|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 17 January 2007
doi: 10.1242/dev.000380
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.
Author for correspondence (e-mail:
a.copp{at}ich.ucl.ac.uk)
Accepted 5 December 2006
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Mouse, Neurulation, Morphogenesis, Neural tube defects, Convergent extension, Planar cell polarity, Chimeras, Embryo culture
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). Cell intercalation in the midline leads to rostrocaudal
extension of the body axis. An essential role in convergent extension has been
assigned to the planar-cell-polarity (PCP) pathway, a highly conserved,
non-canonical Wnt-frizzled-dishevelled signalling cascade. PCP signalling
plays a key role in establishing and maintaining polarity in the plane of
epithelia in Drosophila, and in epithelial and non-epithelial tissues
in vertebrates (Strutt, 2003;
Klein and Mlodzik, 2005).
Both fish and amphibian embryos require an intact PCP pathway for
postgastrulation shaping (Wallingford and
Harland, 2001; Park and Moon,
2002; Darken et al.,
2002; Jessen et al.,
2002). Moreover, in Xenopus, PCP-dependent convergent
extension in the midline is required for neural tube formation following
gastrulation (Wallingford and Harland,
2002; Takeuchi et al.,
2003). In its absence, the neural folds are spaced widely apart,
precluding the apposition and fusion that is required for neural tube closure
(Wallingford and Harland,
2002). A similar failure of initiation of neurulation occurs in
several mouse mutants: loop-tail (Lp), circletail
(Crc), crash (Crsh), protein tyrosine kinase
7 (Ptk7), dishevelled 1 (Dvl1) and
Dvl2 double mutants, and frizzled 3 (Frz3) and
Frz6 double mutants (Greene et
al., 1998; Murdoch et al.,
2001b; Hamblet et al.,
2002; Lu et al.,
2004; Wang et al.,
2006b). This yields the severe defect craniorachischisis (CRN), in
which the neural tube remains open from midbrain to low spine
(Botto et al., 1999;
Copp et al., 1994). CRN
comprises up to 10% of human neural tube defects
(Moore et al., 1997), and
seems likely to result from failure of the initial event of neural tube
closure, as in the mouse (Kirillova et
al., 2000).
All six of the gene loci associated with CRN in the mouse adversely affect
PCP function. The Lp mouse is mutant for Vangl2
(Kibar et al., 2001;
Murdoch et al., 2001a), the
homologue of Drosophila strabismus and van gogh, which
encodes a protein that binds directly to dishevelled, participating in its
recruitment to the cell membrane (Bastock
et al., 2003; Torban et al.,
2004). Crash mice are mutant for Celsr1
(Curtin et al., 2003), the
homologue of Drosophila starry night (also known as
flamingo), which plays a role in assembling the PCP protein complex,
at least in Drosophila (Strutt,
2003). Loss of function of two of the three dishevelled genes
(Dsh-1 and -2), and two of the six frizzled genes
(Frz-3 and -6), also leads to CRN
(Hamblet et al., 2002;
Wang et al., 2006b). Moreover,
mutation of Scrib (also known as Scrb1) in
circletail mice (Murdoch et al.,
2003) and a gene-trap mutation of the tyrosine kinase
Ptk7 (Lu et al.,
2004) generate a CRN phenotype. Although not directly implicated
in the PCP molecular pathway, both Scrb1 and Ptk7 interact
genetically with the other mouse CRN genes
(Murdoch et al., 2001b;
Lu et al., 2004) and produce a
planar-polarity phenotype in the inner ear
(Montcouquiol et al., 2003;
Lu et al., 2004). It is
striking, therefore, that of the more than 100 genes necessary for neural tube
closure in the mouse, all of those with a CRN phenotype disrupt PCP signalling
(Copp et al., 2003).
A defect of convergent extension seems likely to precede the failure of
neural tube closure in mouse PCP mutants, in view of the widely spaced neural
folds described in Lp, Crc, Dvl1;Dvl2 and Ptk7 homozygotes
(Greene et al., 1998;
Murdoch et al., 2003;
Lu et al., 2004;
Wang et al., 2006a), and the
finding of an abnormally short and wide body axis in Lp/Lp and
Dvl1;Dvl2 embryos during late gastrulation
(Wang et al., 2006a). To date,
however, a direct analysis of convergent extension in mouse PCP mutants has
not been reported. In the present study, we vitally labelled pre-neurulation
stage embryos in order to study convergent extension and found that midline
extension is defective in both the axial mesoderm and the neural plate of
Lp/Lp mutants. Analysis of +/+ 
Lp/Lp chimeras
suggested a cell-autonomous defect in the convergence of Lp/Lp cells
towards the neural plate midline along the entire body axis, as well as in
cells recently emerged from the node, the source of axial mesoderm and midline
neural plate in the mouse (Sausedo and
Schoenwolf, 1994; Sulik et
al., 1994). Among possible signalling pathways downstream of the
PCP genes, we found function of RhoA-Rho kinase (ROCK), but not jun N-terminal
kinase (JNK), is necessary for convergent extension and initiation of mouse
neurulation.
|
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). The Crash (Crsh) mutation was originally
detected in the F1 generation of ENU-treated strain 101/H males mated with C3H
females. Crsh genotyping was by PCR amplification of flanking
microsatellites D15Mit159 and D15Mit29
(Curtin et al., 2003). The
Circletail (Crc) mutant arose as described
(Rachel et al., 2000), with
genotyping by PCR amplification of flanking microsatellites D15Mit144 and
D15Mit68 (Murdoch et al.,
2001b). Mice homozygous for the gene trap insertions cordon
bleu (Cobl) and ROSA26 were as described
(Gasca et al., 1995;
Zambrowicz et al., 1997).
Embryo dissection and processing
Embryos were obtained from timed matings; noon on the day of finding a
copulation plug was designated 0.5 days of embryonic development (E0.5).
Pregnant females were killed by cervical dislocation and embryos were
dissected from the uterus in Dulbecco's Modified Eagle's Medium (Gibco, UK)
containing 10% fetal calf serum. Embryos for in situ hybridisation or
histological analysis were washed in phosphate buffered saline (PBS) and fixed
in 4% paraformaldehyde (PFA) in PBS at 4°C overnight, or in Bouin's fluid
at room temperature for several days. Paraffin wax embedding was followed by
sectioning at 8 µm and staining with Haematoxylin and Eosin (H&E).
Embryos homozygous for the cordon bleu gene trap insertion were
stained for bacterial ß-galactosidase (ß-gal) activity as described
(Carroll et al., 2003).
DiI injection into the node of mouse embryos in vitro
DiI
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyaninine
perchlorate; Molecular Probes, USA) was dissolved in 100% ethanol (0.5 mg/ml),
then diluted tenfold in 3 mol/l sucrose. Node and midline injections were
performed at E7.5 (allantoic bud stage) using a hand-held glass micropipette,
while viewing the embryo from its posterior (node injection) or anterior
(injections rostral to the node) surface, on the stage of a Zeiss SV6
stereomicroscope. The pipette tip was inserted into the node or midline region
(Ybot-Gonzalez et al., 2002)
and the DiI solution was released as the pipette was slowly withdrawn. Embryos
were placed immediately into whole embryo culture.
Electroporation of intact mouse embryos
GFP-expressing constructs were pCAß-mGFP6 and
pCAß-IRES-GFP (gifts of Dr Jonathan Gilthorpe), in which GFP
expression is driven by the chick ß-actin promoter under influence of the
cytomegalovirus enhancer (Yaneza et al.,
2002). Both constructs gave identical GFP-labelling results in
embryo electroporation experiments. To prepare a vector expressing
constitutively active RhoA, cDNA was amplified from vector
pGEX-2T-RhoA-Q63L (Self and Hall,
1995) using primers:
5'-GCATATCGATATGGCTGCCATCAGGAAG-3' and
5'-CAATGCGGCCGCTCACAAGATGAGGCAC-3'. The amplified product was
cloned into pGEM-T (Promega, UK) and thence into pCAß-mGFP6 to
generate vector pCAß-RhoA-IRES-GFP, which was verified
by DNA sequencing. Constitutively active ROCK, pCAG-myc-P160 ROCK 
3,
wild-type ROCK, pCAG-myc-P160 ROCK, and an empty control vector, pCAG-myc,
were as described previously (Gutjahr et
al., 2005) (gifts of Dr Shuh Narumiya), and were co-electroporated
with pCAß-IRES-GFP to enable identification of transfected
cells. DNA solution for electroporation contained 4.5 mg/ml
pCAß-mGFP6 or 2.5 mg/ml other vectors, plus 0.1% Fast Green.
E7.5 embryos were placed in PBS in a Petri dish on a stereomicroscope stage
and DNA solution was injected into the amniotic cavity using a hand-held glass
micropipette. Embryos were immediately placed between a pair of gold 5 mm
point electrodes (BTX model 508) attached to a BTX ECM830 electroporator, with
the ventral midline of the caudal embryonic region next to the anode. Current
(five pulses, 50 milliseconds, 15 V) was passed, after which embryos were
placed into culture.
|
).
Transverse sections were prepared following paraffin wax embedding, and cells
of each genotype (ß-gal-positive or ß-gal-negative) were counted in
defined areas of midline or lateral neural plate, with focussing at high
magnification to distinguish the individual cells. Embryos were genotyped
using DNA extracted from yolk sac endoderm, which is entirely host
blastocyst-derived (Beddington and
Robertson, 1989). Yolk sacs were incubated on ice for 1-5 hours in
2.5% pancreatin, 0.5% trypsin in Ca2+ or Mg2+-free PBS,
after which the endodermal and mesodermal layers were peeled apart.
Inhibitor treatment and analysis of embryos after culture
Embryos were cultured (Copp et al.,
1999) with addition of varying concentrations of Y27632 (688000;
Calbiochem, UK) dissolved in sterile distilled water (0.5 to 2 µl of 5
mmol/l stock added per ml of rat serum) or SP600125 (420119; Calbiochem, UK)
dissolved in dimethyl sulfoxide (0.5 to 2.5 µl of 20 mmol/l stock added per
ml of rat serum). At the end of culture, extraembryonic membranes were removed
and somites were counted (Copp et al.,
1999). Embryos were inspected to determine whether neural tube
closure had occurred at the site of Closure 1
(Copp et al., 1994).
DiI-injected or electroporated embryos were flat-mounted, dorsal side upwards
in PBS on a glass microscope slide and photographed with a Leica DC 500
digital camera on a Leica MZ FLIII stereomicroscope.
|
;
Ybot-Gonzalez et al., 2005).
Previously published probes were: JNK1
(Kuan et al., 1999),
Shh (Greene et al.,
1998) and Vangl2
(Doudney et al., 2005). New
cDNA probes were prepared by RT-PCR on total RNA from E10.5 CBA/Ca embryos
using TRIzol reagent (Gibco BRL). Amplified fragments were cloned into the
pGEM-T vector (Promega, UK) and sequenced to confirm identity. Primer
sequences were: Daam1: forward 5'-TGCACTTCAGACAATGGAGC-3'
and reverse 5'-ACTCAGAGGTCTCATAGTCC-3', amplifying a 416 bp
fragment (nucleotides 330 to 745; GenBank NM_172464); RhoA: forward
5'-CTTTATAAGTGATGGCTGCC-3' and reverse
5'-TGGTCTTTGCTGAACACTCC-3', amplifying a 501 bp fragment
(nucleotides 11 to 511; GenBank AF178960); ROCK2: forward
5'-AGAACACCTTAGCAGTGAGG-3' and reverse
5'-TTTGGAACTTTCTGCCTGGG-3', amplifying a 412 bp fragment
(nucleotides 1359 to 1770; GenBank U58513).
RT-PCR analysis
RNA was extracted from E7.5, 8.5 and 9.5 CD1 embryos using the PURESCRIPT
RNA isolation kit (Gentra Systems, UK), DNA was removed with a DNA-free kit
(Ambion, UK), and cDNA was synthesised using a Superscript First Strand
Synthesis System (Invitrogen, UK). PCR products were analysed on ethidium
bromide-stained 1% agarose gels using Hyperladder I (Bioline, UK) as size
markers. Primers were: LIMK1: forward
5'-TACCTTTGGAGACGATACCC-3' and reverse
5'-CCTGATGTACCTGGATTTCC-3', amplifying a 447 bp fragment
(nucleotides 2295 to 2741; GenBank NM_010717); LIMK2: forward
5'-CTGTGGATTGAGATTCTGGG-3' and reverse
5'-TTAGACCCAAAGTCTGAGCC-3', amplifying a 510 bp fragment
(nucleotides 2330 to 2839; GenBank AB008117); ROCK1: forward
5'-CAAGCTTGAAGAGCAACTGC-3' and reverse
5'-CTTGTCTGCTTGTGACTTGG-3', amplifying a 505 bp fragment
(nucleotides 1299 to 1803; GenBank U58512).
Cell culture and immunoblotting
293T cells were cultured by standard methods, plasmids were introduced by
calcium phosphate transfection, and cells were lysed in 50 mmol/l Tris pH 7.5,
150 mmol/l NaCl, 1% Triton X100 containing protease inhibitors (Complete;
Roche). Protein assays were performed on cell extracts and samples containing
10 µg total protein were electrophoresed. Immunoblots were performed as
described previously (Greene et al.,
2002), using an antibody for phosphocofilin (1 in 1000 dilution;
Cell Signalling Technology). Blots were stripped and re-probed with an
antibody to ß-tubulin (1 in 3000 dilution; Santa Cruz Biotechnology
Inc.).
Statistical analysis
Length and width data were subjected to two-way analysis of variance, with
Lp genotype and somite number, or presence or absence of ROCK
inhibitor and somite number as variables. Response to ROCK and JNK inhibitors
(proportion of embryos with open neural tube) was compared between genotypes
by z-test. Response to GFP and RhoA + GFP electroporation (proportion
of embryos with midline extension) was analysed by chi-square test. All
statistics were computed using SigmaStat v.2 (SPSS Inc.).
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
|
|
Length and width measurements of DiI-labelled embryos following the culture period also support a defect in convergent extension (Fig. 2A-C). Embryonic length was significantly greater in somite-matched +/+ embryos compared with Lp/Lp littermates, whereas embryonic width showed the reverse trend: Lp/Lp embryos were significantly wider than +/+ littermates. Lp/+ embryos gave an intermediate result in both cases.
Cell-autonomous defect of convergent extension
In Drosophila, epithelial PCP effects can be cell autonomous, as
in the distal positioning of wing hairs, or non-cell autonomous, as in their
long-range polarisation (Strutt,
2003). To investigate the cell autonomy of defective convergent
extension in the Lp mutant, we constructed chimeras by injecting
wild-type ES cells into blastocysts from Lp/+ x Lp/+
matings. Host blastocysts were also homozygous for the ROSA26 ß-gal
marker, enabling cells of the two genotypes within the chimera to be
distinguished in tissue sections. Cells originating from the host blastocyst
(ß-gal-positive) and from the donor ES cells (ß-gal-negative)
exhibited an apparently random, fine-grained admixture in both the midline and
lateral neural plate of E8 chimeras with +/+ +/+ genotype
(Fig. 3A-C). By contrast, the
midline neural plate of +/+ Lp/Lp chimeras appeared to contain
a lower proportion of ß-gal-positive (Lp/Lp) cells than did
lateral regions (Fig. 3D-F).
Similarly, we observed a predominance of ß-gal-negative cells in the
node-derived notochordal plate of +/+ Lp/Lp chimeras
(Fig. 3D) whereas an even
mixture of ß-gal-positive and -negative cells was apparent in the
notochordal plate of +/+ +/+ chimeras
(Fig. 3A).
|
+/+, +/+ Lp/+ and
+/+ Lp/Lp genotypes (n=3 embryos per genotype).
Individual chimeras are expected to differ from each other in their overall
proportion of ES-cell-derived and blastocyst-derived components. To eliminate
this variation, we subtracted the per cent of ß-gal-negative cells in the
lateral region from that in the midline of each chimera. Hence, a chimera with
similar proportions of the two genotypes in both midline and lateral regions
would score zero, and we found that +/+ +/+ chimeras did indeed yield
values clustered around zero (Fig.
3G). By contrast, +/+ Lp/Lp chimera sections
showed a 15-45% predominance of wild-type cells over Lp/Lp cells in
the midline compared with the lateral neural plate. This effect was present
similarly in caudal, middle and cranial regions of the chimeras.
Interestingly, a wide variation of values was detected in +/+
Lp/+ chimeras. Some sections showed a midline-lateral difference
close to zero, whereas others showed values similar to those found in +/+
Lp/Lp chimeras. We conclude that the colonisation of the
midline by Lp/Lp cells is intrinsically defective and is not rescued
by close juxtaposition to wild-type cells in a chimeric combination.
Mechanism underlying defective midline extension in Lp
Injection of DiI into the wild-type mouse node at E7.5 labels midline
chordamesodermal cells along the whole body axis
(Fig. 1A)
(Beddington, 1994;
Sulik et al., 1994). This is
consistent with a model derived from avian studies in which Hensen's node
`regresses' during gastrulation, laying down the entire notochord
(Catala et al., 1996). In mice,
a notochord is lacking from embryos following surgical or genetic ablation of
the node (Davidson et al.,
1999; Ang and Rossant,
1994; Weinstein et al.,
1994; Ybot-Gonzalez et al.,
2002). Hence, we considered the possibility that the defect of
axial extension in Lp/Lp embryos may result from a defect in the
node. To examine node morphology and gene expression, we bred the cordon
bleu (Cobl) gene trap, which expresses ß-gal specifically
in the node and its derivatives (Carroll et
al., 2003), onto the Lp strain. At E7.5, Lp/Lp
embryos exhibited a ß-gal-positive node that was morphologically
identical to the wild-type node, with closely similar cell numbers
(Fig. 4A-D). By contrast, at
E8.5, we found a markedly broader ß-gal-positive node in Lp/Lp
embryos than in wild type (Fig.
4E,F). The expression domain of sonic hedgehog (Shh) was
also broader in the notochord, and particularly in the node and notochordal
plate, of Lp/Lp embryos compared with +/+ littermates
(Fig. 4G-I). We previously also
found a broader expression domain of brachyury, in the Lp/Lp
primitive streak and node (Greene et al.,
1998). These findings suggest a defect of midline cell
intercalation in Lp, particularly affecting the node and cells
recently emerged from the node.
To determine whether notochordal extension at more rostral levels is also affected in Lp embryos, we injected DiI at midline positions rostral to the E7.5 node (Fig. 4J-L). Rostral injections label a stream of midline notochordal cells extending variable distances along the body axis, often into the cranial region, but not including the node itself (Fig. 4M-O). In Lp/Lp embryos, we observed a similar range of labelling patterns as in wild-type and Lp/+ embryos (Fig. 4P), demonstrating that notochordal extension rostral to the node and notochordal plate are not markedly abnormal in the Lp mutant.
|
; Habas et al.,
2001; Marlow et al.,
2002), and via JNK in Drosophila and Xenopus
(Boutros et al., 1998;
Yamanaka et al., 2002).
Moreover, blockade of ROCK function causes defective neural tube closure in
the chick (Wei et al., 2001).
RT-PCR shows expression of RhoA, ROCK1 and ROCK2 from E7.5
to 9.5 of mouse development, together with targets of ROCK phosphorylation
including LIM kinase (LIMK)-1 and -2
(Maekawa et al., 1999)
(Fig. 5A). In situ
hybridisation at late gastrulation (E8), when convergent extension is
underway, revealed expression of the PCP pathway components Vangl2, Daam1,
RhoA and ROCK2 throughout the embryo
(Fig. 5B-E), whereas at E8.5,
expression of Vangl2 and Daam1 became restricted to the
region of hindbrain and upper spine, where neural tube closure is initiated
(Fig. 5F,G). At this stage,
RhoA, ROCK2 and JNK1 were expressed in domains that overlap
with the PCP genes (compare Fig.
5H-J with F,G). Transcripts are present in both neural tube and
axial and paraxial mesoderm (not shown).
To examine the functional requirement for RhoA and ROCK, or JNK signalling
in mouse convergent extension and closure initiation, we cultured E7.5 +/+ and
Lp/+ embryos for 18 hours in varying concentrations of the ROCK
inhibitor Y27632. Inhibition of ROCK strongly summated with genotype at the
Lp locus, with Lp heterozygotes exhibiting almost 100% open
neural tubes even at the lowest concentration of Y27632 tested (2.5
µmol/l). By contrast, 2.5-5 µmol/l inhibitor had no adverse effect on
neural tube closure in +/+ embryos, with non-closure first observed in 50% of
embryos only at the near-toxic dose of 10 µmol/l
(Fig. 6A;
Table 2). We also identified a
hallmark of Lp homozygotes, a broadened Shh-positive floor plate
(Greene et al., 1998), in both
+/+ and Lp/+ embryos with failed neural tube closure following Y27632
treatment (Fig. 7). Hence, the
developmental defect associated with ROCK-dependent failure of Closure 1
resembled that seen in Lp/Lp, and seems unlikely to be a non-specific
toxic effect.
|
). The PCP mutants Crash and Crc also showed an interaction with ROCK inhibition. Crsh/+ embryos had almost 100% open neural tubes at 2.5 µmol/l Y27632, a concentration that had no effect on +/+ littermates (Fig. 6C). Crc/+ embryos were more resistant: open neural tubes first occurred at 5 µmol/l Y27632 and, even at 10 µmol/l, only 67% of heterozygotes failed to complete Closure 1 (Fig. 6D). This relative resistance of Crc/+ embryos to Y27632 may be an effect of genetic background, as wild-type littermates were also resistant to Y27632. Alternatively, the molecular pathway downstream of Scrb1 may differ subtly from that of the proven PCP genes Vangl2 and Celsr1.
Length and width measurements in Lp embryos treated with Y27632 (Fig. 2D,E) revealed a persistent effect of Lp genotype similar to that of embryos cultured without inhibitor. While Y27632 did not affect embryonic length after culture, embryonic width was significantly greater in all genotypes, suggesting that inhibition of RhoA and ROCK signalling may particularly diminish lateral-to-medial cell movements during gastrulation.
|
), and
providing a likely explanation for the detrimental effect of transfection on
neural tube closure.
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Sulik et
al., 1994). Simultaneously, cells of the overlying neurectoderm
converge and intercalate in the midline at all levels of the neuraxis. In the
present study, vital labelling of the node with DiI, and labelling the midline
neural plate with a GFP-expression vector, showed that convergent extension in
both chordamesoderm and neural plate is defective in Lp/Lp embryos.
The result of this developmental defect is that the midline fails to narrow
and extend in Lp/Lp embryos, unlike in wild-type littermates.
Although the neural folds form and elevate normally, they are spaced widely
apart as result of intervening cells that have not intercalated
(Greene et al., 1998). This
wide spacing of the neural folds precludes initiation of neural tube closure
and leads to severe neural tube defects. It is striking that this mechanism of
neural tube closure disturbance appears to be conserved in Xenopus
embryos in which PCP signalling is disturbed
(Wallingford and Harland,
2002).
DiI injection into the node identifies reduced extension in midline
mesoderm, whereas more rostral labelling of the notochord does not reveal
differences in extension behaviour between Lp mutant and wild-type
embryos. This suggests that, although convergent extension is a key factor in
the narrowing and elongation of the notochordal plate immediately in front of
the node, elongation of the notochord more rostrally is less dependent on
convergent extension. Indeed, morphometric analysis of chick and mouse
gastrulation shows that cell rearrangement is a relatively minor contributor
to notochord elongation at levels rostral to the notochordal plate
(Sausedo and Schoenwolf, 1993;
Sausedo and Schoenwolf, 1994).
Longitudinally oriented mitoses appear to account for the majority of the
elongation observed in the rostral notochord. We suggest that unimpaired
extension of the notochord is the primary factor enabling the limited degree
of axial extension that is achieved by Lp/Lp embryos.
|
), but also
influences earlier cell morphogenesis. For example, neuroepithelial cells
adopt a wedge shape during formation of the median hinge point (MHP) in
response to notochordal influence (Smith
and Schoenwolf, 1989). Hence, faulty midline cell intercalation in
the Lp/Lp neural plate could reflect a disturbance of notochordal
induction. However, Vangl2 and other PCP genes are expressed in the
early neural plate (Kibar et al.,
2001; Murdoch et al.,
2001a; Torban et al.,
2007), consistent with a direct requirement for PCP signalling
during neurectodermal convergent extension. Moreover, our chimera studies
suggest that Lp/Lp neural plate cells are preferentially affected in
convergent extension, compared with wild-type cells. If the neural plate
defect was secondary to faulty notochordal induction, both wild-type and
mutant neural plate cells should be equally affected. It seems likely,
therefore, that convergent extension requires PCP signalling independently in
each germ layer.
The absence of Lp mutant cells from the midline of chimeras could
reflect a role for Vangl2 in cell behaviours other than convergent
extension. For example, differential cell adhesion may underlie the
morphogenetic disturbance in PCP mutant zebrafish
(Ciruna et al., 2006). In
mouse chimeras, however, differentially adhesive cells tend to show marked
spatial segregation (Tam and Rossant,
2003), a phenomenon that we did not observe in +/+
Lp/Lp chimeras. It is also possible that the differential abundance
of Lp mutant and wild-type cells in the midline reflects
proliferative differences, as in zebrafish PCP mutants in which mitotic
orientation is disturbed (Gong et al.,
2004). However, we previously observed no differences in cell
proliferation between Lp/Lp and wild-type neural plate cells
(Gerrelli and Copp, 1997). In
the absence of other likely explanations, therefore, the predominance of
wild-type cells in the midline of +/+ Lp/Lp chimeras supports
a cell-autonomous defect of convergent extension.
PCP signalling via small GTPases regulates intracellular events such as
cytoskeletal remodelling that underlie polarised cell behaviour
(Klein and Mlodzik, 2005). The
apparently cell-autonomous effect on convergent extension in Lp, with
a requirement for RhoA-ROCK signalling, is consistent with this model.
However, both cell-autonomous and non-cell-autonomous features of PCP
signalling have been described in other systems. For example, whereas
mediolateral cell alignment during zebrafish convergent extension requires
both Vangl2 (Stbm), and Rho kinase 2 (Rok2), wild-type cells also fail to
align when transplanted to hosts deficient in Stbm or Rok2
(Marlow et al., 2002;
Jessen et al., 2002).
Conversely, overexpression of Xenopus Vangl2 (Xstbm) prevents cell
intercalation, but can be rescued if overexpressing cells are juxtaposed to
wild-type cells (Goto and Keller,
2002). In both Xenopus and Drosophila,
non-cell-autonomous effects of PCP signalling are generally short-range, often
extending a few cell diameters from the edge of single-genotype patches in
mosaics (Goto and Keller,
2002; Strutt,
2003). By contrast, mutant and wild-type cells were closely
intermingled in our mouse chimeras, without distinct patches of either
genotype. Hence, the conditions necessary to demonstrate non-cell-autonomous
effects may have been absent, so that only cell-autonomous, single-cell
effects were evident.
|
). Despite
their clinical importance, the genetic predisposition to human neural tube
defects is poorly understood. The finding that folic acid can prevent a
proportion of human neural tube defects
(Wald et al., 1991) has
prompted studies to seek sequence variants in genes related to folate
metabolism, which may be associated with human neural tube defects. The gene
encoding 5,10-methylenetetrahydrofolate reductase, a key enzyme in folate
metabolism, has yielded a positive correlation with defects in some
populations (Van der Put et al.,
1997) but, to date, no other genes have been specifically
implicated in predisposition to neural tube defects. The same applies to the
human orthologues of genes required for neural tube closure in the mouse:
although many have been examined, none have so far been implicated in the
causation of human malformations (Boyles et
al., 2005). Among the PCP genes, sequence variants of
VANGL2 and its family member VANLG1 were not found to be
associated with neural tube defects
(Doudney et al., 2005). The
present study suggests that other PCP genes should be considered as candidates
for a role in the aetiology of human neural tube defects.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/4/789/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Clinical Sciences Centre, Hammersmith Campus, Imperial
College London, UK
Present address: Department of Anatomy and Developmental Biology,
University College London, UK
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Ang, S.-L. and Rossant, J. (1994).
HNF-3ß is essential for node and notochord formation in mouse
development. Cell 78,561
-574.[CrossRef][Medline]
Bastock, R., Strutt, H. and Strutt, D. (2003).
Strabismus is asymmetrically localised and binds to Prickle and Dishevelled
during Drosophila planar polarity patterning.
Development 130,3007
-3014.
Beddington, R. S. and Robertson, E. J. (1989).
An assessment of the developmental potential of embryonic stem cells in the
midgestation mouse embryo. Development
105,733
-737.
Beddington, R. S. P. (1994). Induction of a
second neural axis by the mouse node. Development
120,613
-620.[Abstract]
Botto, L. D., Moore, C. A., Khoury, M. J. and Erickson, J.
D. (1999). Neural-tube defects. N. Engl. J.
Med. 341,1509
-1519.
Boutros, M., Paricio, N., Strutt, D. I. and Mlodzik, M.
(1998). Dishevelled activates JNK and discriminates between JNK
pathways in planar polarity and wingless signaling.
Cell 94,109
-118.[CrossRef][Medline]
Boyles, A. L., Hammock, P. and Speer, M. C.
(2005). Candidate gene analysis in human neural tube defects.
Am. J. Med. Genet. C Semin. Med. Genet.
135, 9-23.[Medline]
Carroll, E., Gerrelli, D., Gasca, S., Berg, E., Beier, D., Copp,
A. and Klingensmith, J. (2003). Cordon-bleu is a conserved
gene involved in neural tube formation. Dev. Biol.
262, 16-31.[CrossRef][Medline]
Catala, M., Teillet, M. A., De Robertis, E. M. and Le Douarin,
N. M. (1996). A spinal cord fate map in the avian embryo:
while regressing, Hensen's node lays down the notochord and floor plate thus
joining the spinal cord lateral walls. Development
122,2599
-2610.[Abstract]
Ciruna, B., Jenny, A., Lee, D., Mlodzik, M. and Schier, A.
F. (2006). Planar cell polarity signalling couples cell
division and morphogenesis during neurulation. Nature
439,220
-224.[CrossRef][Medline]
Copp, A., Cogram, P., Fleming, A., Gerrelli, D., Henderson, D.,
Hynes, A., Kolatsi-Joannou, M., Murdoch, J. and Ybot-Gonzalez, P.
(1999). Neurulation and neural tube closure defects.
Methods Mol. Biol. 136,135
-160.
Copp, A. J., Checiu, I. and Henson, J. N.
(1994). Developmental basis of severe neural tube defects in the
loop-tail (Lp) mutant mouse: Use of microsatellite DNA
markers to identify embryonic genotype. Dev. Biol.
165, 20-29.[CrossRef][Medline]
Copp, A. J., Greene, N. D. E. and Murdoch, J. N.
(2003). The genetic basis of mammalian neurulation.
Nat. Rev. Genet. 4,784
-793.[CrossRef][Medline]
Curtin, J. A., Quint, E., Tsipouri, V., Arkell, R. M.,
Cattanach, B., Copp, A. J., Fisher, E. M., Nolan, P. M., Steel, K. P., Brown,
S. D. 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, 1-20.[CrossRef][Medline]
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, B. P., Kinder, S. J., Steiner, K., Schoenwolf, G. C.
and Tam, P. P. L. (1999). Impact of node ablation on the
morphogenesis of the body axis and the lateral asymmetry of the mouse embryo
during early organogenesis. Dev. Biol.
211, 11-26.[CrossRef][Medline]
Doudney, K., Ybot-Gonzalez, P., Paternotte, C., Stevenson, R.
E., Greene, N. D., Moore, G. E., Copp, A. J. and Stanier, P.
(2005). Analysis of the planar cell polarity gene Vangl2 and its
co-expressed paralogue Vangl1 in neural tube defect patients. Am.
J. Med. Genet. A 136,90
-92.[Medline]
Gasca, S., Hill, D. P., Klingensmith, J. and Rossant, J.
(1995). Characterization of a gene trap insertion into a novel
gene, cordon-bleu, expressed in axial structures of the gastrulating
mouse embryo. Dev. Genet.
17,141
-154.[CrossRef][Medline]
Gerrelli, D. and Copp, A. J. (1997). Failure of
neural tube closure in the loop-tail (Lp) mutant mouse:
analysis of the embryonic mechanism. Dev. Brain Res.
102,217
-224.[CrossRef][Medline]
Gong, Y., Mo, C. and Fraser, S. E. (2004).
Planar cell polarity signalling controls cell division orientation during
zebrafish gastrulation. Nature
430,689
-693.[CrossRef][Medline]
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. E., Gerrelli, D., Van Straaten, H. W. M. 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]
Greene, N. D. E., Leung, K. Y., Wait, R., Begum, S., Dunn, M. J.
and Copp, A. J. (2002). Differential protein expression at
the stage of neural tube closure in the mouse embryo. J. Biol.
Chem. 277,41645
-41651.
Gutjahr, M. C., Rossy, J. and Niggli, V.
(2005). Role of Rho, Rac, and Rho-kinase in phosphorylation of
myosin light chain, development of polarity, and spontaneous migration of
Walker 256 carcinosarcoma cells. Exp. Cell Res.
308,422
-438.[CrossRef][Medline]
Habas, R., Kato, Y. and He, X. (2001).
Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires
a novel formin homology protein Daam1. Cell
107,843
-854.[CrossRef][Medline]
Hamblet, N. S., Lijam, N., Ruiz-Lozano, P., Wang, J., Yang, Y.,
Luo, Z., Mei, L., Chien, K. R., Sussman, D. J. and Wynshaw-Boris, A.
(2002). Dishevelled 2 is essential for cardiac outflow tract
development, somite segmentation and neural tube closure.
Development 129,5827
-5838.[CrossRef][Medline]
Jessell, T. M. (2000). Neuronal specification
in the spinal cord: inductive signals and transcriptional codes.
Nat. Rev. Genet. 1,20
-29.[CrossRef][Medline]
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]
Keller, R. (2002). Shaping the vertebrate body
plan by polarized embryonic cell movements. Science
298,1950
-1954.
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]
Kirillova, I., Novikova, I., Augé, J., Audollent, S.,
Esnault, D., Encha-Razavi, F., Lazjuk, G., Attié-Bitach, T. and
Vekemans, M. (2000). Expression of the sonic
hedgehog gene in human embryos with neural tube defects.
Teratology 61,347
-354.[CrossRef][Medline]
Klein, T. J. and Mlodzik, M. (2005). Planar
cell polarization: an emerging model points in the right direction.
Annu. Rev. Cell Dev. Biol.
21,155
-176.[CrossRef][Medline]
Kuan, C. Y., Yang, D. D., Roy, D. R. S., Davis, R. J., Rakic, P.
and Flavell, R. A. (1999). The Jnk1 and Jnk2 protein kinases
are required for regional specific apoptosis during early brain development.
Neuron 22,667
-676.[CrossRef][Medline]
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]
Maekawa, M., Ishizaki, T., Boku, S., Watanabe, N., Fujita, A.,
Iwamatsu, A., Obinata, T., Ohashi, K., Mizuno, K. and Narumiya, S.
(1999). Signaling from Rho to the actin cytoskeleton through
protein kinases ROCK and LIM-kinase. Science
285,895
-898.
Marlow, F., Topczewski, J., Sepich, D. and Solnica-Krezel,
L. (2002). Zebrafish Rho kinase 2 acts downstream of Wnt11 to
mediate cell polarity and effective convergence and extension movements.
Curr. Biol. 12,876
-884.[CrossRef][Medline]
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]
Moore, C. A., Li, S., Li, Z., Hong, S. X., Gu, H. Q., Berry, R.
J., Mulinare, J. and Erickson, J. D. (1997). Elevated rates
of severe neural tube defects in a high-prevalence area in northern China.
Am. J. Med. Genet. 73,113
-118.[CrossRef][Medline]
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 localisation and interaction with the loop-tail mutation.
Genomics 78,55
-63.[CrossRef][Medline]
Murdoch, J. N., Henderson, D. J., Doudney, K., Gaston-Massuet,
C., Phillips, H. M., Paternotte, C., Arkell, R., Stanier, P. and Copp, A.
J. (2003). Disruption of scribble (Scrb1)
causes severe neural tube defects in the circletail mouse.
Hum. Mol. Genet. 12,87
-98.
Park, M. and Moon, R. T. (2002). The planar
cell-polarity gene stbm regulates cell behaviour and cell fate in vertebrate
embryos. Nat. Cell Biol.
4, 20-25.[CrossRef][Medline]
Rachel, R. A., Murdoch, J. N., Beermann, F., Copp, A. J. and
Mason, C. A. (2000). Retinal axon misrouting at the optic
chiasm in mice with neural tube closure defects.
Genesis 27,32
-47.[CrossRef][Medline]
Sausedo, R. A. and Schoenwolf, G. C. (1993).
Cell behaviors underlying notochord formation and extension in avian embryos:
quantitative and immunocytochemical studies. Anat.
Rec. 237,58
-70.[CrossRef][Medline]
Sausedo, R. A. and Schoenwolf, G. C. (1994).
Quantitative analyses of cell behaviors underlying notochord formation and
extension in mouse embryos. Anat. Rec.
239,103
-112.[CrossRef][Medline]
Self, A. and Hall, A. (1995). Purification of
recombinant Rho/Rac/G25K from Escherichia coli. Meth.
Enzymol. 256,3
-10.[Medline]
Smith, J. L. and Schoenwolf, G. C. (1989).
Notochordal induction of cell wedging in the chick neural plate and its role
in neural tube formation. J. Exp. Zool.
250, 49-62.[CrossRef][Medline]
Stanier, P., Henson, J. N., Eddleston, J., Moore, G. E. and
Copp, A. J. (1995). Genetic basis of neural tube defects: the
mouse gene loop-tail maps to a region of Chromosome 1 syntenic with
human 1q21-q23. Genomics
26,473
-478.[CrossRef][Medline]
Strutt, D. (2003). Frizzled signalling and cell
polarisation in Drosophila and vertebrates.
Development 130,4501
-4513.
Strutt, D. I., Weber, U. and Mlodzik, M.
(1997). The role of RhoA in tissue polarity and Frizzled
signalling. Nature 387,292
-295.[CrossRef][Medline]
Sulik, K., Dehart, D. B., Inagaki, T., Carson, J. L., Vrablic,
T., Gesteland, K. and Schoenwolf, G. C. (1994). Morphogenesis
of the murine node and notochordal plate. Dev. Dyn.
201,260
-278.[Medline]
Takeuchi, M., Nakabayashi, J., Sakaguchi, T., Yamamoto, T. S.,
Takahashi, H., Takeda, H. and Ueno, N. (2003). The
prickle-related gene in vertebrates is essential for gastrulation cell
movements. Curr. Biol.
13,674
-679.[CrossRef][Medline]
Tam, P. P. L. and Rossant, J. (2003). Mouse
embryonic chimeras: tools for studying mammalian development.
Development 130,6155
-6163.
Torban, E., Wang, H. J., Groulx, N. and Gros, P.
(2004). Independent mutations in mouse Vangl2 that cause
neural tube defects in looptail mice impair interaction with members
of the Dishevelled family. J. Biol. Chem.
279,52703
-52713.
Torban, E., Wang, H. J., Patenaude, A. M., Riccomagno, M.,
Daniels, E., Epstein, D. and Gros, P. (2007). Tissue,
cellular and sub-cellular localization of the Vangl2 protein during embryonic
development: effect of the Lp mutation. Gene Expr.
Patterns 7,346
-354.[CrossRef][Medline]
Van der Put, N. M. J., Eskes, T. K. A. B. and Blom, H. J.
(1997). Is the common 677C
T mutation in the
methylenetetrahydrofolate reductase gene a risk factor for neural tube
defects? A meta-analysis. Q. J. Med.
90,111
-115.
Wald, N., Sneddon, J., Densem, J., Frost, C., Stone, R. and MRC
Vitamin Study Research Group (1991). Prevention of neural
tube defects: Results of the Medical Research Council Vitamin Study.
Lancet 338,131
-137.[CrossRef][Medline]
Wallingford, J. B. and Harland, R. M. (2001).
Xenopus Dishevelled signaling regulates both neural and mesodermal
convergent extension: parallel forces elongating the body axis.
Development 128,2581
-2592.
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]
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. (2006a). 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. (2006b). 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.
Wei, L., Roberts, W., Wang, L., Yamada, M., Zhang, S. X., Zhao,
Z. Y., Rivkees, S. A., Schwartz, R. J. and Imanaka-Yoshida, K.
(2001). Rho kinases play an obligatory role in vertebrate
embryonic organogenesis. Development
128,2953
-2962.
Weinstein, D. C., Ruiz i Altaba, A., Chen, W. S., Hoodless, P.,
Prezioso, V. R., Jessell, T. M. and Darnell, J. E., Jr
(1994). The winged-helix transcription factor
HNF-3ß is required for notochord development in the mouse
embryo. Cell 78,575
-588.[CrossRef][Medline]
Yamanaka, H., Moriguchi, T., Masuyama, N., Kusakabe, M.,
Hanafusa, H., Takada, R., Takada, S. and Nishida, E. (2002).
JNK functions in the non-canonical Wnt pathway to regulate convergent
extension movements in vertebrates. EMBO Rep.
3, 69-75.[CrossRef][Medline]
Yaneza, M., Gilthorpe, J. D., Lumsden, A. and Tucker, A. S.
(2002). No evidence for ventrally migrating neural tube cells
from the mid- and hindbrain. Dev. Dyn.
223,163
-167.[CrossRef][Medline]
Ybot-Gonzalez, P., Cogram, P., Gerrelli, D. and Copp, A. J.
(2002). Sonic hedgehog and the molecular regulation of neural
tube closure. Development
129,2507
-2517.[Medline]
Ybot-Gonzalez, P., Copp, A. J. and Greene, N. D. E.
(2005). Expression pattern of glypican-4 suggests multiple roles
during mouse development. Dev. Dyn.
233,1013
-1017.[CrossRef][Medline]
Zambrowicz, B. P., Imamoto, A., Fiering, S., Herzenberg, L. A.,
Kerr, W. G. and Soriano, P. (1997). Disruption of overlapping
transcripts in the ROSA ßgeo 26 gene trap strain leads to widespread
expression of ß-galactosidase in mouse embryos and hematopoietic cells.
Proc. Natl. Acad. Sci. USA
94,3789
-3794.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  A. W. Seifert, C. M. Bouldin, K.-S. Choi, B. D. Harfe, and M. J. Cohn Multiphasic and tissue-specific roles of sonic hedgehog in cloacal septation and external genitalia development Development, December 1, 2009; 136(23): 3949 - 3957. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. McNeill Planar Cell Polarity and the Kidney J. Am. Soc. Nephrol., October 1, 2009; 20(10): 2104 - 2111. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. K. Vladar, D. Antic, and J. D. Axelrod Planar Cell Polarity Signaling: The Developing Cell's Compass Cold Spring Harb Perspect Biol, September 1, 2009; 1(3): a002964 - a002964. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tao, M. Suzuki, H. Kiyonari, T. Abe, T. Sasaoka, and N. Ueno Mouse prickle1, the homolog of a PCP gene, is essential for epiblast apical-basal polarity PNAS, August 25, 2009; 106(34): 14426 - 14431. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. Nyholm, S. Abdelilah-Seyfried, and Y. Grinblat A novel genetic mechanism regulates dorsolateral hinge-point formation during zebrafish cranial neurulation J. Cell Sci., June 15, 2009; 122(12): 2137 - 2148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. W. Yen, M. Williams, A. Periasamy, M. Conaway, C. Burdsal, R. Keller, X. Lu, and A. Sutherland PTK7 is essential for polarized cell motility and convergent extension during mouse gastrulation Development, June 15, 2009; 136(12): 2039 - 2048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Massa, D. Savery, P. Ybot-Gonzalez, E. Ferraro, A. Rongvaux, F. Cecconi, R. Flavell, N. D. E. Greene, and A. J. Copp Apoptosis is not required for mammalian neural tube closure PNAS, May 19, 2009; 106(20): 8233 - 8238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nandadasa, Q. Tao, N. R. Menon, J. Heasman, and C. Wylie N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements Development, April 15, 2009; 136(8): 1327 - 1338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhou, H. Y. Kim, and L. A. Davidson Actomyosin stiffens the vertebrate embryo during crucial stages of elongation and neural tube closure Development, February 15, 2009; 136(4): 677 - 688. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Garcia-Garcia, M. Shibata, and K. V. Anderson Chato, a KRAB zinc-finger protein, regulates convergent extension in the mouse embryo Development, September 15, 2008; 135(18): 3053 - 3062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yan, D. Huen, T. Morely, G. Johnson, D. Gubb, J. Roote, and P. N. Adler The multiple-wing-hairs Gene Encodes a Novel GBD-FH3 Domain-Containing Protein That Functions Both Prior to and After Wing Hair Initiation Genetics, September 1, 2008; 180(1): 219 - 228. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nishimura and M. Takeichi Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling Development, April 15, 2008; 135(8): 1493 - 1502. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
