during xenopus gastrulation , mesoderm migrates to the inside of the embryo and moves along the blastocoel roof to establish the three germ layer structure .
this process involves several morphogenetic cell movements including mesendoderm extension and convergent extension . during mesendoderm extension ,
cells migrate along the blastocoel roof in contact with fibronectin ( fn ) fibrils ( winklbauer , 1990 ; davidson et al . , 2002 ) .
in convergent extension , cells are polarized and elongated mediolaterally , then the cells are intercalated .
this movement forms the dorsal mesodermal structure and extends the anteroposterior body axis ( shih and keller , 1992 ; wallingford et al . , 2002 ) .
the noncanonical wnt pathway has been implicated in the regulation of convergent extension ( kuhl , 2002 ; tada et al . ,
one of the intracellular signaling components , xenopus dishevelled ( xdsh ) , plays a pivotal role in this process . when the function of xdsh is inhibited , the polarity of the mesodermal cells is not established normally ( wallingford et al . , 2000 ) . because these cell movements are accompanied by dynamic changes in cell polarity , morphology , and motility , it is very likely that cytoskeletal dynamics are carefully regulated .
we decided to focus on myristoylated alanine - rich c kinase substrate ( marcks ) .
mammalian marcks has been shown to interact with actin ( arbuzova et al . , 2002 ) .
it has been reported that xenopus marcks is expressed maternally and throughout embryogenesis ( ali et al . , 1997 ;
1997 ) , but its role in development was not well understood . here , we report that the loss of marcks function severely impaired gastrulation movements .
marcks regulates the cortical actin formation , cell adhesion , protrusive activity , and cell polarity control during gastrulation .
we further show that marcks is necessary for the protrusive activity regulated by the noncanonical wnt pathway .
these findings show that marcks regulates the cortical actin formation that is requisite for dynamic morphogenetic movements .
to investigate the function of marcks in xenopus development , we conducted loss of function experiments using antisense morpholino oligonucleotides ( mo ) . first , we examined the specificity of marcks mo ( fig .
the mo specifically and effectively inhibited epitope - tagged marcks protein synthesis , leading us to expect that it could inhibit the endogenous marcks protein synthesis .
using marcks mo , we analyzed marcks function in development . when it was injected into the dorsal marginal zone ( dmz ) of four - cell embryos ,
the phenotype of marcks mo was partially rescued by coinjection of marcks mrna ( fig .
as discussed below , however , cell biological effects of marcks mo were efficiently rescued by marcks mrna .
over- and under - expression of marcks may have opposite effects at a cellular level , but both of these effects may negatively influence gastrulation movements .
( a ) both 500 pg of marcks mrna and 5 pmol of marcks mo impaired gastrulation movements , when either was injected into the dorsal marginal region .
( b ) statistical data of the gastrulation - defective phenotype caused by marcks mrna and mo .
( c ) expression of chordin at the gastrula stage , detected by in situ hybridization .
( d ) somites ( left ) and notochord ( right ) were immunostained with 12/101 and mz15 antibodies , respectively .
( e ) 5 pmol of marcks mo was injected into the two dorsal blastomeres at the four - cell stage ; the dmz explants were isolated , and the expression of mesodermal markers was detected by rt - pcr .
( f ) 2 pmol of marcks mo inhibited the activin mrna - induced elongation of animal caps .
it has been reported that marcks - like protein ( xmlp ) is also expressed in xenopus embryo ( zhao et al . , 2001 ) .
although xmlp is similar to marcks ( 23% amino acid identity ) , xmlp - mo injected embryos showed malformations of the anterior axis and eye defect , but the gastrulation defect was not reported ( zhao et al . , 2001 ) .
they seem to play distinct roles in xenopus development . to determine whether this gastrulation defect was caused by a defect in mesodermal differentiation
, we examined the expression of the dorsal mesodermal markers . at the gastrula stage , marcks mo - injected embryos expressed chordin at the same level as control embryos ( fig .
the notochord and somites were formed in the marcks mo - injected embryos , but the extension of these tissues was severely inhibited ( fig .
we also tested the expression of the mesodermal markers in dmz explants by rt - pcr ( fig .
these results indicated that the phenotype was caused , not by a defect in mesoderm differentiation , but by a defect in morphogenetic movements .
next , we tested whether the loss of marcks function affects the animal cap elongation , which mimics convergent extension movements during gastrulation ( fig . 1 f )
. marcks mo blocked the elongation by activin , and it was rescued by coinjecting marcks mrna without the utr , suggesting that marcks is required for convergent extension . during mesodermal convergent extension , the cells become polarized , align mediolaterally , and are then intercalated . to test
how marcks is involved in this process , the convergent extension in dmz explants was observed microscopically .
marcks mo , rhodamine dextran , and mrna encoding membrane - binding venus ( mb - venus ) were coinjected into one of the two dorsal blastomeres ( fig .
mb - venus mrna alone was injected into the other dorsal blastomere . at the gastrula stage ,
these explants adhered to the fn , and convergent extension movements occurred subsequently in the mesoderm ( kinoshita et al .
in the absence of marcks mo , red and nonred cells were polarized and intercalated . in the marcks
mo - injected explants , the nonred cells , which were assumed to lack the mo , were polarized and showed convergent extension .
in contrast , the red cells ( mo - injected cells ) were not polarized and did not participate in the intercalation .
( a ) 5 pmol of marcks mo , rhodamine dextran , and the mrna for 100 pg of mb - venus were coinjected into one of the two dorsal blastomeres at the four - cell stage .
dmz explants were cultured on a cover glass coated with fn , and convergent extension movements were observed .
( b ) control or 5 pmol of marcks mo was coinjected with 100 pg of venus mrna into two blastomeres of four - cell embryos .
dmz explants were cultured on an fn - coated dish until sibling embryos reached the late neurula stage .
marcks mo , control mo , venus mrnas ( green ) , and rhodamine dextran ( red ) were coinjected dorsally as indicated .
cells from the control- and marcks - mo  injected explants were mixed , plated on fn - coated dishes , incubated for 6 h , and fixed in formaldehyde .
cells that did not adhere to the dish were removed by washing five times with pbs .
( d ) 3 pmol of marcks mo inhibited the protrusive activity of cells in dmz explants .
dmz explants were cultured on an fn - coated dish until sibling embryos reached the early neurula stage .
in addition to convergent extension , an important mechanism regulating gastrulation movements is mesendoderm extension ( davidson et al . , 2002 ) .
to test whether marcks is required for this process , dmz explants were cultured on fn - coated dishes according to the method developed by davidson et al .
2 b ) . when venus mrna and the control mo were coinjected , the venus - expressing cells dispersed broadly , and some cells migrated to the front .
we examined 15 explants and confirmed that none of the marcks mo - injected cells reached the leading edge of the migrating mesendoderm .
we examined whether marcks mo affects the adhesion to fn of cells dissociated from the dmz explants .
marcks mo and venus mrna ( green ) or rhodamine dextran ( red ) was injected dorsally ( fig . 2 c ) .
dissociated cells were cultured on fn - coated dishes , and cells that adhered to the dish were counted ( fig .
2 c ) . when marcks mo was coinjected with venus , the adherence of venus - expressing cells was extremely reduced .
in contrast , when marcks mo was coinjected with rhodamine dextran , these red cells rarely adhered to the dish .
a few cells containing marcks mo were found on the dish , but these cells were rounded up and did not spread out on the dish .
the effect of marcks mo on the cell adhesion was rescued by coinjection with marcks mrna ( unpublished data ) .
mesodermal cells had many filopodia - like protrusions when the dmz explants adhered to a fn - coated dish ( fig .
the effect of marcks mo on the protrusive activity was rescued by coinjection with marcks mrna .
thus , marcks is required for the protrusive activity , which may directly correlate with the control of cell adhesion and motility .
the inhibition of cell adhesion and migration on the fn fibrils that cover the blastocoel roof may contribute to the gastrulation defect caused by marcks mo .
the actin - binding domain of mammalian marcks binds to actin filaments and cross - links them in vitro ( hartwig et al . , 1992 ) .
cells expressing marcks - venus were dissociated from dmz explants and cultured on an fn - coated dish .
the cells were then fixed and f - actin was stained with phalloidin . as shown in fig .
( a ) cells expressing 200 pg of marcks - venus mrna were dissociated from dmz explants and plated on a fn - coated cover glass .
( b ) wild - type , ga , and sd mutants were expressed in the dmz explants and observed .
( c ) the actin - binding domain of xenopus moesin was fused with venus ( venus - actin bd ) and expressed in cho cells .
( d ) mo was injected into one blastomere of two - cell embryo with mb - rfp mrna as a tracer .
( e ) western blot with an antiactin antibody ( left ) and coomassie blue ( cbb ) staining ( right ) .
( f ) marcks mo and mb - rfp were coinjected and animal cap cells were immunostained with antiactin antibody .
ga is an unmyristoylated mutant in which the second glycine residue is replaced with alanine .
sd is a pseudophosphorylation mutant whose potential phosphorylation sites were replaced with aspartic acid , which is expected not to bind to actin filaments ( hartwig et al . , 1992 ) .
to detect f - actin , we used the f - actin  binding domain of moesin fused to red fluorescent protein ( rfp ; campbell et al . , 2002 ) , designated rma ( rfp - moesin actin  binding domain ) .
it has been shown biochemically that this domain binds to f - actin ( turunen et al . , 1994 ;
, the corresponding domain of moesin fused with gfp was successfully used to analyze actin dynamics ( dutta et al . , 2002 ) .
we confirmed that our construct ( venus - moesin actin - binding domain ) colocalized with stress fibers and cortical actin stained with phalloidin in cho cells ( fig .
embryonic cells , the rma was localized to the cell cortices , and cytochalasin b treatment , which disrupts actin filaments , dispersed the rma to the cytoplasm ( fig .
when these marcks - venus genes were expressed , the wild - type and ga forms were associated with the plasma membrane and colocalized with rma , but sd was in the cytoplasm . when the cells were treated with cytochalasin b
, ga dispersed to the cytosol with rma , whereas wild - type remained on the membrane .
this result suggested that the association of marcks with the membrane was regulated by two mechanisms , myristoylation and binding to the cortical actin .
we also found that ga and sd mutants did not inhibit gastrulation movement when they were overexpressed ( fig .
these results suggest that both myristoylation and actin biding are required for its function . to test whether marcks regulates cortical actin formation , marcks mo
animal caps were isolated at the late blastula stage , fixed , and stained with phalloidin .
3 d , marcks mo significantly reduced the amount of cortical actin stained by phalloidin .
the amount of actin protein was not affected , however , judging from western blotting and immunocytochemistry using an antipan actin antibody ( fig .
it has been demonstrated that xdsh , an essential cytoplasmic component in this pathway , regulates cell polarity and protrusive activity in dmz cells ( wallingford et al . , 2000 ) .
the wnt pathway activates rhoa and rac ( habas et al . , 2001 , 2003 ) , which have been shown to regulate the protrusive activity ( tahinci and symes , 2003 ) .
thus , the pathway may directly regulate actin cytoskeletal dynamics . to investigate the relationship between the wnt pathway and cortical actin , we examined the localization of xdsh .
cells were dissociated from the dmz explants expressing xdsh - venus , cultured on an fn - coated dish , and stained with phalloidin . as shown in fig .
4 a , xdsh was colocalized with cortical actin , even in the lamellipodial and filopodial protrusions .
marcks is required for the cortical actin dynamics regulated by the noncanonical wnt signaling pathway .
( a ) 250 pg of xdsh - venus mrna was expressed in dmz explants .
( b ) 100 pg of xdshdix - venus mrna was expressed in dmz explants .
( c ) xwnt11 and xfz7 mrnas ( 200 pg each ) were coexpressed in animal cap explants with mb - rfp .
the effect of marcks mo was rescued by coinjection of 200 pg of marcks mrna .
( d ) mb - rfp was injected with or without mrna encoding dominant - negative xwnt-11 ( 2 ng ) .
when rma was expressed during convergent extension , it was located at the tips of elongated mesodermal cells ( fig . 4 b ) .
we showed previously that xdsh - venus was also accumulated in the same region ( kinoshita et al . , 2003 ) .
mammalian dishevelled interacts with actin filament through the nh2-terminal dix domain ( capelluto et al . ,
whether the tip localization of xdsh was due to the interaction between the dix domain and f - actin , we tested the localization of xdsh lacking the dix - domain ( xdshdix ) .
4 b , the xdshdix was located at the tip , indicating that this localization is not due to interaction between f - actin and the dix domain .
this result is consistent with the finding that dishevelleddix can mediate the noncanonical wnt signaling in xenopus and zebrafish ( heisenberg et al . , 2000 ;
the actin depolymerizing reagent , latrunculin a , dispersed both rma and xdshdix to the cytosol .
essentially , the same result was also obtained using cytochalasin b ( unpublished data ) .
these results strongly suggest that xdsh interacts with f - actin either directly or indirectly and mediates the wnt signaling to the actin cytoskeleton . to examine whether the wnt pathway regulates the protrusive activity , we coexpressed xwnt11 and xfz7 ( xenopus frizzled-7 ) in animal cap explants with the membrane - binding rfp .
4 c , the coexpression of xwnt11 and xfz7 dramatically promoted lamellipodia- and filopodia - like protrusions and it was inhibited by xdd1 , a dominant negative xdsh mutant ( sokol , 1996 ; wallingford et al . , 2000 ) .
when marcks mo was coinjected , this activity was severely inhibited ( fig . 4 c ) .
in addition , dorsal mesodermal cells expressing dominant - negative xwnt-11 ( tada and smith , 2000 ) significantly reduced the number of protrusions ( fig . 4 d ) ,
these results strongly suggest that the wnt signaling pathway regulates cortical actin dynamics and that marcks is required for this process . here , we have shown that marcks plays an essential role in regulating cortical actin dynamics in xenopus development .
marcks mo inhibited cell movements , cell shape change , cell adhesion , and interaction with fn probably through the defect it caused in the cortical actin dynamics .
marcks is required not only for gastrulation but also for the neural tube formation . when marcks mo was injected into the dorso - anterior blastomeres of eight - cell embryos to target the neuroectoderm , neural tube closure was severely impaired ( fig .
it has been shown that marcks - deficient mouse shows neural tube closure defect ( stumpo et al .
the regulation of the cortical actin cytoskeleton by marcks may be important for a proper cellular response to signals such as wnt and the fn / integrin pathways .
pkc has been involved in the noncanonical wnt pathway ( sheldahl et al . , 1999 ) and the integrin pathway ( vuori and ruoslahti , 1993 ) .
it would be interesting to determine how the activity of marcks is regulated during development .
procedures for the plasmid construction , rna synthesis and sequences of morpholino oligos were described in the online supplemental material .
the rfp plasmid is a gift from r. tsien ( university of california , san diego , ca ) . in situ
hybridization in xenopus was performed as described by harland ( 1991 ) . for rt - pcr analyses ,
the procedure for whole - mount immunostaining was performed as described in kurata et al .
the antibodies were mz15 for notochord ( a gift from f. watt , imperial cancer research fund , london , uk ) and 12/101 for somites
western blotting was performed using a mouse monoclonal antipan - actin antibody was purchased from neomarkers ( ms-1295-p0 ) . for the animal cap explants ,
marcks mrna or mo was coinjected with 0.5 pg activin mrna into the animal pole of two - cell embryos .
the animal cap was dissected from stage-9 embryos . for dmz explants , mrna or a mo
these explants were cultured in 1 steinberg 's solution until sibling embryos reached stage 17 . to dissociate cells from the explants
, the explants were incubated in the ca - mg  free medium for 2 h. for the cytological observation , explants and dissociated cells were cultured in 1 steinberg 's solution on an fn - coated dish ( 4000030 ; iwakil ) , or on a cover glass coated with fn ( 1 g / cm , f1141 ; sigma - aldrich ) . to stain f - actin ,
cells were fixed in 4% pfa and stained with pbs 0.5% triton x-100 containing a 40-fold dilution of bodipy 581/589 phalloidin ( b-3416 ; molecular probes ) or alexa fluor 488 phalloidin ( a-12379 ; molecular probes ) . for confocal microscopy , images were captured using 510 software ( carl zeiss microimaging , inc . ) .
procedures for the plasmid construction , rna synthesis and sequences of morpholino oligos were described in the online supplemental material .
the rfp plasmid is a gift from r. tsien ( university of california , san diego , ca ) .
in situ hybridization in xenopus was performed as described by harland ( 1991 ) . for rt - pcr analyses ,
the procedure for whole - mount immunostaining was performed as described in kurata et al .
the antibodies were mz15 for notochord ( a gift from f. watt , imperial cancer research fund , london , uk ) and 12/101 for somites
western blotting was performed using a mouse monoclonal antipan - actin antibody was purchased from neomarkers ( ms-1295-p0 ) .
for the animal cap explants , marcks mrna or mo was coinjected with 0.5 pg activin mrna into the animal pole of two - cell embryos .
the animal cap was dissected from stage-9 embryos . for dmz explants , mrna or a mo
these explants were cultured in 1 steinberg 's solution until sibling embryos reached stage 17 . to dissociate cells from the explants
, the explants were incubated in the ca - mg  free medium for 2 h. for the cytological observation , explants and dissociated cells were cultured in 1 steinberg 's solution on an fn - coated dish ( 4000030 ; iwakil ) , or on a cover glass coated with fn ( 1 g / cm , f1141 ; sigma - aldrich ) . to stain f - actin , cells were fixed in 4% pfa and stained with pbs 0.5% triton x-100 containing a 40-fold dilution of bodipy 581/589 phalloidin ( b-3416 ; molecular probes ) or alexa fluor 488 phalloidin ( a-12379 ; molecular probes ) .
for confocal microscopy , images were captured using 510 software ( carl zeiss microimaging , inc . ) .
 myristoylated alanine - rich c kinase substrate ( marcks ) is an actin - binding , membrane - associated protein expressed during xenopus embryogenesis . 
 we analyzed its function in cytoskeletal regulation during gastrulation . 
 here , we show that blockade of its function impaired morphogenetic movements , including convergent extension . 
 marcks was required for control of cell morphology , motility , adhesion , protrusive activity , and cortical actin formation in embryonic cells . 
 we also demonstrate that the noncanonical wnt pathway promotes the formation of lamellipodia- and filopodia - like protrusions and that marcks is necessary for this activity . 
 these findings show that marcks regulates the cortical actin formation that is requisite for dynamic morphogenetic movements . 
Introduction
Results and discussion
Materials and methods
Plasmids, RNA synthesis, and Morpholino oligos
In situ hybridization and RT-PCR analysis
Whole-mount immunostaining and Western blotting
Dissecting explants and cytological observations
Online supplemental material