., Hikita, S. T. and Tolhurst, E. C. (2003). Integrins in the development, function and dysfunction of the nervous system. Front. Biosci. 8,d723 -d750.[Medline]
Defilippi, P., Di Stefano, P. and Cabodi, S. (2006). p130Cas: a versatile scaffold in signaling networks. Trends Cell Biol. 16,257 -263.[CrossRef][Medline]
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.
Dickson, B. J. (2002). Molecular mechanisms of
axon guidance. Science
298,1959
-1964.
Fonseca, P. M., Shin, N. Y., Brabek, J., Ryzhova, L., Wu, J. and Hanks, S. K. (2004). Regulation and localization of CAS substrate domain tyrosine phosphorylation. Cell. Signal. 16,621 -629.[CrossRef][Medline]
Garcia-Alonso, L., Fetter, R. and Goodman, C. S. (1996). Genetic analysis of Laminin A in Drosophila: extracellular matrix containing laminin A is required for ocellar axon pathfinding. Development 122,2611 -2621.[Abstract]
Garton, A. J. and Tonks, N. K. (1999).
Regulation of fibroblast motility by the protein tyrosine phosphatase
PTP-PEST. J. Biol. Chem.
274,3811
-3818.
Godement, P., Wang, L. C. and Mason, C. A. (1994). Retinal axon divergence in the optic chiasm: dynamics of growth cone behavior at the midline. J. Neurosci. 14,7024 -7039.[Abstract]
Gomez, T. M. and Spitzer, N. C. (1999). In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature 397,350 -355.[CrossRef][Medline]
Gomez, T. M., Roche, F. K. and Letourneau, P. C. (1996). Chick sensory neuronal growth cones distinguish fibronectin from laminin by making substratum contacts that resemble focal contacts. J. Neurobiol. 29, 18-34.[CrossRef][Medline]
Hammarback, J. A. and Letourneau, P. C. (1986). Neurite extension across regions of low cell-substratum adhesivity: implications for the guidepost hypothesis of axonal pathfinding. Dev. Biol. 117,655 -662.[CrossRef][Medline]
Hammarback, J. A., McCarthy, J. B., Palm, S. L., Furcht, L. T. and Letourneau, P. C. (1988). Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity. Dev. Biol. 126, 29-39.[CrossRef][Medline]
Hoang, B. and Chiba, A. (1998). Genetic
analysis on the role of integrin during axon guidance in Drosophila.J. Neurosci. 18,7847
-7855.
Honda, H., Oda, H., Nakamoto, T., Honda, Z., Sakai, R., Suzuki, T., Saito, T., Nakamura, K., Nakao, K., Ishikawa, T. et al. (1998). Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas. Nat. Genet. 19,361 -365.[CrossRef][Medline]
Honda, H., Nakamoto, T., Sakai, R. and Hirai, H. (1999). p130(Cas), an assembling molecule of actin filaments, promotes cell movement, cell migration, and cell spreading in fibroblasts. Biochem. Biophys. Res. Commun. 262, 25-30.[CrossRef][Medline]
Hopker, V. H., Shewan, D., Tessier-Lavigne, M., Poo, M. and Holt, C. E. (1999). Growth-cone attraction to netrin-1 is converted to repulsion by laminin-1. Nature 401, 69-73.[CrossRef][Medline]
Huang, J., Sakai, R. and Furuichi, T. (2006).
The docking protein Cas links tyrosine phosphorylation signaling to elongation
of cerebellar granule cell axons. Mol. Biol. Cell
17,3187
-3196.
Huber, A. B., Kolodkin, A. L., Ginty, D. D. and Cloutier, J. F. (2003). Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu. Rev. Neurosci. 26,509 -563.[CrossRef][Medline]
Hynes, R. O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell 110,673 -687.[CrossRef][Medline]
Ivankovic-Dikic, I., Gronroos, E., Blaukat, A., Barth, B. U. and Dikic, I. (2000). Pyk2 and FAK regulate neurite outgrowth induced by growth factors and integrins. Nat. Cell Biol. 2,574 -581.[CrossRef][Medline]
Johansen, J., Halpern, M. E. and Keshishian, H. (1989). Axonal guidance and the development of muscle fiber-specific innervation in Drosophila embryos. J. Neurosci. 9,4318 -4332.[Abstract]
Kuhn, T. B., Schmidt, M. F. and Kater, S. B. (1995). Laminin and fibronectin guideposts signal sustained but opposite effects to passing growth cones. Neuron 14,275 -285.[CrossRef][Medline]
Law, S. F., Zhang, Y. Z., Fashena, S. J., Toby, G., Estojak, J. and Golemis, E. A. (1999). Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain. Exp. Cell Res. 252,224 -235.[CrossRef][Medline]
Letourneau, P. C. (1975a). Cell-to-substratum adhesion and guidance of axonal elongation. Dev. Biol.
2 test). Scale bar: 15 µm in A-D; 15
µm in F,G.