|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 18 January 2006
doi: 10.1242/dev.02230
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Genetics, Cell Biology and Development and Developmental Biology Center, University of Minnesota, Minneapolis, MN 55455, USA.
* Author for correspondence (e-mail: mille380{at}umn.edu)
Accepted 29 November 2005
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Somitogenesis, Morphogenesis, Mesoderm, Ena/VASP, FAK, Integrin, Cadherin, Adhesion, Migration
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Dubrulle and Pourquie, 2004;
Weinmaster and Kintner, 2003).
One output of these signaling events is to initiate changes in cell shape,
motility and adhesion that mediate morphological segmentation
(Kalcheim and Ben-Yair, 2005).
Although the molecular pathways involved in establishing segmental identity
have been studied in some detail, the mechanisms responsible for morphological
segmentation remain elusive.
Previous studies have described the cellular processes that accompany
somite formation in Xenopus
(Hamilton, 1969;
Keller, 2000;
Wilson et al., 1989;
Youn and Malacinski, 1981).
During gastrulation, paraxial mesoderm cells intercalate radially and
mediolaterally to drive anteroposterior extension of the PSM and establish the
notochord/somite boundary (Wilson et al.,
1989). At the end of gastrulation, PSM cells change shape,
lengthening along their mediolateral axis and narrowing along their
anteroposterior axis (Wilson et al.,
1989). Subsequently, blocks of cells in the rostral-most region of
the PSM rotate 90° relative to the anteroposterior axis to generate
somites (Keller, 2000), which
primarily differentiate into mono-nucleate muscle cells of the tadpole
(Chanoine and Hardy, 2003).
Analysis of cell behaviors during segmentation suggest that cells rearrange
independently during rotation (Wilson et
al., 1989; Youn and
Malacinski, 1981), indicating a role for directed migration in
this process. Spatially and temporally coordinated regulation of cell adhesion
is also essential for somitogenesis. Cell-cell adhesion during rotation is
dependent on Type I cadherins (Giacomello
et al., 2002) and somite boundary formation requires the function
of paraxial protocadherin (PAPC) (Kim et
al., 2000). The importance of integrins in Xenopus
somitogenesis is suggested by the expression of several integrins, including
3ß1-, 5ß1-
and 6ß1-integrins, in developing somites,
disruption of somite formation by overexpression of
3-integrin (Meng et al.,
1997) or expression of a dominant negative form of
ß1-integrin (Marsden and DeSimone,
2003), and requirement for integrin function in somite formation
in other vertebrates (Drake et al.,
1992; Goh et al.,
1997; Julich et al.,
2005; Koshida et al.,
2005; Krotoski and
Bronner-Fraser, 1990; Yang et
al., 1993; Zagris et al.,
2004). In addition, presumptive somites become surrounded by a
FN-rich matrix during somitogenesis
(Davidson et al., 2004;
Wedlich et al., 1989) and FN
is required for somitogenesis in mice and zebrafish
(George et al., 1993;
Koshida et al., 2005). FAK, a
crucial signaling molecule activated by integrin-ECM interactions, also
accumulates at somite boundaries and is required for somitogenesis in mice
(Crawford et al., 2003;
Furuta et al., 1995;
Henry et al., 2001;
Hens and DeSimone, 1995),
implicating a potential role for integrin signaling in this process.
Somite formation is dependent on tightly orchestrated morphogenetic
processes, yet little is known about the molecular pathways that coordinate
changes in cell shape, migration and adhesion during somitogenesis. The
Ena/VASP family of actin regulatory proteins function in a variety of cell
types to regulate cell migration and adhesion and these roles are borne out by
the localization of Ena/VASP proteins to focal adhesions and sites of dynamic
membrane reorganization (Krause et al.,
2003; Kwiatkowski et al.,
2003; Sechi and Wehland,
2004). The vertebrate Ena/VASP family comprises three genes, Ena,
vasodilator-stimulated phosphoprotein (VASP) and Ena/VASP-like (Evl). Ena/VASP
proteins are characterized by several protein-protein interaction domains: a
N-terminal EVH1 domain that binds tightly and specifically to a consensus
motif (F/LPPPP) found in a number of proteins, including vinculin, zyxin, RIAM
and lamellipodin (Brindle et al.,
1996; Drees et al.,
2000; Fedorov et al.,
1999; Krause et al.,
2004; Lafuente et al.,
2004); a central, proline-rich domain that binds Profilin
(Gertler et al., 1996;
Reinhard et al., 1995) and SH3
domain proteins such as Abl and nSrc
(Gertler et al., 1995;
Lambrechts et al., 2000); and
a C-terminal EVH2 domain that binds F-actin and mediates multimerization of
Ena/VASP proteins (Bachmann et al.,
1999; Harbeck et al.,
2000; Huttelmaier et al.,
1999). Knockout studies in mice show that Ena/VASP proteins are
required for platelet aggregation, neural tube formation, craniofacial
development and axon guidance (Aszodi et
al., 1999; Hauser et al.,
1999; Lanier et al.,
1999; Menzies et al.,
2004). However, these studies have been hindered by the functional
redundancy of the highly related family members, making it likely that
additional roles for Ena/VASP proteins remain to be uncovered. To overcome the
problem of redundancy, several studies have used dominant-negative proteins to
neutralize the function of all Ena/VASP proteins. This work has revealed
additional roles for Ena/VASP proteins in formation of cell-cell junctions in
epithelial cells (Vasioukhin et al.,
2000), regulation of intercalated disc function in cardiac muscle
(Eigenthaler et al., 2003) and
migration of pyramidal neurons in the cerebral cortex
(Goh et al., 2002).
Furthermore, dominant-negative proteins have also been employed to examine the
mechanism by which Ena/VASP proteins regulate actin dynamics and cell motility
in cultured fibroblasts (Bear et al.,
2000; Bear et al.,
2002).
Previously, we have reported that Xena is expressed throughout the
mesoderm during gastrulation, and that Xena and Xenopus Evl
(Xevl) transcripts are present in the myotome of the tadpole
(Wanner et al., 2005;
Xanthos et al., 2005),
suggesting that Ena/VASP proteins might play a role in somitogenesis and
muscle development in Xenopus. Here, we show that Xena is localized
to cell borders in the PSM and is later enriched at intersomitic and
intermyotomal junctions. Using targeted expression of dominant-negative
proteins that neutralize the function of all Ena/VASP family members, we
demonstrate that Ena/VASP activity is required for somite rotation and
boundary formation. Furthermore, these studies revealed a requirement for
Ena/VASP proteins in FN matrix deposition, spreading of somitic cells on FN
and autophosphorylation of FAK. Finally, we show that FAK is required for
somite formation, FN matrix deposition and localization of Xena to the cell
cortex. Together, these data provide evidence that Ena/VASP proteins and FAK
coordinately regulate somite formation by modulating integrin-dependent
processes during development.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Embryos and microinjections
Xenopus laevis embryos were obtained by fertilization of eggs from
females injected with human chorionic gonadotrophin (Sigma). Eggs were
dejellied in 2% cysteine, cultured in 0.33xMMR
(Sive et al., 2000), and
staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1994).
Capped mRNA for microinjections was synthesized using the SP6 mMessage Machine
kit (Ambion) and embryos were injected in 4% ficoll in 0.33xMMR.
FN spreading and adhesion assays
Somitic cell cultures were prepared as described
(Gomez et al., 2003). Briefly,
the dorsal region of stage 20 embryos were excised, transferred to
Ca2+/Mg2+-free 0.3xMMR, and dissociated for 1
hour. Ectoderm was removed and remaining cells were transferred to FN-coated
coverslips (0.5 µg/ml; Sigma) in 1xSteinberg's
(Sive et al., 2000) and
allowed to adhere for 30 minutes (adhesion assay) or overnight (spreading
assay) at 20°C. Prior to collection, cells were washed three times with
0.3xMMR then fixed in Dent's fixative
(Sive et al., 2000) for 2
hours at 4°C.
Immunofluorescence
Embryos were fixed in Dent's fixative overnight at 4°C. For imaging of
FN, blastocoel roofs were fixed in 2% trichloroacetic acid in PBS overnight at
4°C. Immunostaining was performed with the following antibodies: anti-GFP
(Santa Cruz Biotechnology), anti-Xena
(Xanthos et al., 2005),
anti-Mena (Lebrand et al.,
2004), anti-tenascin (HB1, provided by H. R. Erickson), anti-FN
(4H2) (Ramos and DeSimone,
1996) and anti-FAK (2A7, Upstate Biotechnology). The following
monoclonal antibodies were obtained from the Developmental Studies Hybridoma
Bank: vinculin (VN 3-24), ß1-integrin (8C8), ß-tubulin
(E7) and 12/101. Staining was visualized using Alexa568-conjugated (Molecular
Probes) or Cy2-conjugated (Jackson ImmunoResearch) secondary antibodies. For
imaging of cross-sections, immunostained embryos were incubated overnight in
PBST, and slices were cut with a surgical scalpel. With the exception of BCR
explants and FN adherent cells (mounted in 80% glycerol, 0.5% propylgallate),
all samples were dehydrated, cleared in Murray's clear
(Sive et al., 2000) and
mounted in Sylgard (Dow Corning) wells. Images were captured using a Zeiss
spinning disc microscope and merged images were produced using Adobe
Photoshop. Quantitative analysis of somite area and Xena staining in BCRs was
performed with ImageJ. For analysis if somite area, cross-sectional area of
12/101 positive cells was measured. Results are reported as a ratio of the
area of injected versus uninjected side, or right versus left sides for
controls. For analysis of Xena distribution in BCRs, average pixel intensity
at the membrane (two peak intensities 0.3 µm apart) was compared with the
average pixel intensity of the juxtamembrane region (3.3 µm adjacent to
membrane).
In situ hybridization
In situ hybridization was carried out as described
(Harland, 1991).
Digoxigenin-labeled MyoD (Hopwood
et al., 1989) and PAPC
(Kim et al., 2000) probes were
synthesized using a MAXIScript kit (Ambion). Probes were detected by alkaline
phosphatase-conjugated anti-digoxigenin (Roche) using BM Purple substrate
(Boehringer Mannheim).
Immunoblotting
Protein lysates were prepared by homogenizing explants or embryos in
ice-cold lysis buffer [10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA,
20 mM Na4PO2O7, 1% Triton-X100] supplemented
with phosphatase and protease inhibitors. Homogenates were cleared by
centrifugation at 8000 g for 10 minutes at 4°C. Proteins
were blotted to PVDF membrane and blots were blocked in 5% milk in TBS + 0.1%
Tween (TBSTw) or 5% BSA in TBSTw for phospho-FAK analysis. Blots were probed
with anti-FAK (1:1000, Santa Cruz Biotechnology) or anti--fodrin
antibodies (1:2000) (Giebelhaus et al.,
1987) in 5% milk in TBSTw or anti-FAKpY397 antibodies (1:1000,
BioSource) in 3% BSA in TBSTw overnight at 4°C. Visualization was
performed using HRP-conjugated antibodies (Jackson ImmunoLabs) and enhanced
chemiluminescence (Pierce).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). To
further these studies, we examined the subcellular distribution of Xena using
polyclonal antibodies raised against Xena
(Xanthos et al., 2005) or
monoclonal antibodies raised against mammalian Ena (Mena)
(Lebrand et al., 2004) that
crossreact with Xena (Xanthos et al.,
2005). Both antibodies displayed identical staining patterns, and
anti-Xena staining was blocked by pre-incubation with Xena peptide antigen,
indicating that staining is specific for Xena (data not shown). In the PSM,
Xena localizes to the cell periphery and is enriched at the PSM/notochord
boundary (Fig. 1A). Xena
persists at cell borders during somite rotation and becomes enriched at
intersomitic junctions (Fig.
1B-F). Accumulation of Xena at presumptive intersomitic boundaries
in anterior regions of the embryo is first observed prior to rotation at
presumptive somite boundaries in the rostral-most region of the PSM.
Enrichment of Xena appears coincident with the deposition of FN at these sites
(Fig. 1C-E, arrow indicates
formed boundary, arrowheads indicate newest forming boundary). Xena and FN
often colocalize at nascent boundaries
(Fig. 1E), although their
staining patterns are sometimes discontinuous in a single focal plane. By
contrast, Xena accumulation at intersomitic junctions in posterior regions
appears to coincide with the formation of nascent boundaries during somite
rotation (Fig. 1F). The
differences in the timing of Xena accumulation at somite boundaries in
anterior versus posterior regions suggests that different mechanisms may be at
work to mediate somite formation in these two regions, as appears to be the
case in zebrafish (Julich et al.,
2005; Koshida et al.,
2005). As development proceeds, Xena is found at cell-cell
contacts within somites, continues to be enriched at intersomitic junctions,
and later is enriched at intermyotomal junctions in tadpoles (Figs
1,
2). Xena colocalizes at
intersomitic and intermyotomal junctions with components of cell-matrix
adhesion complexes, including ß1-integrin
(Fig. 2A-F), vinculin
(Fig. 2G-L), FAK
(Fig. 2M-O) and tenascin (data
not shown).
|
).
Xvasp transcripts are provided maternally and are present throughout
embryogenesis (data not shown). However, in situ hybridization analyses show
that Xvasp mRNA does not appear to be expressed in the PSM, somites
or myotome (data not shown). RT-PCR analysis of Ena/VASP expression in adult
muscle reveals the presence of Xena transcripts in thigh and pectoral
muscle, whereas Xevl and Xvasp transcripts are present at
low levels in adult thigh and pectoral muscle (data not shown).
Dominant negative inhibition of Ena/VASP function during embryogenesis
The expression patterns of Xena and Xevl suggest that
Ena/VASP proteins may play a role in somite and/or muscle development in
Xenopus. To test this hypothesis, we neutralized the function of all
Ena/VASP proteins using a dominant-negative construct containing four repeats
of the EVH1-binding motif (FPPPP) linked to the ActA mitochondrial targeting
sequence (FP4-mito). This construct takes advantage of the highly
specific binding of the EVH1 domain to the FPPPP ligand
(Carl et al., 1999;
Niebuhr et al., 1997) and has
previously been shown to redirect Ena/VASP proteins from their normal
localization to the surface of the mitochondria, effectively blocking the
function of all Ena/VASP proteins in cultured cells
(Bear et al., 2000;
Bear et al., 2002) and mouse
embryos (Goh et al., 2002).
Importantly, this dominant-negative approach in conjunction with targeted
injection of mRNAs into early Xenopus embryos allows for
tissue-specific inhibition of all Ena/VASP family members, thereby alleviating
potential problems with functional redundancy observed in mice. A similar
construct containing a mutated binding motif (APPPP; AP4-mito)
shows a substantially lower affinity for EVH1 binding
(Bear et al., 2000) and serves
as a control. Both constructs are tagged with EGFP to allow visualization.
The efficacy of the FP4-mito and AP4-mito proteins in
Xenopus was tested by injecting capped mRNA (500 pg) encoding these
proteins unilaterally into four-cell stage Xenopus embryos just
vegetal to the equator and ventral to the second cleavage furrow, which
resulted in mosaic expression of the proteins almost exclusively in the
somites and myotome. Injected embryos were raised to stage 22, fixed and
co-stained for Xena and GFP. In cells expressing FP4-mito, Xena was
not visible at the cell periphery and instead was restricted to the cell body,
where it co-localized with FP4-mito-GFP
(Fig. 3A-C). Adjacent cells
that did not express FP4-mito protein retained normal, cortical
localization of Xena. AP4-mito expression had little effect on Xena
(Fig. 3D-F), causing only a
mild and incomplete mis-localization of Xena when AP4-mito was
present at very high levels. Mis-localization of Xena by FP4-mito
was also observed in the blastocoel roof (BCR) of stage 12 embryos and the
myotome (data not shown), and thus is predicted to neutralize Ena/VASP
activity throughout embryonic development. These results, together with
previously published reports (Bear et al.,
2000; Bear et al.,
2002; Goh et al.,
2002), demonstrate that the FP4-mito dominant negative
provides an effective means to neutralize Ena/VASP function during
development.
|
|
), we
first analyzed whether neutralization of Ena/VASP activity impaired
gastrulation movements of the PSM. Development of the PSM was assessed by
immunostaining for ß-tubulin, which stains cortical microtubules and
facilitates visualization of cell morphology. We found that the distribution
and morphology of cells in the PSM was unaffected by either
AP4-mito or FP4-mito expression
(Fig. 4A,B), suggesting that
Ena/VASP function is not required for cell shape changes or movements that
generate the PSM. To corroborate these findings, we used in situ hybridization
to assess the expression of two markers of the PSM, MyoD
(Hopwood et al., 1989) and
PAPC (Kim et al.,
2000). No difference in the intensity or distribution of these
markers was observed in uninjected, AP4-mito- or
FP4-mito-injected embryos (Fig.
4C-H), indicating that specification and patterning of the PSM is
not dependent on Ena/VASP function.
Ena/VASP function is required for somitogenesis
To address whether Ena/VASP proteins are required for somitogenesis,
FP4-mito and AP4-mito expressing embryos were analyzed
for defects in somite formation by immunostaining for the somite/muscle marker
12/101 (Fig. 5),
ß1-integrin (Fig. 6) and
FAK (data not shown). We found that somites of FP4-mito injected
embryos appeared disorganized with cells adopting random orientations
(Fig. 5A,B,E, 93.9%,
n=33; Fig. 6A-C).
FP4-mito expression also led to abnormal somite boundary formation,
evidenced by the disruption of ß1-integrin
(Fig. 6A-C) and FAK (data not
shown) staining, the presence of irregular somite borders, and the failure of
cells to extend from one end of the somite to the other. Conversely, a
significantly lower percentage of AP4-mito-injected embryos showed
only a mild disruption in somite morphology
(Fig. 5C-E; 26.9%,
n=26), which correlated with weak mis-localization of Xena at sites
of high AP4-mito protein. In addition, neither ß1-integrin
(Fig. 6D-F) nor FAK (data not
shown) localization was disrupted by AP4-mito. We were unable to
examine potential changes in the actin cytoskeleton at high resolution because
of the relative opacity of embryonic Xenopus cells and the
requirement that embryos and explants are dehydrated and cleared prior to
imaging, which precludes the use of phalloidin and many commercial antibodies
for visualization of actin.
Further analysis of the defects associated with Ena/VASP inhibition
revealed that the phenotype caused by Ena/VASP inhibition manifested as an
expansion in somite area, as measured from digital tracings of cross-sectional
images of 12/101 staining (Fig.
5F-I). Somite expansion was quantified by calculating the ratio
between somite areas of injected and uninjected sides of the embryo. We found
a statistically significant increase in somite area in
FP4-mito-injected embryos compared with both AP4-mito
injected and uninjected embryos (Fig.
5J). Closer examination revealed that much of this expansion was
due to an increase in the number of cells that lay parallel or at oblique
angles to the plane of the section, instead of the appropriate perpendicular
orientation. Similar defects in somitogenesis were observed in embryos
expressing an EVH1-GFP dominant-negative protein
(Eigenthaler et al., 2003;
Vasioukhin et al., 2000),
providing additional evidence that the phenotype is specific to inhibition of
Ena/VASP function (data not shown).
|
|
;
Koshida et al., 2005;
Wedlich et al., 1989) and its
deposition is required for somitogenesis in mice and zebrafish
(George et al., 1993;
Julich et al., 2005;
Koshida et al., 2005). Thus,
we examined FN localization to gain information about how Ena/VASP proteins
might regulate somite formation. Immunostaining sections of stage 20 embryos
for FN revealed a tendency of FP4-mito-expressing PSM and somites
to separate from the overlying dorsal epidermis and/or adjacent neural tube
(Fig. 7A,B, arrows; 53.8%,
n=26). In affected embryos, the FN matrix between somites and
adjacent neural tube or notochord appeared fragmented and poorly defined
(bracket in Fig. 7A), and an
apparent shredding of the FN matrix was often observed between somites
(arrowheads in Fig. 7A). This
phenotype was rarely seen in AP4-mito-injected embryos
(Fig. 7C,D; 7.1%,
n=14), and never observed in uninjected controls (n=14; data
not shown). Furthermore, within sections where intersomitic junctions were
discernible, we observed gaps in the FN matrix at somite borders of
FP4-mito-injected embryos (Fig.
7A, asterisks; 68.2%, n=22), whereas similar gaps were
observed at a much lower frequency in AP4-mito (30.8%,
n=13) and uninjected embryos (14.3%, n=14).
Ena/VASP function is required for FN matrix assembly
Given the effect of Ena/VASP inhibition on the FN matrix, we used integrin
5ß1-dependent FN fibril assembly on the blastocoel roof (BCR) as
an assay to test whether Ena/VASP function is required for integrin-dependent
FN matrix assembly. During gastrulation, cells lining the inner surface of the
BCR assemble a dense FN matrix (Winklbauer
and Stoltz, 1995), the formation of which can be blocked by
antibodies raised against 5- and
ß1-integrin, or FN
(Davidson et al., 2002;
Marsden and DeSimone, 2001;
Ramos and DeSimone, 1996).
Thus, this event provides a simple model with which to investigate the
regulation of integrin-mediated FN fibrillogenesis. In uninjected embryos, FN
covered the BCR in a dense matrix (Fig.
7E; n=10). This pattern was recapitulated in BCRs
expressing AP4-mito, although in areas of high GFP expression a
mild thinning was observed (Fig.
7F, n=8). By contrast, all FP4-mito explants
displayed a marked disruption of the FN matrix that coincided with regions of
FP4-mito expression (Fig.
7G, n=17). We also found that gastrulation was delayed in
FP4-mito-expressing embryos, suggesting that disruption of the FN
matrix affects mesendoderm migration across the BCR during gastrulation. Thus,
Ena/VASP function is required for FN fibrillogenesis in vivo.
|
). To investigate whether Ena/VASP proteins modulate adhesion
of somitic cells to FN, stage 20 dorsal explants from uninjected,
AP4-mito or FP4-mito-injected embryos were dissociated,
and triplicate samples of cells were allowed to adhere to FN coated coverslips
for 30 minutes. The number of adherent cells was then counted before and after
washing. This analysis revealed a slight, but nonsignificant, decrease in
adhesion of FP4-mito expressing cells compared with
AP4-mito and uninjected cells (data not shown).
|
|
|
; Henry et al.,
2001; Hens and DeSimone,
1995) and is required for FN fibrillogenesis in cultured mammalian
cells (Ilic et al., 2004).
Given these results, we tested whether FAK activation, as assessed by levels
of autophosphorylation at tyrosine 397, is blocked by inhibition of Ena/VASP
activity. Protein lysates prepared from uninjected stage 20 dorsal explants or
explants expressing either AP4-mito or FP4-mito were
blotted and probed with anti-FAK pY397 antibodies. We found that expression of
FP4-mito caused a significant decrease in the levels of
phospho-FAK, when compared with AP4-mito or uninjected control
explants (Fig. 9).
|
; Ilic et al.,
1998; Richardson et al.,
1997; Richardson and Parsons,
1996; Sieg et al.,
1999; Slack et al.,
2001; Taylor et al.,
2001). The relative expression levels and activity of FRNK were
first examined by western blot analysis, which demonstrated that injection of
500 pg of FRNK mRNA resulted in expression of FRNK at levels similar to that
of endogenous FAK (Fig. 10A).
We also found that FRNK expression resulted in a significant decrease in
autophosphorylation of FAK (Fig.
10B), indicating that FRNK is an effective inhibitor of FAK
activity in vivo. To test the requirement for FAK in somitogenesis, 500 pg of FRNK mRNA was injected unilaterally at the four-cell stage into regions fated to become somites. FRNK expression led to defects in somite rotation, evidenced by the presence of misoriented cells and disruption of intersomitic boundaries (Fig. 10C; 83%, n=18). Next, we tested whether FAK is required for FN matrix assembly in the BCR. We found that in contrast to uninjected or GFP-injected BCRs (Fig. 10D; n=9), FRNK expression blocked FN matrix assembly in the BCR (Fig. 10E; n=12). Thus, FAK is required for somite formation and FN matrix deposition in Xenopus. In addition, the similarity of the phenotypes observed in FRNK and FP4-mito-injected embryos supports the idea that FAK and Ena/VASP function in a common pathway to regulate FN matrix assembly and somitogenesis in Xenopus.
FAK modulates Xena localization
To further explore the relationship between Ena/VASP and FAK, we examined
whether FAK regulates the subcellular distribution of Xena. In these
experiments, 500 pg of FRNK mRNA was injected into the animal pole region of
two-cell stage embryos, animal caps were harvested at stage 10 and Xena
localization was determined by confocal microscopy. In GFP-injected animal
caps, Xena is enriched and tightly localized to the cell cortex
(Fig. 11A; n=5),
whereas in FRNK-injected caps Xena displays a more diffuse cortical staining
pattern (Fig. 11B;
n=7). Comparison of the ratios of pixel intensities at membrane
versus juxtamembrane regions of representative cells from control and
FRNK-injected animal caps demonstrate that inhibition of FAK results in a
significant decrease in membrane-associated Xena staining
(Fig. 11C-E;
GFP=1.26±0.08, FRNK=1.11±0.05, n=50 cells per
treatment). Overall levels of Xena were not affected by FRNK expression,
indicating that the observed redistribution is not due to altered levels of
Xena (data not shown). These data suggest that FAK regulates Xena localization
and provides further evidence that Ena/VASP proteins and FAK functionally
interact in vivo.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
How might Ena/VASP proteins and FAK regulate integrin-dependent adhesion
and migration during somitogenesis? The most obvious role for Ena/VASP
proteins is as a molecular link between cell-surface integrins and the actin
cytoskeleton. Previous studies have shown that Ena/VASP proteins localize to
focal adhesions and bind F-actin, as well as several components of integrin
adhesion complexes, including vinculin and zyxin
(Brindle et al., 1996;
Drees et al., 1999). Thus,
Ena/VASP proteins are appropriately positioned to act as a key regulatory link
between integrins and the actin cytoskeleton to orchestrate changes in
adhesive strength and cell motility during somitogenesis. By modulating the
link between integrins and actin, Ena/VASP proteins could modulate integrin
clustering and formation of juxtamembrane adhesion complexes. Such a role for
Ena/VASP may also explain how Ena/VASP proteins regulate FAK
autophosphorylation, as FAK activity is dependent on integrin clustering and
targeting of FAK to focal adhesions (Hagel
et al., 2002; Shen and
Schaller, 1999).
It is widely accepted that FAK transmits signals to a variety of targets to
govern focal adhesion remodeling associated with changes in cell adhesion and
movement (Schlaepfer et al.,
2004). Thus, the phenotypes caused by inhibiting FAK function
support the idea that integration of integrin-dependent events by FAK is
required for somitogenesis in Xenopus. Specifically, FAK might
facilitate transduction of integrin signals into local changes in cell
motility, adhesive strength and FN matrix assembly/patterning. Interestingly,
we found that FAK activity correlates with localization of Xena to the cortex
of cells in the BCR, although it is not known whether Xena is a direct or
indirect target of FAK. These data support the idea that Xena and FAK
functionally interact during somitogenesis and predict that Xena would be
enriched at sites where FAK activity is high. In agreement with this notion,
Xena and FAK co-localize at somite boundaries and phosphorylated (active) FAK
is enriched at intersomitic boundaries
(Crawford et al., 2003;
Henry et al., 2001).
A second potential mechanism by which Ena/VASP proteins and FAK might
regulate cell behaviors during somitogenesis is through inside-out activation
of integrin adhesion and FN matrix assembly. Inside-out regulation of integrin
activity is an important mechanism underlying changes in cell adhesion and
movements that drive morphogenesis
(Coppolino and Dedhar, 2000;
Miranti and Brugge, 2002).
Studies in Xenopus have shown that developmentally regulated changes
in integrin activity govern a variety of morphogenetic behaviors, including
initiation of gastrulation movements, spreading of mesodermal cells on FN and
FN matrix assembly (Marsden and DeSimone,
2003; Na et al.,
2003; Ramos and DeSimone,
1996; Ramos et al.,
1996). Here, we show that Ena/VASP and FAK are required for FN
matrix assembly in the BCR and inhibition of Ena/VASP function leads to
disruption of the FN matrix surrounding somites and blocks spreading of
somitic cells on FN. Our interpretation of these results is that Ena/VASP
proteins and FAK mediate inside-out regulation of integrin activity during
somitogenesis.
A number of studies have shown that inside-out activation of integrins and
integrin-mediated FN fibrillogenesis is dependent on an intact actin
cytoskeleton (Pankov et al.,
2000; Wu et al.,
1995; Zaidel-Bar et al.,
2003). Thus, it seems likely that the underlying cause of
defective FN matrix assembly in Ena/VASP and FAK inhibited embryos is
disruption of cytoskeletal organization and linkages between the actin
cytoskeleton and cell-surface integrins. Consistent with this idea,
FAK-/- cells display defects in actin stress fiber organization and
integrin-mediated FN matrix assembly and patterning
(Ilic et al., 2004). Likewise,
loss of Ena/VASP function would be predicted to disrupt actin dynamics leading
to dysregulation of integrin activity. In support of this idea, roles for
Ena/VASP proteins in the regulation of integrin-mediated adhesion have been
reported, although these studies reveal that the function of Ena-VASP proteins
in cell adhesion may be cell-type dependent. In osteoclasts, VASP function was
found to correlate with vß3-integrin adhesion and redistribution
of VASP was linked to increased cell motility
(Yaroslavskiy et al., 2005).
Ena/VASP activity also correlates with T-cell receptor-mediated actin
remodeling and integrin activation in lymphocytes
(Griffiths and Penninger,
2002; Krause et al.,
2000). Moreover, Dictyostelium cells lacking VASP show
defects in cell migration that are attributed to the inability of VASP-null
cells to properly adhere to the substratum
(Han et al., 2002). However,
knockout studies in mice have shown that VASP negatively regulates
IIbß3-integrin activity adhesion in platelets
(Aszodi et al., 1999;
Hauser et al., 1999). Thus, a
clear connection exists between Ena/VASP, FAK and integrins, although further
studies are required to elucidate the precise mechanisms by which Ena/VASP and
FAK regulate integrin activity during somitogenesis.
If Ena/VASP proteins and FAK work through integrins to control somite
formation, then one would expect that loss of integrin function would be
associated with defects in migration and adhesion during somitogenesis.
Consistent with this idea, studies in several systems have demonstrated a
requirement for integrins in somite formation or maintenance
(Drake et al., 1992;
Goh et al., 1997;
Julich et al., 2005;
Koshida et al., 2005;
Krotoski and Bronner-Fraser,
1990; Yang et al.,
1993; Zagris et al.,
2004). Furthermore, studies in Xenopus have shown that
expression of a dominant-negative form of ß1-integrin blocks FN matrix
assembly and results in marked defects in somite formation
(Marsden and DeSimone, 2003).
In addition, inhibition of 5-integrin with a function-blocking antibody
results in abnormal segmentation and the loss of intersomitic boundaries (B.
Hoffstrom and D. DeSimone, personal communication). Loss of 5-integrin
function, however, does not appear to affect somite rotation, as most cells
appear to orient themselves properly with their long axis parallel to the
anteroposterior axis. The observation that initial somite morphogenesis
appears to occur normally following loss of 5-integrin function is
consistent with genetic studies in zebrafish showing that itga5 is not
required for somite formation, but is required for maintenance of somite
boundaries (Julich et al.,
2005; Koshida et al.,
2005). These data contrast phenotypes associated with inhibition
of Ena/VASP, FAK and ß1-integrin
(Marsden and DeSimone, 2003)
where somite formation is impaired. One potential explanation for these
differences would be that additional -integrin subunits, such as
3- or 6-integrin, might play essential roles in somite
formation.
Additional mechanisms by which Ena/VASP proteins could govern somitogenesis
that are consistent with our data include regulation of polarized protrusive
activity and cell-cell adhesion. During somite formation in Xenopus,
cells display polarized protrusive behavior which is thought to help drive
rotation (Wilson et al.,
1989). Ena/VASP proteins are known to bind the barbed-ends of
actin filaments to promote actin polymerization and filopodia formation at the
leading edge, activities that could contribute to the protrusive behavior of
cells during somite formation. In addition, the localization of Xena to
cell-cell contacts in the PSM and somitic mesoderm suggests a potential role
for Ena/VASP proteins in cell-cell adhesion during somitogenesis. Evidence
from several model systems underscores the importance of cadherin-based
adhesion in somite formation (Giacomello
et al., 2002; Horikawa et al.,
1999; Kim et al.,
2000; Linask et al.,
1998) and of Ena/VASP proteins in modulating cadherin function
(Grevengoed et al., 2003;
Grevengoed et al., 2001;
Vasioukhin et al., 2000). In
Xenopus, inhibition of cadherin function results in misorientation of
cells and overall disorganization of the myotome
(Giacomello et al., 2002),
defects similar to those caused by neutralization of Ena/VASP function. These
observations leave open the possibility that Ena/VASP proteins are required
for modulating cell-cell adhesion during somite formation. Interestingly,
Marsden and DeSimone (Marsden and
DeSimone, 2003) have shown that integrins regulate cadherin
adhesion during gastrulation, raising the possibility that Ena/VASP proteins
may indirectly regulate cadherin function during somitogenesis by influencing
integrin activity. Addressing potential roles for Ena/VASP proteins in
regulating protrusive activity and cell-cell adhesion during somitogenesis
will be one of our next challenges.
The results presented in this paper indicate that Ena/VASP proteins and FAK
are key components of the molecular machinery that drives somite formation in
Xenopus. Moreover, our data indicates an important role for Ena/VASP
proteins and FAK in the modulation of integrin activity during somitogenesis.
Despite differences in the cellular behaviors that accompany somitogenesis
among vertebrates, the molecular pathways that control morphological
segmentation appear to be conserved
(Holley and Nusslein-Volhard,
2000; Keller,
2000; Pourquie,
2000; Pourquie,
2001; Stickney et al.,
2000). In particular, the dynamic regulation of integrin-mediated
adhesion and migration appears to play crucial roles in coordinating cell
behaviors during somitogenesis (Drake et
al., 1992; Goh et al.,
1997; Julich et al.,
2005; Koshida et al.,
2005; Krotoski and
Bronner-Fraser, 1990; Yang et
al., 1993; Zagris et al.,
2004). Thus, our studies help set the stage for future experiments
that will be needed to determine the precise molecular mechanisms regulating
somite formation.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aszodi, A., Pfeifer, A., Ahmad, M., Glauner, M., Zhou, X. H., Ny, L., Andersson, K. E., Kehrel, B., Offermanns, S. and Fassler, R. (1999). The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function. EMBO J. 18,37 -48.[CrossRef][Medline]
Aulehla, A. and Herrmann, B. G. (2004).
Segmentation in vertebrates: clock and gradient finally joined.
Genes Dev. 18,2060
-2067.
Bachmann, C., Fischer, L., Walter, U. and Reinhard, M.
(1999). The EVH2 domain of the vasodilator-stimulated
phosphoprotein mediates tetramerization, F-actin binding, and actin bundle
formation. J. Biol. Chem.
274,23549
-23557.
Bear, J. E., Loureiro, J. J., Libova, I., Fassler, R., Wehland, J. and Gertler, F. B. (2000). Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101,717 -728.[CrossRef][Medline]
Bear, J. E., Svitkina, T. M., Krause, M., Schafer, D. A., Loureiro, J. J., Strasser, G. A., Maly, I. V., Chaga, O. Y., Cooper, J. A., Borisy, G. G. et al. (2002). Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109,509 -521.[CrossRef][Medline]
Brindle, N. P., Holt, M. R., Davies, J. E., Price, C. J. and Critchley, D. R. (1996). The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin. Biochem. J. 318,753 -757.[Medline]
Carl, U. D., Pollmann, M., Orr, E., Gertlere, F. B., Chakraborty, T. and Wehland, J. (1999). Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands. Curr. Biol. 9, 715-718.[CrossRef][Medline]
Chanoine, C. and Hardy, S. (2003). Xenopus muscle development: from primary to secondary myogenesis. Dev. Dyn. 226,12 -23.[CrossRef][Medline]
Coppolino, M. G. and Dedhar, S. (2000). Bi-directional signal transduction by integrin receptors. Int. J. Biochem. Cell Biol. 32,171 -188.[CrossRef][Medline]
Crawford, B. D., Henry, C. A., Clason, T. A., Becker, A. L. and
Hille, M. B. (2003). Activity and distribution of paxillin,
focal adhesion kinase, and cadherin indicate cooperative roles during
zebrafish morphogenesis. Mol. Biol. Cell
14,3065
-3081.
Davidson, L. A., Hoffstrom, B. G., Keller, R. and DeSimone, D. W. (2002). Mesendoderm extension and mantle closure in Xenopus laevis gastrulation: combined roles for integrin alpha(5)beta(1), fibronectin, and tissue geometry. Dev. Biol. 242,109 -129.[CrossRef][Medline]
Davidson, L. A., Keller, R. and DeSimone, D. W. (2004). Assembly and remodeling of the fibrillar fibronectin extracellular matrix during gastrulation and neurulation in Xenopus laevis. Dev. Dyn. 231,888 -895.[CrossRef][Medline]
Drake, C. J., Davis, L. A., Hungerford, J. E. and Little, C. D. (1992). Perturbation of beta 1 integrin-mediated adhesions results in altered somite cell shape and behavior. Dev. Biol. 149,327 -338.[CrossRef][Medline]
Drees, B. E., Andrews, K. M. and Beckerle, M. C.
(1999). Molecular dissection of zyxin function reveals its
involvement in cell motility. J. Cell Biol.
147,1549
-1560.
Drees, B., Friederich, E., Fradelizi, J., Louvard, D., Beckerle,
M. C. and Golsteyn, R. M. (2000). Characterization of the
interaction between zyxin and members of the Ena/vasodilator-stimulated
phosphoprotein family of proteins. J. Biol. Chem.
275,22503
-22511.
Dubrulle, J. and Pourquie, O. (2004). Coupling
segmentation to axis formation. Development
131,5783
-5793.
Eigenthaler, M., Engelhardt, S., Schinke, B., Kobsar, A.,
Schmitteckert, E., Gambaryan, S., Engelhardt, C. M., Krenn, V., Eliava, M.,
Jarchau, T. et al. (2003). Disruption of cardiac Ena-VASP
protein localization in intercalated disks causes dilated cardiomyopathy.
Am. J. Physiol. Heart Circ. Physiol.
285,H2471
-H2481.M
Fedorov, A. A., Fedorov, E., Gertler, F. and Almo, S. C. (1999). Structure of EVH1, a novel proline-rich ligand-binding module involved in cytoskeletal dynamics and neural function. Nat. Struct. Biol. 6,661 -665.[CrossRef][Medline]
Furuta, Y., Ilic, D., Kanazawa, S., Takeda, N., Yamamoto, T. and Aizawa, S. (1995). Mesodermal defect in late phase of gastrulation by a targeted mutation of focal adhesion kinase, FAK. Oncogene 11,1989 -1995.[Medline]
George, E. L., Georges-Labouesse, E. N., Patel-King, R. S., Rayburn, H. and Hynes, R. O. (1993). Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 119,1079 -1091.[Abstract]
Gertler, F. B., Comer, A. R., Juang, J. L., Ahern, S. M., Clark,
M. J., Liebl, E. C. and Hoffmann, F. M. (1995). enabled, a
dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine
kinase, encodes an Abl substrate with SH3 domain-binding properties.
Genes Dev. 9,521
-533.
Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J. and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87,227 -239.[CrossRef][Medline]
Giacomello, E., Vallin, J., Morali, O., Coulter, I. S., Boulekbache, H., Thiery, J. P. and Broders, F. (2002). Type I cadherins are required for differentiation and coordinated rotation in Xenopus laevis somitogenesis. Int. J. Dev. Biol. 46,785 -792.[Medline]
Giebelhaus, D. H., Zelus, B. D., Henchman, S. K. and Moon, R.
T. (1987). Changes in the expression of alpha-fodrin during
embryonic development of Xenopus laevis. J. Cell Biol.
105,843
-853.
Gilmore, A. P. and Romer, L. H. (1996). Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions dec
