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First published online 10 July 2006
doi: 10.1242/dev.02473
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1 Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue,
New York, NY 10021, USA.
2 Biochemistry, Cell and Molecular Biology Program, Weill Graduate School of
Medical Sciences, Cornell University, 445 East 69th Street, New York, NY
10021, USA.
* Author for correspondence (e-mail: k-anderson{at}sloankettering.edu)
Accepted 5 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Morphogenesis, Cell migration, Axis specification, Nap1, WAVE, Mouse embryo
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gastrulation in the mouse, which establishes the three definitive germ
layers from the single-cell layer of the epiblast, consists of a sequence of
interdependent morphogenetic movements. Gastrulation initiates at E6.5 with
the formation the primitive streak at one position on the circumference of the
proximal epiblast; the position of the primitive streak defines the future
posterior of the embryo. At the primitive streak, cells of the epiblast
undergo an epithelial-to-mesenchymal transition (EMT), exit the primitive
streak, enter either the mesodermal or endodermal germ layers, and then
migrate anteriorly to form the definitive endoderm and mesoderm germ layers
(Tam and Behringer, 1997
). The
EMT depends on Fgf signaling, which leads to the transcriptional
downregulation of E-cadherin as cells traverse the primitive streak, which
allows them to migrate away from the primitive streak in the mesodermal and
endodermal layers (Ciruna and Rossant,
2001). The position and time that mesoderm cells move through the
primitive streak is crucial for their fate: extraembryonic and cardiac
mesoderm exit the primitive streak first, followed by paraxial mesoderm, which
is generated by the middle primitive streak, and the node and notochord, which
arise from the anterior primitive streak
(Tam and Behringer, 1997).
It has been proposed that the position of the primitive streak in the
posterior epiblast depends on the earlier movement of a group of
extraembryonic cells, the anterior visceral endoderm (AVE). At the onset of
gastrulation, the AVE overlies the anterior epiblast at the boundary between
embryonic and extraembryonic regions. The AVE cells secrete Cerl, Lefty1 and
Dkk1, which inhibit the Nodal and Wnt pathways that are required for primitive
streak formation; this restricts the position of the primitive streak to a
single site opposite the AVE (Lu et al.,
2001). The cells that will become the AVE originate in the
visceral endoderm at the distal tip of the egg cylinder at E5.0 when they
begin to express characteristic molecular markers, including the transcription
factors Hex and Hesx1. Between E5.5 and E6.0 the AVE cells migrate from their
initial distal position to the proximal limit of the epiblast
(Thomas et al., 1998).
Time-lapse imaging of Hex-GFP-expressing AVE cells has demonstrated that
AVE cells migrate actively through the visceral endoderm: they become
polarized and extend filopodia in the direction of migration and appear to
move in response to a directional cue
(Srinivas et al., 2004).
Embryological ablation and transplantation experiments suggest that
extraembryonic ectoderm may be the source of chemotactic signals that direct
AVE migration (Rodriguez et al.,
2005). In addition, inhibition of Nodal or Wnt signaling can
reorient the direction of AVE migration
(Kimura-Yoshida et al., 2005;
Yamamoto et al., 2004), and
the Nodal inhibitor Lefty1 and Wnt3 are asymmetrically
expressed in the visceral endoderm at the time of AVE migration
(Rivera-Pérez and Magnuson,
2005; Takaoka et al.,
2006). However, the molecular mechanisms and signals that provide
the force and directionality of AVE migration in the normal embryo are not
clear.
Cell migration depends on reorganization of the actin cytoskeleton that is
mediated, in part, by proteins of the related WASP and WAVE families. WASP is
auto-regulated by an inhibitory domain that prevents interaction with the
ARP2/3 actin-nucleating complex. Binding of CDC42 to WASP relieves WASP
autoinhibition, allowing WASP to bind and activate ARP2/3, and promote
formation of filopodia at the leading edge of migrating cells. WAVE proteins
lack an auto-inhibitory domain and purified WAVE activates ARP2/3
constitutively (Bompard and Caron,
2004). WAVE is regulated by a complex of the Sra1, Nap1, Abi1 and
HSPC300 proteins, which mediates responses to Rac (which binds Sra1), as well
as to Nck (which binds Nap1) and Abl (which binds Abi1)
(Echarri et al., 2004;
Eden et al., 2002;
Gautreau et al., 2004;
Innocenti et al., 2004;
Kobayashi et al., 1998;
Steffen et al., 2004). Because
the activities of Nck, Rac and Abl are regulated by intercellular signals, the
components of the WAVE complex couple extracellular cues to the formation of
lamellipodia at the leading edge of migrating cells.
The WAVE complex is crucial for diverse aspects of morphogenesis during
development, including chemotactic movement of Dictyostelium amoebae
(Blagg et al., 2003;
Ibarra et al., 2006), axon
pathfinding in Drosophila (Hummel
et al., 2000) and spreading of the surface ectoderm in C.
elegans (Soto et al.,
2002). However, the roles of the WAVE complex in the morphogenetic
events required for early vertebrate development have not been studied. Two
murine WAVE genes, WAVE1 and WAVE2, are expressed
in the early embryo and appear to have overlapping functions in early
development, as both single mutants survive beyond midgestation without gross
morphological defects (Soderling et al.,
2003; Yamazaki et al.,
2003; Yan et al.,
2003). Abi2 mutants are viable and do not have
morphological defects (Grove et al.,
2004), whereas mutants in Abi1, either of the two
Sra genes and Hspc300 have not been described.
Here, we demonstrate that Nap1, the only member of its gene family expressed in the early mouse embryo, is essential for specific aspects of early morphogenesis. We confirm that Nap1 is required for the stability of WAVE and the membrane localization of WAVE complex proteins in embryonic cells. Based on the phenotypes of mutant embryos, we find that Nap1 and WAVE play specific roles in the regulation of migration of the mesoderm and endoderm, and in neural tube closure. In addition, we find that Nap1 regulates anteroposterior axis formation because it is required for normal polarization and migration of cells in the AVE. Our findings demonstrate that proper AVE migration is required for localized expression of the signals that determine the position of the primitive streak.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Nap1khlo mutant mice were genotyped
based on linkage to flanking SSLP markers (D2MIT94 and D2SKI328;
http://mouse.ski.mskcc.org/).
Phenotypic analysis was carried out in congenic C3HeB/FeJ animals. Two ES cell
lines (XE068 and XE133) carrying gene trap insertions in the Nap1
gene were obtained from BayGenomics. Both lines contained identical insertions
504 base pairs downstream of exon 24 of Nap1. Mice derived from both
ES cell clones produced identical phenotypes and are collectively referred to
as Nap1GT. Nap1GT mice were genotyped
by PCR using primers to detect the neo gene (Jackson Laboratories;
IMR013, IMR014, IMR015 and IMR016) or using primers that specifically
amplified the wild-type (P1, 5'-CCAGATGGCTGCACTCTTTA-3'; P2,
5'-CGTTCCTGAGAGGACAGAGC-3') or Nap1GT (P1, P3,
5'-TCTAGGACAAGAGGGCGAGA-3') allele. The Hex-GFP transgene
was detected by PCR using primers specific to GFP (Jackson
Laboratories; IMR872, IMR1416).
Genetic mapping of khlo
The khlo mutation was mapped between D2SKI308 and
D2SKI324
(http://mouse.ski.mskcc.org)
in a backcross panel of 911 opportunities for recombination between C57BL/6J
and C3HeB/FeJ or CAST/EiJ. Candidate genes in the critical interval were
identified based on physical map information from Ensembl and the Celera
Discovery System, and sequenced from RT-PCR products amplified from E8.5
Nap1khlo and wild-type C57BL/6J RNA samples. The
Nap1 transcript amplified from four out of four
Nap1khlo mutant embryos contained a T to C transition
mutation at nucleotide 50 of the Nap1-coding region.
Analysis of khlo mutant embryos
In situ hybridization, immunofluorescence and X-gal staining were carried
out as described (Eggenschwiler and
Anderson, 2000). Embryos for histological analysis were fixed in
4% paraformaldehyde in the decidual tissue, embedded in paraffin and sections
were taken every 8 µm. Sections were stained with Hematoxylin and Eosin
according to standard protocols.
Western blotting
Embryos were lysed in RIPA buffer with a protease inhibitor cocktail
(Roche). Total protein concentration was estimated by Bradford assay and
approximately 20 µg of total embryo extract was subject to western
blotting. Bands were detected with ECL Plus (Amersham), and band intensities
were measured from digital scans of films using ImageJ software (NIH).
Explant analysis
Primary explant cultures of nascent mesoderm, epiblast and primitive streak
were generated as described (Burdsal et
al., 1993; Ciruna and Rossant,
2001). The explants were cultured for 24-48 hours on fibronectin
(BD Biosciences). Mesoderm migration in primitive streak explants was measured
on digital photomicrographs using ImageJ software (NIH).
Analysis of Hex-GFP expression
Embryos from Nap1khlo/+; Hex-GFP/+
intercrosses were dissected at E6.0 and fixed in 4% paraformaldehyde for one
hour on ice. GFP-positive embryos were counterstained with
rhodamine-phalloidin (Molecular Probes) and scanned on an inverted Leica TCS
SP2 confocal microscope. Image stacks were assembled and analyzed using
Volocity software (Improvision).
Antibodies and reagents
Polyclonal anti-Nap1 antiserum was raised against a C-terminal peptide
(CHAVYKQSVTSSA), as described (Eden et
al., 2002; Kitamura et al.,
1996; Steffen et al.,
2004). Anti-Abi1 monoclonal antibody was a generous gift from G.
Scita (Milan). Polyclonal antiserum against Sra1 was a generous gift from T.
Stradal (Braunschweig). Commercial antibodies used were: anti-E-cadherin
(Sigma), anti-cortactin (Upstate), anti-WAVE1 (BD Transduction Laboratories)
and anti-
-tubulin (Sigma). Fluorescent secondary antibodies were from
Jackson Laboratories and Molecular Probes. HRP-conjugated secondary antibodies
were from Zymed and Amersham. Filamentous actin was detected with
rhodamine-conjugated phalloidin (Molecular Probes) and nuclei were visualized
with DAPI (Sigma).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Mutants arrested development prior to embryonic turning,
with only 5-6 pairs of small somites when wild-type littermates had 8-12 pairs
of somites (Fig. 1A,B). At
E8.5, khlo embryos accumulated a mass of disorganized mesenchymal
cells at the primitive streak, the source of new mesoderm and endoderm
(Fig. 1D,
Fig. 2B). This phenotype is
seen in embryos with defects in migration of mesoderm away from the primitive
streak (Bladt et al., 2003;
Meyers et al., 1998;
Xue et al., 2001), suggesting
that the khlo mutation affected cell migration. The two khlo
heart primordia failed to fuse to form a single heart tube (cardia bifida;
Fig. 1H,
Fig. 2D), and the underlying
endoderm failed to close to form the foregut pocket
(Fig. 2D). Closure of neural
ectoderm into a tube failed completely in khlo embryos
(Fig. 1B; data not shown).
Because most mesodermal and neural cell types were specified normally in
khlo embryos (Fig.
1B,D,F,H; see also Fig. S1 in the supplementary material), it
appeared that the mutant phenotypes were the result of defects in cell and
tissue movements. In addition to these morphogenetic defects, a fraction of
khlo embryos showed duplications of the anteroposterior body axis of
varying severity (Fig. 3B,C,E;
see below).
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) into Nap1 (Nap1GT) that should
encode a truncated protein fused to ß-geo
(Fig. 4C,D). Embryos homozygous
for the gene trap allele, as well as khlo/Nap1GT
embryos, arrested at E9.0 with phenotypes similar to that of khlo
(Fig. 4F; data not shown). The
similar embryonic phenotypes caused by the two alleles and their failure to
complement confirmed that the early lethality and morphological abnormalities
of the mutants were the result of the loss of Nap1 activity. The
phenotypes of Nap1GT homozygous and
Nap1GT/Nap1khlo embryos were slightly weaker
than that of Nap1khlo homozygotes (see below), which
suggested that Nap1GT retained some wild-type activity.
Consistent with this hypothesis, we detected a small amount of normally
spliced Nap1 transcript in RNA samples from
Nap1GT homozygous embryos by RT-PCR (data not shown).
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Nap1 is essential for stability and membrane localization of the WAVE complex in embryonic cells
Previous RNA interference studies in cultured cells indicated that both
Nap1 and its blood cell-specific homolog Hem1 are required for the stability
of WAVE proteins (Steffen et al.,
2004; Weiner et al.,
2006). Although we did not detect WAVE2 protein in early wildtype
embryos (data not shown), three isoforms of WAVE1 were observed in extracts
from E8.5 wild-type embryos. WAVE1 levels were reduced more than 10-fold in
Nap1khlo mutant embryo extracts (0.06±0.03,
n=5), and by approximately twofold in Nap1khlo
heterozygous embryo extracts (0.44±0.09, n=5,
Fig. 6A); thus, Nap1 is
required for WAVE stability in the early embryo.
The WAVE complex localizes to lamellipodia, and this membrane localization
is essential for its activity (Innocenti
et al., 2004; Steffen et al.,
2004). RNA interference experiments have indicated that Nap1 is
required for membrane localization of the WAVE complex components Sra1 and
Abi1 (Steffen et al., 2004).
In primary cultures of wild-type E7.5 mesodermal cells, Sra1 and Abi1
localized to the leading edge of migrating cells
(Fig. 6B,D). By contrast, Sra1
and Abi1 could not be detected on the surface of Nap1khlo
mutant mesodermal cells (Fig.
6C,E). We observed a low level of Sra1 and Abi1 surface
localization in Nap1GT cells (data not shown), consistent
with the slightly milder phenotype of Nap1GT embryos.
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Nap1 mutant mesodermal cells lack normal lamellipodia and fail to polarize
Because WAVE proteins regulate the reorganization of the actin cytoskeleton
required for cell migration, we assessed the organization of the actin
cytoskeleton in cells isolated from Nap1khlo embryonic
tissue layers. The epiblast (the epithelial layer of the early embryo) and
mesoderm layers were isolated from E7.5 embryos, cultured in conditions where
they retain their epithelial and mesenchymal character
(Burdsal et al., 1993), and
stained with phalloidin to visualize the actin cytoskeleton. The actin
cytoskeleton of Nap1khlo mutant epiblast cells was
indistinguishable from that of wild-type cells
(Fig. 6F,G). By contrast,
phalloidin staining showed that mesoderm cells from
Nap1khlo embryos were more compact than wild-type mesoderm
cells, and had a collapsed network of stress fibers surrounding the nucleus
(Fig. 6H,I). Wild-type
mesodermal cells were polarized and had large lamellipodia, as visualized by
staining for cortactin, an actin-binding protein that is enriched in
lamellipodia (Weed et al.,
2000) (Fig. 6J). By
contrast, Nap1khlo mesodermal cells were surrounded by
many short protrusions, had only small lamellipodia, and lacked clear polarity
(Fig. 6K). Thus, mesodermal
cells isolated from Nap1khlo mutants lacked the actin
organization required for efficient polarized cell migration, similar to the
cellular phenotypes seen in cultured WAVE2 mutant fibroblasts
(Yan et al., 2003), or in cell
lines depleted for Nap1, Hem1, Sra1 or Abi1 activity by small interfering RNAs
(Innocenti et al., 2004;
Steffen et al., 2004;
Weiner et al., 2006).
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). Wildtype primitive streak explants produced
E-cadherin-negative mesenchymal cells that migrated away from the epiblast and
traveled approximately 350 µm over 24 hours
(Fig. 7A). By contrast,
mesenchymal cells produced by Nap1khlo mutant primitive
streaks downregulated E-cadherin, but failed to migrate and accumulated
beneath the epiblast (Fig. 7B).
Thus, Nap1 was absolutely required for mesoderm migration in culture.
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Although cardiac mesoderm cells were present in the two lateral cardiac anlagen of Nap1khlo embryos, these primordia failed to move ventrally to fuse in a single heart tube, which resulted in cardia bifida (Fig. 1H). Despite the abnormal position of Nap1khlo hearts, the myocardial and endocardial tissue layers were organized normally (Fig. 2D) and the hearts could beat. Thus, the tissue reorganization required to generate the layered organization of the heart does not depend on Nap1, whereas the movement of the heart primordia to the midline does require Nap1.
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), and closure of the foregut is required for fusion of the
lateral heart primordia (Constam and
Robertson, 2000; Roebroek et
al., 1998). Thus, the cardia bifida seen in
Nap1khlo mutant embryos may be secondary to delayed
endoderm migration.
Axis duplications in Nap1 mutant embryos
Although most of the phenotypes of Nap1 mutant embryos could be
attributed to defects in morphogenesis, approximately one quarter of
Nap1 mutants had a striking defect in patterning of the body axis.
Primitive streak-derived structures were duplicated in 16% of E8.5 embryos
homozygous for Nap1khlo (13 out of 82) and 8% of
Nap1GT homozygotes (7 out of 86). In half of these embryos
(6 out of 13 for Nap1khlo and 4 out of 7 for
Nap1GT), only the allantois, the most posterior embryonic
structure, was duplicated (Fig.
3B). In the other half, all primitive streak-derived structures,
including the node and the notochord, were duplicated
(Fig. 3C). In addition to these
embryos, some Nap1khlo homozygotes (3 out of 45) had a
second allantois located anterior to the head
(Fig. 3E).
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) or
because of abnormal specification of the primitive streak
(Pöpperl et al., 1997;
Shawlot and Behringer, 1995).
When assayed prior to formation of the node, approximately 20% (3 out of 16)
of E7.5 Nap1khlo embryos expressed the primitive streak
marker brachyury (T) ectopically in two stripes or in a ring at the
proximal end of the epiblast (Fig.
9A-C). Formation of the primitive streak requires Wnt3, and
Wnt3 expression in the posterior epiblast and adjacent visceral
endoderm is the earliest known marker for the posterior pole of the embryonic
axis (Rivera-Pérez and Magnuson,
2005). At E6.5, Wnt3 expression was restricted to the
posterior epiblast of wild-type embryos, but was expanded around the embryonic
circumference in 
40% (3 out of 7) Nap1khlo mutants
(Fig. 9D,E). These results
indicate that the axis duplications in Nap1khlo embryos
were caused by the failure to restrict primitive streak formation to a single
site.
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). Because Nap1 is required for cell migration and for the
restriction of the primitive streak to the posterior, we tested whether Nap1
was required for migration of the AVE. Cerl and Hex are
expressed in overlapping but distinct populations of AVE cells
(Yamamoto et al., 2004). The
AVE, marked by either Cerl or Hex, failed to reach its
normal proximal position at the embryonic/extraembryonic boundary in 50%
(6 out of 11) of the Nap1khlo embryos examined at E6.5 and
E7.5 (Fig. 10A-D). Using a
Hex-GFP transgene (Srinivas et
al., 2004), we were able to follow the behavior of individual AVE
cells. By E6.0, all of the GFP-expressing cells had left the distal tip of the
wild-type embryos (Fig.
10E,G), but in half (4 out of 8) of the
Nap1khlo embryos the majority of the Hex-GFP-expressing
cells failed to migrate away from the distal pole
(Fig. 10E,F), although some
Hex-GFP-expressing cells reached the presumptive anterior
(Fig. 10F). Additionally, in
one embryo in which the majority of the GFP-expressing cells migrated to the
presumptive anterior side of the embryo, a few GFP-positive cells were present
on the posterior side of the embryo (Fig.
10H). We therefore conclude that Nap1 is required for efficient
directional migration of the AVE and that inefficient migration of the AVE is
the cause of the axis duplications seen in Nap1 mutant embryos.
Cells of the wild-type AVE extend polarized processes during migration
(Srinivas et al., 2004), and
we had seen that Nap1 was required for cellular polarity in explanted
mesodermal cells. We therefore hypothesized that the defective AVE movement in
Nap1khlo mutants could be due to a defect in polarization
of the AVE cells. Using the Hex-GFP transgene
(Srinivas et al., 2004) to
mark the AVE, we confirmed that AVE cells in wild-type E6.0 embryos were
elongated and polarized in the direction of migration
(Fig. 10I). By contrast,
Hex-GFP-expressing cells in all Nap1khlo mutant embryos
examined (n=8), regardless of their position along the proximodistal
axis, were more round and had no obvious leading-trailing polarity
(Fig. 10J), a phenotype
similar to that seen in explanted mesodermal cells
(Fig. 6I,K). These data
demonstrate that Nap1 is required for the polarization of AVE cells
and that this polarity is required for efficient migration of the AVE.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although Nap1 is essential for survival of the embryo, not all
morphogenetic events are disrupted in Nap1khlo embryos.
For example, Nap1, and therefore the regulated activity of WAVE, are
absolutely required for the cell shape changes that allow neural tube closure.
By contrast, the morphogenetic events that shape the notochord, the layers of
the heart and the EMT during gastrulation appear to be normal in
Nap1khlo embryos. Most cell migration events in
Nap1khlo mutants are disrupted, although to varying
degrees. Migration of AVE cells, which move as individuals, was consistently
disrupted, although some AVE cells arrive at the correct final destination.
Migration of the definitive endoderm, which migrates as an epithelial sheet,
is severely retarded. Migration of the mesoderm, which migrates as loosely
connected groups of cells, was moderately retarded in vivo. Among the
mesodermal subtypes, only the paraxial mesoderm showed significant disruption:
only a few, small somites were specified in mutant embryos. As the paraxial
mesoderm is the last mesodermal type to move through the primitive streak
(Tam et al., 2001), the defect
in paraxial mesoderm may represent the cumulative effects of slightly delayed
mesoderm migration throughout the course of gastrulation; alternatively,
paraxial mesoderm may be uniquely dependent on Nap1-mediated signals for
migration.
In none of these cases (migration of the AVE, endoderm and mesoderm) does
loss of Nap1 completely block cell migration in vivo. This finding is similar
to that seen with mutations in Dictyostelium NapA, PirA and
Scar, which encode the homologs of Nap1, Sra1 and WAVE
(Blagg et al., 2003;
Ibarra et al., 2006).
NapA, PirA and Scar mutant cells are motile and can move
towards a chemoattractant, but they move more slowly and fail to orient
efficiently towards the chemoattractant. Thus, as in Dictyostelium,
embryonic cell migrations are regulated both by WAVE and by other components
that collaborate with WAVE to promote directional migration.
Tissue-specific regulation of WAVE
The phenotypes of mouse mutants that lack Nck, Abl or
Rac, the components that act upstream of the WAVE complex, suggest
that different signals regulate the WAVE complex in the different cell types.
Embryos that lack both Nck1 and Nck2 arrest at approximately
E9.0 and have an external morphology similar to that of
Nap1khlo mutants (Bladt
et al., 2003). This phenotypic similarity suggests that receptor
tyrosine kinase signaling, mediated by Nck, could be the central regulator of
the WAVE complex during mesoderm and endoderm migration. The neural tube of
Nck1; Nck2 double mutants closes in the trunk, in contrast to the
completely open neural tube of Nap1khlo embryos, which
suggests that Nck does not regulate neural tube closure. However, embryos that
lack two Abl genes, Abl and Arg, completely fail to
close the neural tube (Koleske et al.,
1998). Thus, the Abl kinases are good candidates to act upstream
of WAVE in neural tube closure. Rac1 is required for survival of the
nascent mesoderm (Sugihara et al.,
1998), precluding analysis of its role in germ layer migration or
neural tube closure. Migration of the AVE has not been analyzed in
Rac1 mutants or in Nck1; Nck2 or Abl; Arg double
mutant embryos, so it is not clear which of these upstream regulators control
the WAVE complex in the AVE.
Cell migration is required for specification of a single body axis and acts upstream of Wnt3
Because movement of the AVE correlates with primitive streak formation, it
has been hypothesized that AVE movement determines primitive streak position
and orientation of the anteroposterior axis. However, all the genes that have
been previously shown to be required for AVE migration, such as Cripto,
Otx2 and Lim1, regulate the expression and/or activity of the
Wnt and Nodal signals that control the primitive streak fate
(Ding et al., 1998;
Kinder et al., 2001;
Perea-Gomez et al., 2001). As
a result, the phenotypes of these mutants do not formally distinguish between
the possibility that AVE migration is required to specify the position of the
primitive streak and the possibility that the same signaling molecules act
twice to control AVE migration and primitive streak formation
(Ding et al., 1998;
Perea-Gomez et al., 2001;
Shawlot et al., 1998). Because
Nap1 is dedicated to the regulation of the cytoskeleton, the coupled
Nap1khlo phenotypes of disrupted AVE migration and axis
duplication demonstrate that movement of the AVE is required for normal
positioning of the primitive streak.
The nature of the earliest events that define the position of the
anteroposterior axis is still controversial. Recent data have suggested that
Wnt3 expression in the posterior visceral endoderm may precede AVE
migration (Rivera-Pérez and
Magnuson, 2005) and that overexpression of Wnts can block AVE
migration (Kimura-Yoshida et al.,
2005). These observations suggested that the direction of AVE
migration is a consequence of a pre-existing anteroposterior polarization of
Wnt signaling. However, the AVE migrates normally in Wnt3 mutant
embryos (Liu et al., 1999). In
addition, our data demonstrate that AVE movement is crucial for the
restriction of Wnt3 expression to the posterior of the embryo. We
therefore argue that AVE migration begins before Wnt signaling is localized
and that other, non-Wnt, signals initiate the polarized migration of the AVE
cells. Our findings would, however, be consistent with the possibility that
once AVE movement is initiated, localized Wnt signals reinforce the direction
of AVE migration.
While AVE migration and the domain of Wnt3 expression appear to be
directly coupled, additional events must regulate the formation of the
primitive streak and the definitive anteroposterior axis. Although we find
that the degree of migration of the AVE is correlated to the domain of
Wnt3 expression, the frequency of axis duplication at E8.5 was
significantly lower than the frequency of AVE migration defects (13 out of 82
versus 9 out of 18; 
2, P
0.01). We infer that the
expanded Wnt3 domain represents the region that is competent to form
a primitive streak, but additional regulative events restrict primitive streak
formation to a single site in all but the most severely affected mutants.
Consistent with these observations, embryological experiments in the chick
have demonstrated the existence of a streak-derived inhibitor that prevents
ectopic streak formation (Bertocchini et
al., 2004).
We propose that several mechanisms act in concert to insure formation of a single primitive streak and, therefore, a single anteroposterior body axis. The migration of AVE cells is regulated by Nap1 and also by Nap1-independent mechanisms. AVE migration controls the size of the domain competent to form a primitive streak, marked by Wnt3 expression. If the AVE migrates slowly or does not reach the correct final position, a feedback loop is initiated from the primitive streak that prevents the specification of an ectopic primitive streak. Only when more than one mechanism fails, is it possible for two stable primitive streaks to form and direct the specification of more than one body axis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/16/3075/DC1
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ACKNOWLEDGMENTS |
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