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First published online 13 August 2008
doi: 10.1242/dev.022897
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1 Molecular Biology and Genetics Department, Cornell University, Ithaca, NY
14853, USA.
2 Sloan Kettering Institute, New York, NY 10021, USA.
* Author for correspondence (e-mail: garciamj{at}cornell.edu)
Accepted 10 July 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Axis elongation, Convergent extension, Definitive endoderm, Morphogenesis, Mouse development
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Extensive studies on intact embryos and tissue explants
using time-lapse imaging have confirmed that coordinated cell rearrangements
mediate convergent extension in fish and frog embryos
(Concha and Adams, 1998;
Davidson and Keller, 1999;
Elul and Keller, 2000;
Jessen et al., 2002;
Keller and Tibbetts, 1989;
Tahinci and Symes, 2003;
Wallingford et al., 2000;
Wilson and Keller, 1991).
Non-canonical Wnt signaling is required for convergent extension in
Xenopus and zebrafish (reviewed by
Tada et al., 2002). Genetic
and experimental disruptions of this signaling pathway, such as loss of
function mutations in zebrafish van gogh-like 2 (vangl2;
also known as trilobite)
(Hammerschmidt et al., 1996;
Jessen et al., 2002), or
overexpression of mutated forms of Dishevelled in Xenopus
(Goto and Keller, 2002;
Moon et al., 1993;
Tada and Smith, 2000;
Wallingford et al., 2000),
cause characteristic convergent extension defects, such as a short
anterior-posterior axis, a wide notochord and a broad, open neural tube. Other
genetic pathways are also important for convergent extension in zebrafish,
including BMP gradients (von der Hardt et
al., 2007), the zinc-finger protein Bloody fingers
(Sumanas et al., 2005) and the
ERR
orphan nuclear receptor (Bardet
et al., 2005).
In the mouse, the morphogenetic events that create the elongated
anterior-posterior body axis are not well understood. Elongation of the mouse
embryo takes place during late gastrulation [embryonic day (E) 7.5-9.0], when
extensive cell rearrangements/movements generate the germ layers and organ
primordia (Kinder et al.,
1999). As these cells reorganize and migrate, the embryo grows
dramatically, from 
600 cells at pregastrula stages (E6.0) to nearly
14,000 at neurulation (E8.5) (Lawson,
1999). Recent time-lapse imaging studies showed that cell
intercalation takes place in the axial midline of mouse embryos during the
lengthening of the node along the anterior-posterior axis
(Yamanaka et al., 2007).
However, the importance of convergent extension movements to elongation of
other embryonic tissues is not clear, in part owing to a lack of analysis of
cell behavior during these stages.
Mouse mutants that lack components of the non-canonical Wnt signaling
pathway show some of the features characteristic of Xenopus and
zebrafish embryos with disrupted convergent extension, including a wide
notochord and open neural tube (Greene et
al., 1998; Kibar et al.,
2001; Murdoch et al.,
2001a). It has been proposed that defects in axial mesendoderm
extension in mouse Vangl2 [also known as loop-tail (Lp)]
mutant embryos are caused by defective midline cell intercalation in the node
area (Ybot-Gonzalez et al.,
2007). Although it is clear that non-canonical Wnt signaling
contributes to the elongation of the mammalian embryo
(Wallingford et al., 2002;
Wang, J. et al., 2006), the
phenotypes of mouse mutants that lack non-canonical Wnt signaling are not as
severe as those of their zebrafish mutant counterparts. For example,
elongation and convergence of non-axial mesoderm is not as severely affected
in mouse Vangl2 embryos (Greene
et al., 1998; Kibar et al.,
2001; Murdoch et al.,
2001a) as in zebrafish vangl2 mutants
(Hammerschmidt et al., 1996;
Jessen et al., 2002), even
though the mutations disrupt orthologous genes. Mouse mutants that lack
non-canonical Wnt signaling die at birth with severe neurulation defects and
disruption of planar cell polarity (PCP) in inner ear hair cells
(Curtin et al., 2003;
Montcouquiol et al., 2003;
Wang, Y. et al., 2006), but
their trunk length is similar to that of wild-type littermates and the
contribution of PCP defects to mouse axis elongation is not clear. To date,
the results suggest that convergent extension mechanisms controlled by
non-canonical Wnt signaling are important for elongation of some embryonic
tissues such as the notochord
(Ybot-Gonzalez et al., 2007),
but the differences between mouse and zebrafish Vangl2 mutant
phenotypes argue that other pathways and/or mechanisms contribute to the
elongation of non-axial tissues in the mouse embryo.
Here we report the identification and characterization of Chato, a novel KRAB zinc-finger protein required for mammalian convergent extension. Two independent recessive mutant alleles of chato cause morphogenetic defects similar to those of fish and frog embryos with defective convergent extension, including a shorter and wider body axis, open neural tube and mediolaterally expanded somites. To evaluate whether chato regulates convergent extension mechanisms similar to those seen in fish and frogs, we measured changes in the length and width of wild-type and mutant embryonic tissues during early development. Because of the relative simplicity of its morphogenetic movements, we focused our analysis on the definitive endoderm layer, the precursor of the gut. Morphometric analysis of wild-type embryos shows that the definitive endoderm narrows and elongates during embryogenesis and that convergent extension of this tissue is mediated by cell rearrangements. In chato mutants, the definitive endoderm is wider and cell rearrangements do not take place. Genetic experiments indicate that Chato regulates convergent extension events through a novel pathway independent of non-canonical Wnt signaling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kasarskis et al.,
1998). chato was analyzed in C3H/FeJ, CAST/Ei and
129Sv/ImJ genetic backgrounds. Nodal-lacZ and Lp mice were
obtained from Dr Elizabeth J. Robertson
(Collignon et al., 1996b) and
from Jackson Labs (LPT/LeJ strain) respectively. Lp mice were
outcrossed to C3H/FeJ and genotyped with D1Mit36 and D1Mit149 SSLP
markers.
Physical mapping and sequencing of candidate genes
Genetic mapping of Zfp568chato was performed by linkage
analysis of 981 opportunities for recombination with SSLP markers
(www.informatics.jax.org
and
http://mouse.ski.mskcc.org).
Physical map information was obtained from Ensembl
(http://www.ensembl.org/Mus_musculus/index.html).
cDNAs of all candidate genes in the chato interval (Zfp27, Zfp74, Zfp568, Zfp14, Zfp82 and Zfp260) were amplified by RT-PCR (Superscript One-Step RT-PCR, Invitrogen) using RNA from E8.5 chato and C57BL/6J (control) embryos. Amplification products were sequenced. A mutation, T to C, was identified at codon 64 of the Zfp568 ORF. This point mutation generated an MspI restriction fragment length polymorphism that was used to confirm linkage with chato embryos and carrier animals. No mutations were found in any of the other genes in the interval.
Characterization of the Zfp568RRU161 allele
BayGenomics RRU161 gene trap creates an abnormal splicing between the first
coding exon of Zfp568 and a splicing acceptor site present in the
gene-trap vector
(http://www.genetrap.org).
RRU161 completely disrupts the normal splicing of Zfp568, as
tested by RT-PCR of homozygote RRU161 embryos using primers located in the
first and second coding exons of Zfp568. The RRU161 gene-trap fusion
protein contains 11 amino acids from Zfp568 followed by 19 amino acids that do
not contain any recognizable functional domains (β-galactosidase coding
sequence was out of frame).
Analysis of mutant embryos
Embryos were dissected in PBS containing 0.4% BSA at different stages as
assessed by presence of vaginal plugs in mothers. Whole-mount RNA in situ
hybridization and staining for β-galactosidase activity were performed as
described (Belo et al., 1997;
Nagy, 2003).
Embryos used for length and width measurements were fixed in 4% paraformaldehyde at 4°C for 8-10 hours, then washed and photographed in PBS (dehydration was avoided to prevent shrinkage of embryos). Measurements were taken with Axiovision AC Zeiss software on pictures of the same magnification.
For immunohistochemistry and TUNEL, embryos were cryosectioned (8-10 µm)
as previously described (Garcia-Garcia and
Anderson, 2003). Antibodies used were anti-E-cadherin (cadherin 1)
(Sigma) at 1/250 and anti-phospho-histone H3 (Ser10) (Upstate) at 1/250. TUNEL
was performed using the ApopTag Detection Kit (Chemicon). As positive controls
for TUNEL, we used sections treated with DNase I.
Cell counts were collected from embryos processed through Ttr in situ hybridization, embedding, cryosectioning (8 µm) and counterstaining with Fast Red. Data plots and statistical analysis of measurements were performed using Excel software (Microsoft). Statistical significance was calculated using two-tailed t-tests with Prism software (GraphPad).
Scanning electron microscopy was performed at Sloan-Kettering and Cornell Imaging Facilities using Jeoul and Hitachi 4500 microscopes. Samples were fixed overnight in PBS containing 2.5% glutaraldehyde, washed in PBS, dehydrated in ethanol and processed for critical-point drying and gold-palladium coating.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; see Materials and methods). chato mutant embryos
arrested by E9.0 and remained unturned, with a short anterior-posterior body
axis and an open gut tube (Fig.
1; see Fig. S1A,B in the supplementary material).
Analysis of mesodermal tissues in chato embryos showed that
defects in axis elongation were accompanied by a failure of cells to properly
localize with respect to the midline. Analysis of Twist1 expression,
which marks somites and lateral plate mesoderm
(Quertermous et al., 1994),
showed that these mesodermal tissues were located further away from the
midline of chato embryos than in wild-type littermates
(Fig. 1A,B). Expression of a
Nodal-lacZ reporter (Collignon et
al., 1996b) also showed that the lateral plate mesoderm in
chato mutants was shorter and wider than in wild-type embryos
(Fig. 1C,D). Somitic mesoderm
was specified in all chato mutants, but it showed defects in
morphogenesis (Fig. 1A,B,E,F).
Many chato embryos (n=61/184) showed condensed somites that
were mediolaterally expanded and narrow in the anterior-posterior axis, as
shown by expression of Meox1
(Candia et al., 1992)
(Fig. 1E,F and
Fig. 3C-E). Mesodermal
precursors of the heart, which arise from lateral positions, failed to migrate
and fuse at the midline of all chato mutants and remained in two
separate domains at both sides of the embryo as shown by expression of the
heart marker Nkx2.5 (Fig.
1G,H) (Lints et al.,
1993); this cardia bifida phenotype is presumably responsible for
the death of the embryos at E9.5-10. Altogether, these mesodermal defects are
similar to those seen in zebrafish embryos in which convergent extension is
disrupted (Matsui et al.,
2005), but are different than those of mouse non-canonical Wnt
pathway mutants.
Morphogenetic defects in the chato neural plate and notochord
Epithelial tissues in chato embryos also had morphogenetic
defects. The chato headfolds failed to fuse to form a neural tube
(Fig. 2A-G). In the open neural
plate, markers of specific cell-type populations, such as Krox20
(Egr2) (Wilkinson et al.,
1989), were expressed in domains that were narrow along the
anterior-posterior axis and laterally expanded when compared with wild-type
littermates (Fig. 2A,B), a
phenotype similar to zebrafish vangl2 mutants
(Jessen et al., 2002). The
neural tube also failed to close normally at more-posterior positions of the
anterior-posterior axis. In some chato mutants, it completely failed
to close (55%, Fig. 2E),
whereas in others it remained open only at some locations (45%,
Fig. 2D), as visualized by
expression of the pan-neural marker Sox2
(Collignon et al., 1996a).
Failure to close the neural tube is a characteristic phenotype of zebrafish
and Xenopus convergent extension conditions
(Darken et al., 2002;
Goto and Keller, 2002;
Wallingford and Harland,
2002), as well as of mouse mutants in components of non-canonical
Wnt signaling (Lp; Fig.
2H) (reviewed by Copp et al.,
2003).
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). The
floor plate ventral hinge was morphologically normal in chato mutants
(Fig. 2F,G). In addition, other
markers of territories along the dorsal-ventral neural axis, including
Shh (Echelard et al.,
1993), Foxa2 (Ruiz i
Altaba et al., 1993) and Olig2
(Zhou et al., 2001), were
expressed in regions comparable to those of wild-type littermates (not
shown).
chato embryos also showed other phenotypic differences from
non-canonical Wnt signaling mutants. The notochord, a mesendoderm-derived
tissue, is wider in fish, frog and mouse embryos in which the activity of this
pathway is disrupted (Goto and Keller,
2002; Greene et al.,
1998; Hammerschmidt et al.,
1996). Analysis of brachyury (T) expression
(Wilkinson et al., 1990) in
whole-mount chato embryos at E8.5 revealed that the notochord was
disrupted and was wider than in wild-type littermates in some regions, but
narrower or absent in other positions (Fig.
2I,J). In transverse sections, analysis of T expression
indicated that the characteristic notochord rod present in wild-type embryos
at these stages had not been formed in chato mutants and, instead,
the notochord was still part of the mesendoderm layer
(Fig. 2F,G). Therefore,
although the notochord irregularities of chato mutants indicate
defects in the reorganization of this tissue, these defects are different than
those of non-canonical Wnt signaling mutants
(Fig. 2H).
chato does not genetically interact with non-canonical Wnt signaling mutants
To assess whether chato affected the activity of the non-canonical
Wnt pathway, we tested for genetic interactions between chato and
Lp. Mouse mutant embryos that lack Lp (Vangl2) display some of the
hallmarks of convergent extension mutants, including a wider notochord and
failure to close the neural tube (Greene
et al., 1998; Murdoch et al.,
2001a). Lp mutants show strong genetic interactions with
other mutations that affect non-canonical Wnt signaling. For example, embryos
that are doubly heterozygous for Lp and scribbled [Scrib;
also known as circletail (Crc)] (Lp/+; Crc/+)
(Murdoch et al., 2001b) or for
Lp and Ptk7 (Lp/+; Ptk7/+)
(Lu et al., 2004), as well as
Lp+/-; Dvl1+/-;
Dvl2-/- embryos (Wang,
J. et al., 2006), all show the same neural tube closure defects
seen in Lp homozygous embryos. By contrast, we found that
Lp+/-; chato+/- double heterozygous
animals were viable and fertile and had the curled tail typical of Lp
heterozygotes (see Fig. S2 in the supplementary material). We also mated
double heterozygous carriers to obtain more-severe mutant combinations and
evaluated their phenotypes in mesoderm, neural tube and notochord. We did not
observe any modification of the Lp mutant phenotype in embryos
lacking one copy of chato (Lp-/-;
chato+/-). Similarly, the chato mutant phenotype
did not change in the absence of one copy of Lp
(Lp+/-; chato-/-). Lp-chato
double mutant embryos (Lp-/-;
chato-/-) showed characteristics of both chato
and Lp mutants, including elongated somites and an open neural tube
(see Fig. S2 in the supplementary material). The lack of genetic interaction
between the two mutants does not support a role of chato in
non-canonical Wnt signaling.
To further test whether chato interferes with non-canonical Wnt
signaling, we assayed expression of components of this pathway in
chato mutants. We found that Vangl1, Vangl2, Celsr1,
frizzled 3 (Fzd3), Dvl1, Dvl2 and Prickle1 were all
expressed in chato mutants (see Fig. S3A-H in the supplementary
material; data not shown) in the same tissues as in wild-type control embryos
(see Fig. S3A-H in the supplementary material)
(Crompton et al., 2007;
Torban et al., 2006).
Reciprocally, chato expression was unaltered in Lp mutants
(see Fig. S3I,J in the supplementary material). Since none of our experiments
supports an interaction between chato and non-canonical Wnt
signaling, we speculate that the morphogenetic defects of chato and
Lp mutants might arise through different molecular mechanisms.
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290 genes
(Urrutia, 2003). Members of
this family contain a variable number of zinc-finger domains, which are
believed to provide DNA-binding specificity to different targets
(Gebelein and Urrutia, 2001),
and one or several KRAB domains, which have strong transcriptional repressor
activity (Margolin et al.,
1994).
The missense mutation in the chato allele causes a Leu to Pro
change in the first of the two KRAB domains of Zfp568
(Fig. 3A,B). This change maps
to a highly conserved position within the KRAB domain required for
transcriptional repression in COS-1 cells
(Margolin et al., 1994). To
confirm that mutation of Zfp568 is responsible of the chato
mutant phenotype and to test whether the missense mutation disrupted activity
of Zfp568 completely, we generated mutant mice from the BayGenomics gene-trap
clone RRU161. This gene-trap insertion generates a truncated Zfp568 protein of
11 amino acids that lacks all functional domains, and should represent a null
allele of Zfp568 (Fig.
3A). Both Zfp568chato/Zfp568RRU161
and Zfp568RRU161 homozygous embryos recapitulated the
chato phenotype (Fig.
3C-F). Thus, the complementation test indicated that the
ENU-induced chato mutation is a null allele of Zfp568.
Zfp568 (chato) showed a broad expression pattern during embryogenesis (Fig. 3G-L). At E7.5, chato was expressed in all cell types as assessed by in situ hybridization in whole-mount embryos and in sections (Fig. 3G,H). At later stages, expression was also ubiquitous in extraembryonic and embryonic tissues (Fig. 3J,L). Expression was highest in the extraembryonic ectoderm (Fig. 3G,J, arrowheads).
chato mutants fail to undergo convergent extension of definitive endoderm
Characterization of the cellular basis of the chato axis
elongation defects was complicated by the architecture of the E8.5 mouse
embryo, which consists of several cellular layers, some of which (e.g. the
neuroepithelium) are folded. Compared with other germ layers, we found that
the simple epithelial structure of the definitive endoderm made it amenable to
straightforward and reliable analysis during the stages of axis elongation.
Definitive endoderm cells arise from the primitive streak during gastrulation
and form an epithelial monolayer that is continuous with the extraembryonic
visceral endoderm (VE) on the exterior of the embryo after E8.0 (reviewed by
Lewis and Tam, 2006).
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). At
E7.5, some VE cells were still present in the embryonic region
(Fig. 4A,B, arrowhead). After
E8.0 (0-somite stage), the definitive (Ttr-negative) endoderm covered
the entire embryonic region (Fig.
4C-J). Posterior views of wild-type embryos marked with
Ttr revealed that the definitive endoderm narrowed between E8.0 and
E9.5 (Fig. 4). Measurements of
definitive endoderm in wild-type embryos showed that the total length of the
definitive endoderm increased 50% between 0-somite and 10-somite stage embryos
(Fig. 4K, blue columns;
Fig. 5H). At the same stages,
definitive endoderm width, measured as the lateral distance across the center
of the embryo (Fig. 4H, red
line), decreased 2.7-fold (Fig.
4K, green columns; Fig.
5H). These measurements demonstrate that elongation of the
definitive endoderm of the wild-type mouse embryo is accompanied by narrowing
of the tissue, and thus definitive endoderm undergoes convergent
extension. At early E8.5 (2- to 4-somite stage), the length of the chato definitive endoderm was not significantly different from that of wild-type littermates (P=0.31), but its width was 1.23-fold that of the wild type (P=0.019, Fig. 5F,H). At the 5- to 7-somite stage, the definitive endoderm of chato mutants was 14% shorter (P=0.031) and twice as wide (P=0.0002) as that of wild-type embryos of the same stage (Fig. 5E,F,H). The length and width measurements indicated that the chato mutant endoderm grew in both dimensions (Fig. 5E-H). However, the length-to-width ratio (LWR) of chato embryonic endoderm did not significantly change between the 2- to 4-somite and the 5- to 7-somite stages (P=0.23, Fig. 5G, red columns), whereas the wild-type LWR more than doubled (P=0.0007, Fig. 5G, gray columns). By the 5- to 7-somite stage, the LWR of wild-type definitive endoderm was 2.6-fold greater than that of chato mutants (P=0.0022, Fig. 5G). Thus, convergent extension of the mouse definitive endoderm requires the activity of the Chato protein.
Elongation and narrowing of the wild-type definitive endoderm is coupled to cell rearrangements
Convergent extension in zebrafish and Xenopus embryos depends on
cell rearrangements, including mediolateral cell intercalation and polarized
cell migration, which contribute to a decrease in the number of cells across
the width of the embryo and to an increase in the number of cells along the
anterior-posterior axis (reviewed by
Wallingford et al., 2002). We
therefore evaluated variations in the number of cells across the width of the
mouse definitive endoderm to assess the contribution of cell rearrangements to
convergent extension of the mouse endoderm.
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A decrease in the number of cells across the width of the definitive
endoderm could be the result of mediolateral cell intercalation. However, this
number could also be influenced by proliferation, apoptosis and delamination
of cells from the primitive streak. To evaluate the contribution of cell
proliferation, we assayed the frequency of mitotic cells in transverse
sections of the definitive endoderm using phospho-histone H3 antibodies
(Fig. 7A,C, green signal).
Between E8.0 (0 somites) and E9.0 (10 somites), the definitive endoderm
contained 0-3 proliferating cells per section at all levels along the
anterior-posterior axis where the gut remained open (n=21 embryos/380
sections, Fig. 7A,C and data
not shown). By contrast, other embryonic tissues, such as the mesoderm or
neuroepithelia, showed a higher mitotic index
(Fig. 7A,C). Our results
confirm previous reports indicating that the definitive endoderm is a
relatively quiescent tissue during early developmental stages
(Tremblay and Zaret, 2005) and
indicate that the rate of proliferation in the endoderm plays a minor role in
the growth of the definitive endoderm during these stages. We did not observe
any apoptotic cells in the definitive endoderm at any of the stages analyzed
(data not shown). Delamination of cells from the primitive streak plays
important roles in the growth of the definitive endoderm at gastrulation
stages (Lewis and Tam, 2006).
Therefore, during the stages of anterior-posterior axis elongation, the number
of cells in the endoderm might increase owing to the continued delamination of
cells from the primitive streak, with a minor contribution from cell
proliferation. Because we did not observe apoptosis in the endoderm, we
conclude that cellular rearrangements (mediolateral cell intercalation or
polarized cell migration) must be responsible for the observed decrease in the
number of cells across the width of the definitive endoderm and for the
decrease in the width of the tissue.
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70% more cells across the width of the definitive endoderm than
wild-type embryos (P<0.0001,
Fig. 6A,B), paralleling the
increased width of the definitive endoderm in chato mutants
(Fig. 5B,D). The rate of cell
proliferation (n=3 embryos/56 sections) in the definitive endoderm of
chato mutants was similar to that of the wild type
(Fig. 7B-D). Also, no apoptosis
was observed in the chato mutant endoderm and we did not detect any
abnormality in the delamination of definitive endoderm from the primitive
streak or in the migration of definitive endoderm cells at gastrulation (see
Fig. S1C-F in the supplementary material). Therefore, we conclude that the
definitive endoderm is wide in chato mutants because normal function
of the chato gene is required for the cells to rearrange into a
longer, narrower structure. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Yamanaka et al., 2007;
Ybot-Gonzalez et al., 2007).
Based on embryo morphology and the pattern of expression of molecular markers,
chato mutants appear to have global defects in elongation of the body
axis, with abnormalities in the neural plate, paraxial mesoderm, lateral plate
mesoderm and definitive endoderm.
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14-somite stage embryos, when
definitive endoderm closes to form the gut tube. From the 0- to 10-somite
stages, the width of the wild-type definitive endoderm narrows 2.6-fold (from
820 to 310 mm); at the same time it elongates 2-fold. Although delamination of
cells from the primitive streak probably contributes to the elongation of the
definitive endoderm, the cell rearrangements that we observed are likely to
account for the narrowing of the definitive endoderm and to contribute to the
anterior-posterior elongation of this tissue. By contrast, the definitive
endoderm does not narrow in chato embryos. The most dramatic change
in dimensions of the definitive endoderm of wild-type embryos occurs between
the 2- to 4-somite and 5- to 7-somite stages, when the length-to-width ratio
more than doubles; at the same stages, the length-to-width ratio of the
chato definitive endoderm does not change significantly. In parallel
with the abnormal dimensions of the tissue, the chato mutation
disrupts cell rearrangements in the definitive endoderm. We therefore conclude
that the cell rearrangements that depend on Chato are responsible for
convergent extension of the definitive endoderm.
The mechanisms underlying the cell rearrangements of convergent extension
have been studied in vertebrate and invertebrate embryos. Mediolateral cell
intercalation mediates the elongation of Xenopus embryos and animal
cap explants (Elul et al.,
1997; Keller and Tibbetts,
1989; Wilson and Keller,
1991), polarized cell migration is also important for zebrafish
convergent extension (Concha and Adams,
1998; Jessen et al.,
2002; Warga and Kimmel,
1990), and germ band elongation of Drosophila embryos is
propelled by the directional generation and resolution of multicellular
rosettes (Bertet et al., 2004;
Blankenship et al., 2006). One
or more of these mechanisms may mediate convergent extension of the mouse
definitive endoderm. Because mouse definitive endoderm has an epithelial
organization, where cells are held together by adherent apical complexes
(Fig. 7, E-cadherin in red), we
favor the hypothesis that mediolateral cell intercalation and/or multicellular
rosettes, rather than cell migration, mediate definitive endoderm convergent
extension. The development of new methods that enable observation of live
mouse embryos at cellular resolution will be required to elucidate the precise
mechanisms involved.
Chato is likely to act in convergent extension of all germ layers
Although our studies of convergent extension in chato focused on
the definitive endoderm, the chato phenotype suggests that it also
acts in other tissues to regulate convergent extension. Both chato
lateral plate and somitic mesoderm are shorter in the anterior-posterior axis
and wider in the mediolateral dimension than in wild-type embryos, similar to
zebrafish convergent extension mutants
(Hammerschmidt et al., 1996;
Jessen et al., 2002). The
neural plate in chato fails to close, which could be due to defects
in cell rearrangement in this tissue layer. Because chato is broadly
expressed, it seems likely that it acts autonomously in these tissues to
control cell rearrangements. It is, however, possible that convergent
extension of the definitive endoderm is required for the migration and/or
reorganization of epithelial and mesenchymal tissues.
Most chato mutants (n=156/184) also show extraembryonic defects, including a ruffled VE (see Fig. S1A,B in the supplementary material). It is therefore possible that these extraembryonic defects could influence the reorganization of definitive endoderm, epithelial and mesenchymal tissues in chato mutants. However, the defects in embryonic morphogenesis precede the appearance of extraembryonic phenotypes in chato mutants (see Fig. S1C-F in the supplementary material). In addition, 16% of E8.5 chato mutants do not show obvious extraembryonic abnormalities but have strong convergent extension phenotypes. Therefore, we favor the hypothesis that the embryonic and extraembryonic defects in chato embryos represent distinct, autonomous requirements for chato. Further experiments assessing the phenotype of chato chimeric embryos or using conditional alleles will define the tissue requirements of this novel KRAB zinc-finger protein.
The role of the Chato KRAB zinc-finger protein in morphogenesis
The chato mutation defines the role of a novel KRAB zinc-finger
protein in mammalian convergent extension. Although KRAB domain zinc-finger
proteins represent one of the largest gene families in mammals
(Urrutia, 2003), only a few
mutants have been described. These mutants affect diverse processes, including
fertility, pigmentation and embryonic growth
(Casademunt et al., 1999;
Krebs et al., 2003), but Chato
is the first member of this family shown to be required for embryonic
morphogenesis. Although the high degree of sequence conservation among members
of the family suggests that the genes might be functionally redundant, the
severity and specificity of the chato phenotype indicates that some
KRAB domain proteins have distinct functions.
The KRAB domain seems to be a relatively recent evolutionary feature, as it
has only been found in the genomes of tetrapod vertebrates
(Urrutia, 2003)
(www.ensembl.org).
Nevertheless, the C-terminal zinc-finger-containing region of chato
shows homology to genes found in other animals. The closest homolog of
chato in Drosophila is crooked legs
(crol), with 39% identity and 53% similarity to the Chato zinc-finger
domain. crol mutant pupae die with twisted legs that fail to elongate
(D'Avino and Thummel, 1998).
Although the zebrafish genome does not encode any KRAB domain proteins,
morpholinos that disrupt the activity of the zinc-finger gene bloody
fingers (blf) display shortened and widened axial tissue due to
defective convergent extension (Sumanas et
al., 2005). Blf and Chato share similar zinc-finger domains, but,
based on synteny, it is unlikely that Blf is the zebrafish ortholog of Chato.
Therefore, it is possible that Chato, Crol and Blf derived from a common
ancestral zinc-finger protein that controlled tissue elongation during
morphogenesis.
Our results suggest that the Chato KRAB zinc-finger protein acts through a
molecular pathway that is independent of non-canonical Wnt signaling. Although
mutations in both the mouse chato and non-canonical Wnt signaling
genes affect convergent extension, their phenotypes are fundamentally
different. The defects in axis elongation in the chato mesoderm are
more profound than those reported in mouse non-canonical Wnt signaling mutants
(Figs 1,
2)
(Greene et al., 1998;
Wang, J. et al., 2006). Most
clearly, our analysis shows that chato mutants fail to close the gut
endoderm and fail to undergo convergent extension in the gut, phenotypes that
are not present in Lp mutants (data not shown). By contrast,
Lp mutants have more-dramatic defects in neural tube closure and in
convergent extension of the notochord than chato mutants
(Greene et al., 1998;
Wang, J. et al., 2006;
Ybot-Gonzalez et al., 2007). A
specific role for non-canonical Wnt signaling in morphogenesis of axial
tissues is supported by the high level of expression of Vangl2 and
Vangl1 in the mouse neural tube
(Torban et al., 2006;
Torban et al., 2008).
Altogether, the observations suggest that Chato and non-canonical Wnt
signaling act in different tissues and regulate convergent extension through
different molecular mechanisms.
Because chato mutants are blocked in both definitive endoderm
convergent extension and the accompanying cell rearrangements, we conclude
that these cell rearrangements drive convergent extension of the mammalian
endoderm. KRAB zinc-finger proteins are believed to act as transcriptional
repressors (Bellefroid et al.,
1991), so Chato might regulate the transcription of genes that
regulate specific aspects of cytoskeleton dynamics, components of the
extracellular matrix (ECM) or chemotactic clues. Because mutations in mouse
genes that have global effects on cytoskeleton organization or the ECM
(Garcia-Garcia and Anderson,
2003; George et al.,
1993; Rakeman and Anderson,
2006) cause phenotypes dramatically different from those of
chato mutants, we infer that chato controls cellular
processes that are specific to convergent extension.
Chato might act in a common molecular pathway with Hand1 and Yap65
Although the molecular mechanisms that implement Chato function remain to
be discovered, additional information might come from the analysis of two
other mouse mutants with phenotypes similar to chato. Mutants that
lack Hand1, which encodes a bHLH transcription factor, arrest
development at the 9- to 14-somite stage, fail to close the gut endoderm, have
a kinked neural plate and show extraembryonic defects similar to those of
chato embryos (Firulli et al.,
1998). Loss of mouse Yap65 (Yap1), which encodes
a protein with a proline-rich domain, WW domains, SH3 binding motifs, a
coiled-coil and a PDZ binding motif, also causes phenotypes similar to
chato (Morin-Kensicki et al.,
2006). Studies similar to those described here could test whether
these mutants have convergent extension defects in epithelia, mesenchyme and
endoderm and whether cell rearrangements underlie the Hand1 and
Yap1 abnormalities. Future experiments will be able to test whether
chato, Hand1 and Yap1 act in a common biochemical process
that regulates convergent extension in the mouse.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/18/3053/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bardet, P. L., Horard, B., Laudet, V. and Vanacker, J. M. (2005). The ERRalpha orphan nuclear receptor controls morphogenetic movements during zebrafish gastrulation. Dev. Biol. 281,102 -111.[CrossRef][Medline]
Bellefroid, E. J., Poncelet, D. A., Lecocq, P. J., Revelant, O.
and Martial, J. A. (1991). The evolutionarily conserved
Kruppel-associated box domain defines a subfamily of eukaryotic multifingered
proteins. Proc. Natl. Acad. Sci. USA
88,3608
-3612.
Belo, J. A., Bouwmeester, T., Leyns, L., Kertesz, N., Gallo, M., Follettie, M. and De Robertis, E. M. (1997). Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech. Dev. 68, 45-57.[CrossRef][Medline]
Bertet, C., Sulak, L. and Lecuit, T. (2004). Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation. Nature 429,667 -671.[CrossRef][Medline]
Blankenship, J. T., Backovic, S. T., Sanny, J. S., Weitz, O. and Zallen, J. A. (2006). Multicellular rosette formation links planar cell polarity to tissue morphogenesis. Dev. Cell 11,459 -470.[CrossRef][Medline]
Candia, A. F., Hu, J., Crosby, J., Lalley, P. A., Noden, D., Nadeau, J. H. and Wright, C. V. (1992). Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos. Development 116,1123 -1136.[Abstract]
Casademunt, E., Carter, B. D., Benzel, I., Frade, J. M., Dechant, G. and Barde, Y. A. (1999). The zinc finger protein NRIF interacts with the neurotrophin receptor p75(NTR) and participates in programmed cell death. EMBO J. 18,6050 -6061.[CrossRef][Medline]
Cereghini, S., Ott, M. O., Power, S. and Maury, M. (1992). Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles. Development 116,783 -797.[Abstract]
Collignon, J., Sockanathan, S., Hacker, A., Cohen-Tannoudji, M., Norris, D., Rastan, S., Stevanovic, M., Goodfellow, P. N. and Lovell-Badge, R. (1996a). A comparison of the properties of Sox-3 with Sry and two related genes, Sox-1 and Sox-2. Development 122,509 -520.[Abstract]
Collignon, J., Varlet, I. and Robertson, E. J. (1996b). Relationship between asymmetric nodal expression and the direction of embryonic turning. Nature 381,155 -158.[CrossRef][Medline]
Concha, M. L. and Adams, R. J. (1998). Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis. Development 125,983 -994.[Abstract]
Copp, A. J., Greene, N. D. and Murdoch, J. N. (2003). The genetic basis of mammalian neurulation. Nat. Rev. Genet. 4,784 -793.[CrossRef][Medline]
Crompton, L. A., Du Roure, C. and Rodriguez, T. A. (2007). Early embryonic expression patterns of the mouse Flamingo and Prickle orthologues. Dev. Dyn. 236,3137 -3143.[CrossRef][Medline]
Curtin, J. A., Quint, E., Tsipouri, V., Arkell, R. M., Cattanach, B., Copp, A. J., Henderson, D. J., Spurr, N., Stanier, P., Fisher, E. M. et al. (2003). Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse. Curr. Biol. 13,1129 -1133.[CrossRef][Medline]
D'Avino, P. P. and Thummel, C. S. (1998). crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis. Development 125,1733 -1745.[Abstract]
Darken, R. S., Scola, A. M., Rakeman, A. S., Das, G., Mlodzik, M. and Wilson, P. A. (2002). The planar polarity gene strabismus regulates convergent extension movements in Xenopus. EMBO J. 21,976 -985.[CrossRef][Medline]
Davidson, L. A. and Keller, R. E. (1999). Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergent extension. Development 126,4547 -4556.[Abstract]
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75,1417 -1430.[CrossRef][Medline]
Elul, T. and Keller, R. (2000). Monopolar protrusive activity: a new morphogenic cell behavior in the neural plate dependent on vertical interactions with the mesoderm in Xenopus. Dev. Biol. 224,3 -19.[CrossRef][Medline]
Elul, T., Koehl, M. A. and Keller, R. (1997). Cellular mechanism underlying neural convergent extension in Xenopus laevis embryos. Dev. Biol. 191,243 -258.[CrossRef][Medline]
Firulli, A. B., McFadden, D. G., Lin, Q., Srivastava, D. and Olson, E. N. (1998). Heart and extra-embryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1. Nat. Genet. 18,266 -270.[CrossRef][Medline]
Garcia-Garcia, M. J. and Anderson, K. V. (2003). Essential role of glycosaminoglycans in Fgf signaling during mouse gastrulation. Cell 114,727 -737.[CrossRef][Medline]
Garcia-Garcia, M. J., Eggenschwiler, J. T., Caspary, T., Alcorn,
H. L., Wyler, M. R., Huangfu, D., Rakeman, A. S., Lee, J. D., Feinberg, E. H.,
Timmer, J. R. et al. (2005). Analysis of mouse embryonic
patterning and morphogenesis by forward genetics. Proc. Natl. Acad.
Sci. USA 102,5913
-5919.
Gebelein, B. and Urrutia, R. (2001).
Sequence-specific transcriptional repression by KS1, a
multiple-zinc-finger-Kruppel-associated box protein. Mol. Cell.
Biol. 21,928
-939.
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]
Goto, T. and Keller, R. (2002). The planar cell polarity gene strabismus regulates convergence and extension and neural fold closure in Xenopus. Dev. Biol. 247,165 -181.[CrossRef][Medline]
Greene, N. D., Gerrelli, D., Van Straaten, H. W. and Copp, A. J. (1998). Abnormalities of floor plate, notochord and somite differentiation in the loop-tail (Lp) mouse: a model of severe neural tube defects. Mech. Dev. 73,59 -72.[CrossRef][Medline]
Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A., Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P., Heisenberg, C. P. et al. (1996). Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio. Development 123,143 -151.[Abstract]
Jessen, J. R., Topczewski, J., Bingham, S., Sepich, D. S., Marlow, F., Chandrasekhar, A. and Solnica-Krezel, L. (2002). Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements. Nat. Cell Biol. 4, 610-615.[Medline]
Kasarskis, A., Manova, K. and Anderson, K. V.
(1998). A phenotype-based screen for embryonic lethal mutations
in the mouse. Proc. Natl. Acad. Sci. USA
95,7485
-7490.
Keller, R. and Tibbetts, P. (1989). Mediolateral cell intercalation in the dorsal, axial mesoderm of Xenopus laevis. Dev. Biol. 131,539 -549.[Medline]
Kibar, Z., Vogan, K. J., Groulx, N., Justice, M. J., Underhill, D. A. and Gros, P. (2001). Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tube mutant Loop-tail. Nat. Genet. 28,251 -255.[CrossRef][Medline]
Kinder, S. J., Tsang, T. E., Quinlan, G. A., Hadjantonakis, A. K., Nagy, A. and Tam, P. P. (1999). The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo. Development 126,4691 -4701.[Abstract]
Krebs, C. J., Larkins, L. K., Price, R., Tullis, K. M., Miller,
R. D. and Robins, D. M. (2003). Regulator of sex-limitation
(Rsl) encodes a pair of KRAB zinc-finger genes that control sexually dimorphic
liver gene expression. Genes Dev.
17,2664
-2674.
Lawson, K. A. (1999). Fate mapping the mouse embryo. Int. J. Dev. Biol. 43,773 -775.[Medline]
Lewis, S. L. and Tam, P. P. (2006). Definitive endoderm of the mouse embryo: formation, cell fates, and morphogenetic function. Dev. Dyn. 235,2315 -2329.[CrossRef][Medline]
Lints, T. J., Parsons, L. M., Hartley, L., Lyons, I. and Harvey, R. P. (1993). Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119,419 -431.[Abstract]
Lu, X., Borchers, A. G., Jolicoeur, C., Rayburn, H., Baker, J. C. and Tessier-Lavigne, M. (2004). PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates. Nature 430,93 -98.[CrossRef][Medline]
Margolin, J. F., Friedman, J. R., Meyer, W. K., Vissing, H.,
Thiesen, H. J. and Rauscher, F. J. (1994). Kruppel-associated
boxes are potent transcriptional repression domains. Proc. Natl.
Acad. Sci. USA 91,4509
-4513.
Matsui, T., Raya, A., Kawakami, Y., Callol-Massot, C.,
Capdevila, J., Rodriguez-Esteban, C. and Izpisua Belmonte, J. C.
(2005). Noncanonical Wnt signaling regulates midline convergence
of organ primordia during zebrafish development. Genes
Dev. 19,164
-175.
Montcouquiol, M., Rachel, R. A., Lanford, P. J., Copeland, N. G., Jenkins, N. A. and Kelley, M. W. (2003). Identification of Vangl2 and Scrb1 as planar polarity genes in mammals. Nature 423,173 -177.[CrossRef][Medline]
Moon, R. T., Campbell, R. M., Christian, J. L., McGrew, L. L., Shih, J. and Fraser, S. (1993). Xwnt-5A: a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis. Development 119,97 -111.[Abstract]
Morin-Kensicki, E. M., Boone, B. N., Howell, M., Stonebraker, J.
R., Teed, J., Alb, J. G., Magnuson, T. R., O'Neal, W. and Milgram, S. L.
(2006). Defects in yolk sac vasculogenesis, chorioallantoic
fusion, and embryonic axis elongation in mice with targeted disruption of
Yap65. Mol. Cell. Biol.
26, 77-87.
Murdoch, J. N., Doudney, K., Paternotte, C., Copp, A. J. and
Stanier, P. (2001a). Severe neural tube defects in the
loop-tail mouse result from mutation of Lpp1, a novel gene involved in floor
plate specification. Hum. Mol. Genet.
10,2593
-2601.
Murdoch, J. N., Rachel, R. A., Shah, S., Beermann, F., Stanier, P., Mason, C. A. and Copp, A. J. (2001b). Circletail, a new mouse mutant with severe neural tube defects: chromosomal localization and interaction with the loop-tail mutation. Genomics 78, 55-63.[CrossRef][Medline]
Nagy, A. (2003). Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Quertermous, E. E., Hidai, H., Blanar, M. A. and Quertermous,
T. (1994). Cloning and characterization of a basic
helix-loop-helix protein expressed in early mesoderm and the developing
somites. Proc. Natl. Acad. Sci. USA
91,7066
-7070.
Rakeman, A. S. and Anderson, K. V. (2006). Axis
specification and morphogenesis in the mouse embryo require Nap1, a regulator
of WAVE-mediated actin branching. Development
133,3075
-3083.
Ruiz i Altaba, A., Prezioso, V. R., Darnell, J. E. and Jessell, T. M. (1993). Sequential expression of HNF-3 beta and HNF-3 alpha by embryonic organizing centers: the dorsal lip/node, notochord and floor plate. Mech. Dev. 44, 91-108.[CrossRef][Medline]
Sumanas, S., Zhang, B., Dai, R. and Lin, S. (2005). 15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation. Dev. Biol. 283,85 -96.[CrossRef][Medline]
Tada, M. and Smith, J. C. (2000). Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development 127,2227 -2238.[Abstract]
Tada, M., Concha, M. L. and Heisenberg, C. P. (2002). Non-canonical Wnt signalling and regulation of gastrulation movements. Semin. Cell Dev. Biol. 13,251 -260.[CrossRef][Medline]
Tahinci, E. and Symes, K. (2003). Distinct functions of Rho and Rac are required for convergent extension during Xenopus gastrulation. Dev. Biol. 259,318 -335.[CrossRef][Medline]
Torban, E., Wang, H. J., Patenaude, A. M., Riccomagno, M., Daniels, E., Epstein, D. and Gros, P. (2006). Tissue, cellular and sub-cellular localization of the Vangl2 protein during embryonic development: effect of the Lp mutation. Gene Expr. Patterns 7,346 -354.
Torban, E., Patenaude, A. M., Leclerc, S., Rakowiecki, S.,
Gauthier, S., Andelfinger, G., Epstein, D. J. and Gros, P.
(2008). Genetic interaction between members of the Vangl family
causes neural tube defects in mice. Proc. Natl. Acad. Sci.
USA 105,3449
-3454.
Tremblay, K. D. and Zaret, K. S. (2005). Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues. Dev. Biol. 280, 87-99.[CrossRef][Medline]
Urrutia, R. (2003). KRAB-containing zinc-finger repressor proteins. Genome Biol. 4, 231.[CrossRef][Medline]
von der Hardt, S., Bakkers, J., Inbal, A., Carvalho, L., Solnica-Krezel, L., Heisenberg, C. P. and Hammerschmidt, M. (2007). The Bmp gradient of the zebrafish gastrula guides migrating lateral cells by regulating cell-cell adhesion. Curr. Biol. 17,475 -487.[CrossRef][Medline]
Wallingford, J. B. and Harland, R. M. (2002). Neural tube closure requires Dishevelled-dependent convergent extension of the midline. Development 129,5815 -5825.[CrossRef][Medline]
Wallingford, J. B., Rowning, B. A., Vogeli, K. M., Rothbächer, U., Fraser, S. E. and Harland, R. M. (2000). Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405,81 -85.[CrossRef][Medline]
Wallingford, J. B., Fraser, S. E. and Harland, R. M. (2002). Convergent extension: the molecular control of polarized cell movement during embryonic development. Dev. Cell 2, 695-706.[CrossRef][Medline]
Wang, J., Hamblet, N. S., Mark, S., Dickinson, M. E., Brinkman,
B. C., Segil, N., Fraser, S. E., Chen, P., Wallingford, J. B. and
Wynshaw-Boris, A. (2006). Dishevelled genes mediate a
conserved mammalian PCP pathway to regulate convergent extension during
neurulation. Development
133,1767
-1778.
Wang, Y., Guo, N. and Nathans, J. (2006). The
role of Frizzled3 and Frizzled6 in neural tube closure and in the planar
polarity of inner-ear sensory hair cells. J. Neurosci.
26,2147
-2156.
Warga, R. M. and Kimmel, C. B. (1990). Cell
movements during epiboly and gastrulation in zebrafish.
Development 108,569
-580.
Wilkinson, D. G., Bhatt, S., Chavrier, P., Bravo, R. and Charnay, P. (1989). Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse. Nature 337,461 -464.[CrossRef][Medline]
Wilkinson, D. G., Bhatt, S. and Herrmann, B. G. (1990). Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343,657 -659.[CrossRef][Medline]
Wilson, P. and Keller, R. (1991). Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants. Development 112,289 -300.[Abstract]
Yamanaka, Y., Tamplin, O. J., Beckers, A., Gossler, A. and Rossant, J. (2007). Live imaging and genetic analysis of mouse notochord formation reveals regional morphogenetic mechanisms. Dev. Cell 13,884 -896.[CrossRef][Medline]
Ybot-Gonzalez, P., Savery, D., Gerrelli, D., Signore, M.,
Mitchell, C. E., Faux, C. H., Greene, N. D. and Copp, A. J.
(2007). Convergent extension, planar-cell-polarity signalling and
initiation of mouse neural tube closure. Development
134,789
-799.
Zhou, Q., Choi, G. and Anderson, D. J. (2001). The bHLH transcription factor Olig2 promotes oligodendrocyte differentiation in collaboration with Nkx2.2. Neuron 31,791 -807.[CrossRef][Medline]
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