© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 3153-3158 doi:10.1242/dev.111427
RESEARCH REPORT
Vangl-dependent planar cell polarity signalling is not required for
neural crest migration in mammals
Sophie E. Pryor1, Valentina Massa1,*, Dawn Savery1, Philipp Andre2, Yingzi Yang2, Nicholas D. E. Greene1 and
Andrew J. Copp1,‡
The role of planar cell polarity (PCP) signalling in neural crest (NC)
development is unclear. The PCP dependence of NC cell migration
has been reported in Xenopus and zebrafish, but NC migration has not
been studied in mammalian PCP mutants. Vangl2Lp/Lp mouse
embryos lack PCP signalling and undergo almost complete failure of
neural tube closure. Here we show, however, that NC specification,
migration and derivative formation occur normally in Vangl2Lp/Lp
embryos. The gene family member Vangl1 was not expressed in NC
nor ectopically expressed in Vangl2Lp/Lp embryos, and doubly
homozygous Vangl1/Vangl2 mutants exhibited normal NC migration.
Acute downregulation of Vangl2 in the NC lineage did not prevent
NC migration. In vitro, Vangl2Lp/Lp neural tube explants generated
emigrating NC cells, as in wild type. Hence, PCP signalling is not
essential for NC migration in mammals, in contrast to its essential role
in neural tube closure. PCP mutations are thus unlikely to mediate
NC-related birth defects in humans.
KEY WORDS: Cell migration, Embryo, Mouse, Neural crest, Neural
tube, Planar cell polarity
INTRODUCTION
The neural crest (NC) is a transient cell population that delaminates
from the dorsal neural tube and migrates extensively, generating a
variety of cell types (Kulesa et al., 2010; Sauka-Spengler and
Bronner-Fraser, 2008). NC emigration is closely coordinated
spatiotemporally with closure of the neural tube, and some genes
[e.g. AP2α (Tfap2a), Cecr2, Pax3, Zic2] (Harris and Juriloff, 2007)
are necessary for both embryonic events. Signalling via the planar
cell polarity (PCP) pathway is required for neural tube closure in
vertebrates, and recently PCP mutations were reported in human
neural tube defects (Juriloff and Harris, 2012). However, the role of
PCP signalling in NC migration, particularly in mammals, remains
unresolved.
The PCP pathway is an evolutionarily conserved, non-canonical
Wnt-frizzled-dishevelled signalling cascade. The vertebrate
homologues of Drosophila ‘core’ PCP genes regulate many
developmental processes, including convergent extension (CE) cell
1
Newlife Birth Defects Research Centre, Institute of Child Health, University College
2
London, 30 Guilford Street, London, WC1N 1EH, UK. Genetic Disease Research
Branch, National Human Genome Research Institute, 49 Convent Drive, MSC 4472,
Bethesda, MD 20892, USA.
*Present address: Department of Health Sciences, University of Milan, Via A.
Di Rudinì, 8, Milan, Italy.
‡
Author for correspondence (a.copp@ucl.ac.uk)
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Received 14 April 2014; Accepted 15 June 2014
movements in embryonic axis elongation, orientation of
mechanosensory hair cells in the cochlea, and the arrangement of
fur, feathers and scales (Seifert and Mlodzik, 2007).
In Xenopus embryos, disruption of PCP signalling (Dsh-DEP+ or
dominant-negative Wnt11 mRNA) inhibited cranial NC migration
in vivo and in vitro (De Calisto et al., 2005). Similar findings were
reported with the PCP-associated gene PTK7 (Shnitsar and
Borchers, 2008), and NC migration defects were also observed in
zebrafish treated with Dsh-DEP+ or wnt5 morpholino (Matthews
et al., 2008). Knockdown of Strabismus (Vangl2 orthologue)
inhibited Xenopus NC migration similarly to Dsh-DEP+ (CarmonaFontaine et al., 2008), whereas a milder NC migration phenotype
was observed in the trilobite (vangl2) zebrafish mutant (Matthews
et al., 2008).
It is unclear whether PCP signalling is essential for mammalian
NC migration. NC-related anomalies comprise up to 20% of
clinically important human birth defects (Bolande, 1974; Dolk
et al., 2010), so it is important to ascertain whether PCP mutations
are a likely cause. Here, we examined NC migration in mice lacking
Vangl1/2 function. Loop-tail (Lp) is a dominant-negative allele of
the core PCP gene Vangl2 that abrogates PCP signalling (Kibar
et al., 2001; Murdoch et al., 2001; Song et al., 2010; Yin et al.,
2012). Vangl2Lp homozygotes fail almost completely in neural tube
closure due to defective CE in midline neural plate and axial
mesoderm (Ybot-Gonzalez et al., 2007). They also display defects
of cochlea organisation, heart morphogenesis, lung and kidney
branching and reproductive system development – all attributed to
severely disrupted PCP function (Montcouquiol et al., 2003; Torban
et al., 2007; vandenBerg and Sassoon, 2009; Yates et al., 2010a, b).
We find no defects in NC migration in Vangl1/2 mutant embryos,
either in vivo or in vitro, arguing strongly that PCP signalling is not
essential for early NC development in mammals.
RESULTS
NC specification and migration are normal in Vangl2Lp/Lp
embryos
The specification of NC cells was detected by whole-mount in situ
hybridisation (WISH) for Sox9, a marker of premigratory NC
(Cheung and Briscoe, 2003). Sox9-positive NC cells were visible
along the mid-dorsal aspect of the embryonic day (E) 9.5 wildtype neural tube and, similarly, on the tips of the open neural folds
in stage-matched Vangl2Lp/Lp embryos (supplementary material
Fig. S1A-F).
Migrating NC cells were detected by WISH for Erbb3, a neuregulin
receptor tyrosine kinase (Garratt et al., 2000). Both wild-type and
stage-matched Vangl2Lp/Lp embryos at E9.5 showed streams of
cranial NC cells migrating from the hindbrain towards branchial
arches 1 and 2, and around the optic vesicles (Fig. 1A,B,D,E). Erbb3positive trunk NC cells were delaminating from the neuroepithelium
and migrating ventrally (Fig. 1C,F). Later in development, NC cell
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DEVELOPMENT
ABSTRACT
RESEARCH REPORT
Development (2014) 141, 3153-3158 doi:10.1242/dev.111427
emigration from the trunk neural tube also appeared closely
comparable in wild-type and Vangl2Lp/Lp embryos (supplementary
material Fig. S1G-T).
A similar NC migration pattern was detected by fluorescent
lineage labelling in both Vangl2+/+; Wnt1-Cre/YFP and Vangl2Lp/Lp;
Wnt1-Cre/YFP embryos. At E8.5, YFP-positive NC cells had
colonised the forebrain, peri-ocular region and branchial arches
1 and 2 (Fig. 1G-I,K-M), and migrating NC cells were present
at heart-level in both genotypes (Fig. 1J,N). Closely comparable
patterns of NC cell distribution were present later in development at
different axial levels (supplementary material Fig. S2A-H). No
significant differences were found in the number of migrating YFPpositive NC cells in Vangl2+/+ and Vangl2Lp/Lp embryos at E9, E9.5
or E10.5 (supplementary material Fig. S2I). Analysis of embryos at
E10.5, both by Erbb3 WISH (supplementary material Fig. S1U-BB)
and Wnt1-Cre/YFP labelling (supplementary material Fig. S2J-Y),
also revealed very similar NC cell distribution and patterning of
NC-derived structures. We conclude that specification, migration and
tissue colonisation by NC is normal in Vangl2Lp/Lp mutants that fail in
neural tube closure.
Vangl1 does not compensate for loss of Vangl2 during NC
migration
We examined whether the gene family member Vangl1 could
compensate for loss of Vangl2, thereby ensuring normal NC
migration. Vangl1 is a highly conserved, structurally similar
paralogue of Vangl2 (Torban et al., 2004) and the only other
known mammalian orthologue of Drosophila Strabismus (Van
3154
Gogh). Both Vangl1 and Vangl2 proteins interact physically with
mammalian dishevelled (Torban et al., 2004). Moreover, Vangl1
interacts genetically with Vangl2 during neurulation (Torban et al.,
2008), with a more severe phenotype in Vangl1/Vangl2 double
homozygotes than in Vangl2Lp/Lp (Song et al., 2010).
Vangl1 expression was detected solely in the ventral
neuroepithelium of E8.5 Vangl2+/+ and Vangl2Lp/Lp embryos,
from the level of hindbrain to low spine (Fig. 2A,E). In both
genotypes, Vangl1 transcripts could not be detected in the upper
hindbrain, midbrain (Fig. 2B,F) or edges of the trunk neural
folds (Fig. 2C,D,G,H), which are all sites of Erbb3-positive NC
cell origin (Fig. 2I-L). Vangl2 expression also showed no overlap
with Erbb3, but rather exhibited generalised neural tube
expression, overlapping with Vangl1 only at the ventral midline
(Fig. 2M-P). Later in neurulation, Vangl1 expression remained
distinct from Erbb3 along the body axis, with no evidence of
ectopic expression in Vangl2Lp/Lp embryos (supplementary
material Fig. S3).
To test experimentally whether Vangl1 may compensate for
Vangl2 disruption in NC migration, we bred mice doubly
homozygous for Vangl1 and Vangl2 loss of function (Song et al.,
2010). The pattern of Erbb3-positive NC cell migration was very
similar at both E8.5 and E9.5 in normally developing controls
(Vangl1gt/+; Vangl2Δ/+; Fig. 3A,C-E) and in doubly homozygous
mutants (Vangl1gt/gt; Vangl2Δ/Δ; Fig. 3B,F-H), despite the
entirely open neural tube in the latter embryos. We conclude
that Vangl gene function is not required for mouse NC migration
in vivo.
DEVELOPMENT
Fig. 1. Normal pattern of NC cell migration in Vangl2Lp/Lp mouse
embryos. Migrating NC detected by Erbb3 mRNA expression
(A-F) and YFP expression regulated by Wnt1-Cre (G-N). Wild-type
(+/+; A-C,G-J) and Vangl2Lp/Lp (Lp/Lp; D-F,K-N) embryos at early E9.5
(13-14 somites) both exhibit NC cells colonising forebrain, peri-ocular
region (A,D, arrows) and upper branchial arches (ba). Transverse
sections show branchial arch colonisation (B,E,H,I,L,M) and migration
from closed (+/+) and open (Lp/Lp) neural tube (arrows in C,F,J,N). da,
dorsal aorta. Scale bars: 500 µm in A,D; 200 µm in B,C,E,F; 250 µm in
G,K; 100 µm in H-J,L-N.
RESEARCH REPORT
Development (2014) 141, 3153-3158 doi:10.1242/dev.111427
Constitutional absence of Vangl2-dependent PCP signalling
in loop-tail embryos could stimulate a compensatory mechanism
(e.g. activation of a Vangl2-independent pathway) in the NC or
surrounding tissue, allowing normal NC migration (Fig. 3I, i). To
address this, we produced Vangl2Lp/flox; Wnt1-Cre/YFP embryos in
which Vangl2 expression was ablated specifically in the NC
lineage. We reasoned that acute ablation of Vangl2 should prevent
any compensatory mechanism from arising, and so lead to NC
migration defects (Fig. 3I, ii). Fluorescently labelled NC cells were
detected in E9.5 Vangl2Lp/flox; Wnt1-Cre/YFP embryos in a pattern
indistinguishable from that of controls (Fig. 3J-O). WISH for
Erbb3 revealed no difference between conditional mutants and
controls (data not shown). We conclude that the normal pattern of
NC migration observed in Vangl2Lp/Lp embryos is unlikely to arise
from a compensatory mechanism masking a role for Vangl2 in
NC migration.
outgrowth area did not differ between Vangl2+/+, Vangl2Lp/+ and
Vangl2Lp/Lp genotypes at either 24 or 48 h (Fig. 4B). Double
immunostaining confirmed that the majority of YFP-positive NC
cells also expressed the NC cell marker p75 (Ngfr – Mouse Genome
Informatics) (supplementary material Fig. S4D,E).
In Xenopus NC outgrowths, leading edge cells extended large,
polarised lamellipodia whereas those with defective PCP signalling
failed to polarise (Carmona-Fontaine et al., 2008; De Calisto et al.,
2005). In mouse Vangl2+/+ and Vangl2Lp/Lp explants, we observed
both highly polarised YFP-expressing cells at the leading edge as
well as non-polarised cells (Fig. 4C; supplementary material Fig.
S4C). The proportion of cells polarised in the direction of migration
did not differ significantly between genotypes (Fig. 4D,E), nor did
the distance migrated by leading edge NC cells from the central
explant (Fig. 4F). Together, these data demonstrate that loss of
function of the core PCP gene Vangl2 does not impair NC cell
migration in vitro.
Vangl2Lp/Lp NC cells migrate normally in vitro
DISCUSSION
Migration of Xenopus NC was inhibited after disruption of PCP
signalling (De Calisto et al., 2005). By contrast, we observed
comparable in vitro outgrowth of migratory cells from Vangl2+/+ and
Vangl2Lp/Lp neural tube explants (supplementary material Fig. S4A,B).
YFP-positive premigratory NC cells were initially detected along the
dorsal margin of neural tube/fold explants from Vangl2+/+ and
Vangl2Lp/Lp embryos expressing Wnt1-Cre/R26R-YFP. After 24 h,
similar numbers of YFP-positive migratory cells had emerged from the
explants of both genotypes (Fig. 4A). The percentage increase in
In contrast to Xenopus and zebrafish, where Wnt/PCP signalling is
required for NC migration (Carmona-Fontaine et al., 2008;
Matthews et al., 2008), we could detect no abnormality of NC
development in Vangl1/2 mouse mutants with severe PCP defects.
NC migration disorders are typically associated with anomalies of
craniofacial development and cardiac outflow tract (OFT) septation,
but neither defect is observed in Vangl2Lp/Lp fetuses (Henderson
et al., 2001). Pairwise loss of mouse dishevelled genes Dvl1/2 and
Dvl2/3 does cause cardiac OFT defects but cardiac NC migration
Acute ablation of Vangl2 function in the NC lineage
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DEVELOPMENT
Fig. 2. Vangl1 is not expressed in wild-type NC,
nor ectopically in Vangl2Lp/Lp mutants. WISH in
intact embryos shown from dorsal (A,E,I,M) and right
lateral (A′,E′,I′,M′) views, and in sections (B-D,F-H,
J-L,N-P) at levels indicated by dashed lines in A,E,I,
M. Vangl1 mRNA expression is confined to midline
neuroepithelium, from hindbrain to low trunk (arrows),
in E8.5 wild-type embryos (+/+; A-D). There is no
ectopic expression in Vangl2Lp/Lp embryos (Lp/Lp;
E-H) nor overlap with Erbb3-positive NC (I-L). Vangl2
is expressed throughout the neuroepithelium (M-P),
overlapping with Vangl1 only in midline cells, and not
overlapping with Erbb3. Scale bars: 200 µm.
RESEARCH REPORT
Development (2014) 141, 3153-3158 doi:10.1242/dev.111427
Fig. 3. Normal NC migration in Vangl1/2 double
mutants and after acute Vangl2 downregulation in
the NC lineage. (A-H) Control (A,C-E; Vangl1gt/+;
Vangl2Δ/+) and double-mutant (B,F-H; Vangl1gt/gt;
Vangl2Δ/Δ) embryos exhibit normal migration of Erbb3positive cranial NC (E8.5; A,B) and cranial/trunk NC
(E9.5; C-H). Acute NC downregulation of Vangl2 to test
for a possible compensatory mechanism in Vangl2Lp/Lp
embryos (I) reveals identical YFP-positive NC migration
in control (J-L; Vangl2+/flox; Wnt1-Cre) and
downregulation (M-O; Vangl2Lp/flox; Wnt1-Cre) E9.5
embryos. Arrows indicate comparable streams of NC
cells migrating from the trunk neural tube in both
genotypes. Scale bars: 200 µm in A; 500 µm in C,F;
100 µm in J-O.
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Neurocristopathies are congenital malformations involving
defective NC development (Bolande, 1974). These include
craniofacial anomalies, gut innervation defects and disorders of
cardiac OFT septation, which occur in ∼5 per 1000 births (Dolk
et al., 2010). Environmental factors (e.g. alcohol, retinoic acid) are
relatively minor causes of birth defects [0.12 cases per 1000 births
(Dolk et al., 2010)] and genetic factors are likely to be quantitatively
more significant. Core PCP genes have been implicated in human
neural tube closure defects (Juriloff and Harris, 2012) and, given the
close spatiotemporal relationship between neurulation and NC
development, PCP genes might be considered as strong candidates
for congenital neurocristopathies. Our findings, however, argue that
PCP genes are an unlikely cause of NC-related birth defects.
Instead, attention should be focused on other groups of genes, such
as those regulating the guidance of migrating NC cells and the
differentiation of NC derivatives.
MATERIALS AND METHODS
Mouse strains and embryos
Animal studies were performed according to the UK Animals (Scientific
Procedures) Act 1986 and the Medical Research Council’s Responsibility in
the Use of Animals for Medical Research (July 1993). Experimental
embryos were generated from strains: Vangl2Lp/+ (CBA/Ca background)
(Ybot-Gonzalez et al., 2007), Wnt1-Cre (Jiang et al., 2000) crossed with
R26R-EYFP (Srinivas et al., 2001), doubly heterozygous Vangl1gt/+;
Vangl2Δ/+ mice (Song et al., 2010) and Vangl2flox/flox (gift from Deborah
Henderson, Institute of Genetic Medicine, Newcastle University, UK). See
supplementary material methods for breeding schemes and genotyping.
Noon after overnight mating was designated E0.5. Embryos were dissected
at E8.5-10.5 in Dulbecco’s Modified Eagle’s Medium (DMEM) containing
10% fetal calf serum (FCS). Whole-mount YFP expression was visualised
by direct fluorescence. Embryos for WISH or immunohistochemistry were
fixed in 4% paraformaldehyde (PFA) in PBS at 4°C overnight.
DEVELOPMENT
has been described in these mice as either normal (Etheridge et al.,
2008) or disrupted via a disorder of Wnt/β-catenin signalling
(Hamblet et al., 2002). Canonical Wnt/β-catenin signalling is
known to be required for NC migration in mice (Ikeya et al., 1997).
We conclude that the PCP dependence of NC development is not
universal among vertebrates.
Several lines of evidence indicate that PCP signalling is abrogated
in Vangl2Lp/Lp mice. Vangl2 recruits all three dishevelled family
members to the plasma membrane (Torban et al., 2004) as part of
the asymmetric localisation of PCP protein complexes needed for
signal transduction. Membrane localisation is lost in Vangl2Lp/Lp
embryos (Torban et al., 2007). Moreover, the Vangl2Lp allele acts as
a dominant negative in the female reproductive tract and brain
ependymal cells (Guirao et al., 2010; vandenBerg and Sassoon,
2009). Stronger neural tube and inner ear phenotypes occur in looptail mice than in Vangl2 knockouts, supporting a dominant-negative
effect of the Vangl2Lp allele (Song et al., 2010; Yin et al., 2012).
This is likely to result from disrupted trafficking from endoplasmic
reticulum to plasma membrane, which affects the Vangl2Lp protein
(Merte et al., 2010) and other PCP proteins in Vangl2Lp/Lp mice
(Yin et al., 2012).
We could not detect functional redundancy between Vangl1 and
Vangl2 in relation to NC migration. Moreover, acute downregulation
of Vangl2 in the NC lineage did not suggest a compensatory
mechanism in mice with constitutional lack of Vangl2. Vangl2 is
expressed at the mRNA level in the mouse neural tube but not in
migrating NC cells. Similarly, mRNAs for other core PCP
components, including Celsr1 (Formstone and Little, 2001; Shima
et al., 2002) and Dvl1 (Gray et al., 2009), are not detected in NC.
Hence, our finding of normal NC migration in loop-tail mice is
consistent with the absence of PCP signalling in NC cells after
emigration from the neural tube.
RESEARCH REPORT
Development (2014) 141, 3153-3158 doi:10.1242/dev.111427
Fig. 4. NC cells migrate similarly from
Vangl2+/+ and Vangl2Lp/Lp explant
cultures. YFP-positive NC are initially
(0 h; A) on the dorsal margin of Vangl2+/+;
Wnt1-Cre/YFP (+/+) explants and on
Vangl2Lp/Lp; Wnt1-Cre/YFP (Lp/Lp) neural
fold tips. Cells emerge in similar numbers (at
24 h; A), with no difference in outgrowth area
(P=0.91, one-way ANOVA; B). Leading edge
NC cells (anti-GFP/YFP; DAPI) are
polarised (C, arrows) or non-polarised
(C, arrowheads). Analysis of leading edge
cells (D) reveals no difference between
genotypes in the proportion of polarised cells
nor in the mean distance migrated (P=0.42,
E; P=0.21, F; one-way ANOVA). bf, bright
field. Error bars indicate s.e.m. At least three
explants were studied per genotype and time
point. Scale bars: 200 µm in A; 50 µm in C.
WISH, immunohistochemistry and immunocytochemistry
Author contributions
WISH was performed on a minimum of five embryos per probe and per
genotype. Digoxygenin-labelled RNA probes for Erbb3, Vangl1 and Vangl2
were as described (Doudney et al., 2005; Henderson et al., 2001). Hybridised
embryos were embedded in 2% agarose in PBS and vibratome-sectioned at
50 µm thickness before mounting in Mowiol (Sigma). Immunohistochemistry
utilised 7 µm wax sections; primary and secondary antibodies are listed in
supplementary material methods. Sections and explants were mounted using
Vectashield medium with DAPI (Vector Labs).
S.E.P., N.D.E.G. and A.J.C. designed the experiments; S.E.P. and V.M. performed
the experiments; D.S., P.A. and Y.Y. performed the mouse crosses; S.E.P.,
N.D.E.G. and A.J.C. analysed the data and wrote the manuscript.
Statistical analysis
Statistical tests were performed using SigmaStat (Systat) version 3.5.
Acknowledgements
The authors declare no competing financial interests.
Supplementary material
flox
mice.
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Competing interests
This work was supported by the Wellcome Trust [grants 083361, 087259, 087525] and
Medical Research Council [grant G0801124]. Deposited in PMC for immediate release.
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.111427/-/DC1
Neural tube explant culture
We thank Prof. Deborah Henderson for providing Vangl2
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DEVELOPMENT
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Development (2014) 141, 3153-3158 doi:10.1242/dev.111427
3158
SUPPLEMENTARY METHODS
Mouse breeding and genotyping
Mice heterozygous for the loop-tail mutation (Vangl2Lp/+; CBA/Ca background) were
intercrossed to generate wild-type (Vangl2+/+) and homozygous mutant (Vangl2Lp/Lp)
embryos. Vangl2Lp genotyping was performed using the Crp microsatellite DNA marker, as
described (Copp et al., 1994).
To generate embryos in which the NC cells and descendants were fluorescently
labelled, crosses were performed between Wnt1-Cre (Jiang et al., 2000) and R26R-EYFP
reporter (Srinivas et al., 2001) mice. Wnt1-Cre; YFP offspring were bred with Vangl2Lp/+
mice and the Vangl2Lp/+; Wnt1-Cre/YFP offspring were intercrossed to generate Vangl2+/+;
Wnt1-Cre/YFP
and Vangl2Lp/Lp; Wnt1-Cre/YFP embryos for analysis.
Doubly homozygous Vangl1gt/gt; Vangl2∆/∆ embryos were produced by matings
between doubly heterozygous Vangl1gt/+; Vangl2∆/+ mice, which were bred and genotyped as
described previously (Song et al., 2010).
To ablate Vangl2 specifically in the NC lineage, Vangl2Lp/+; Wnt1-Cre/YFP mice were
crossed with Vangl2flox/flox animals (gift of Prof. Deborah Henderson). The experimental
offspring were Vangl2Lp/flox; Wnt1-Cre or Vangl2Lp/flox; Wnt1-Cre/YFP (i.e. Vangl2Lp/– in the
Wnt1-positive NC cells, but Vangl2Lp/+ in all other tissues). Control offspring were Vangl2+/flox;
Wnt1-Cre (or Vangl2+/flox; Wnt1-Cre/YFP), Vangl2+/flox and Vangl2Lp/flox.
NC cell counts
Sections from Vangl2+/+; Wnt1-Cre/YFP and Vangl2Lp/Lp; Wnt1-Cre/YFP embryos were
analysed at the level of the developing heart (n= at least 4 sections per genotype), trunk
(n=3 sections per genotype) and foregut/trachea (n=5 sections per genotype). Sections
were matched for body level between genotypes and total migrating YFP-labelled NC cells
were counted in each section.
Analysis of NC cell migration in vitro
The rate of cell migration over 2 days was assessed by measuring the area of outgrowth
(outer dotted lines in Fig. S4B) as a percentage increase of the area covered by the central
1
Development | Supplementary Material
explant tissue at 24 and 48 h (inner dotted lines; at least three samples measured per
genotype and time point). This allowed for any variation due to differences in the original size
of the explant, or to pieces of the tissue detaching and floating away during the first few
hours of culture. Migration rate was analysed by measuring the distance travelled by YFPpositive leading edge NC cells from the periphery of the central explant tissue. Polarity of NC
cells at the leading edge was assessed by measuring the distribution of cell area relative to
the direction of migration, with ‘forward’ defined as the direction away from the central mass
of neuroepithelial tissue. The cell area within six circular sectors, centred on the nucleus,
was measured, allowing each cell to have designated ‘front’, ‘sides’ and ‘back’ based on the
direction in which the majority of the area was distributed (Fig. 4D). Cells that did not display
a majority of at least 5% in any direction were designated ‘none’. Cells which were polarised
in the direction of forward migration were defined as those with the majority of their area
distributed towards the ‘front’. Migration rate and polarity analysis were performed on at least
64 cells from three explants for each genotype. All area and distance measurements were
performed using ImageJ software.
Antibodies
Primary antibodies: Chicken polyclonal anti-GFP/YFP (Abcam, ab13970; dilutions 1:100 for
sections; 1:250 for explant cultures); rabbit polyclonal anti-P75 (Santa Cruz, sc-8317; dilution
1:200). Secondary antibodies: Fluorescein-labelled goat anti-chicken IgY (Aves Labs, F-1005;
dilution 1:200); goat anti-chicken IgG-Alexa488 (Invitrogen, A11039; dilution 1:400; goat antirabbit IgG-Alexa568 (Invitrogen, A21069; dilution 1:400).
Supplementary references
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.
Jiang, X.B., Rowitch, D.H., Soriano, P., McMahon, A.P. and Sucov, H.M. (2000). Fate of the
mammalian cardiac neural crest. Development 127, 1607-1616.
Song, H., Hu, J., Chen, W., Elliott, G., Andre, P., Gao, B. and Yang, Y. (2010). Planar cell
2
Development | Supplementary Material
polarity breaks bilateral symmetry by controlling ciliary positioning. Nature 466, 378-382.
Srinivas, S., Watanabe, T., Lin, C.S., William, C.M., Tanabe, Y., Jessell, T.M. and
Costantini, F. (2001). Cre reporter strains produced by targeted insertion of EYFP and ECFP
into the ROSA26 locus. BMC Dev. Biol. 1, 4.
3
Development | Supplementary Material
Pryor et al: Supplementary Figures Figure S1 NC specification, migration and derivative formation appear normal in Vangl2Lp/Lp embryos. Whole‐mount in situ hybridization for pre‐migratory NC marker Sox9 (A‐F) and migratory NC marker Erbb3 (G‐BB) in wild‐type (+/+; A, C, E, G‐K, Q, R, U‐X) and Vangl2Lp/Lp (Lp/Lp; B, D, F, L‐P, S, T, Y‐BB) embryos. (A‐F) Sox9 labels pre‐migratory E9.5 NC cells as far caudally as recently formed somites (arrows in A, B), with no difference between genotypes. NC cells are specified on open neural fold tips in Lp/Lp (* in F). (G‐P) Erbb3 marks a rostral‐caudal progression of E9.5 trunk NC cells emigrating from the neural tube (arrows in G, L). Sections confirm closely similar NC migration patterns in both genotypes (H‐K and M‐P). (Q‐T) Streams of NC cells migrate similarly in the trunk of E10.5 +/+ and Lp/Lp embryos. (U‐BB) Erbb3 expression in E10.5 NC‐derived trigeminal (v), facio‐acoustic (vii/viii), glossopharyngeal (ix), and vagal (x) cranial ganglia (U, V, Y, Z) and in dorsal root ganglia (drg; W, AA) of +/+ and Lp/Lp embryos. Emerging NC are visible in low trunk region of both genotypes (arrows in X, BB) Abbreviations: ba, branchial arch; da, dorsal aorta; f, facial NC; ov, optic vesicle. Scale bars: 500 µm (A‐F, G, L, Q, S, U‐BB); 200 µm (H‐K, M‐P, R, T). 1 Development | Supplementary Material
Figure S2 Fluorescent labelling of NC reveals normal migration and derivative formation in Vangl2Lp/Lp embryos. NC and its YFP‐expressing descendants in Vangl2+/+; Wnt1‐Cre/YFP (+/+; A‐D, J‐
Q) and Vangl2Lp/Lp; Wnt1‐Cre/YFP (Lp/Lp; E‐H, R‐Y) embryos. (A‐H) E9.5 embryos showing NC colonization, similarly in +/+ and Lp/Lp, of upper and lower branchial arches (arrows: A, E), and forebrain/hindbrain (B, F). NC cells are emigrating from neural tube (arrows: C, D, G, H) and colonizing regions lateral to foregut and around paired aortae (arrowheads: C, D, G, H). (I) Number of migrating NC cells (mean + SEM) does not differ between genotypes for each region analysed (heart level, p = 0.183; trunk, p = 0.446; foregut/trachea, p = 0.664). (J‐Y) E10.5 embryos showing YFP‐positive NC cells within the nasal process, branchial arches, branchial pouches (arrowheads in J, R) and dorsal root ganglia (arrows in J, R). Despite the widely open neural folds in Lp/Lp, no differences in distribution of YFP‐
expressing NC cells are observed between genotypes in sections. NC derivatives are detected sub‐epidermally (K, S), around the developing eye (L, T), and in the branchial pouches (M, U). NC cells are migrating into the developing heart (arrows in N, V) and around the foregut (asterisks in O, W). YFP‐positive dorsal root ganglia are normally sized in Lp/Lp (arrows in P, X, also Q, Y). Auto‐
fluorescent blood cells appear yellow. Scale bars: 100 µm (A‐H, K‐Q, S‐Y), 500 µm (J, R). 2 Development | Supplementary Material
Figure S3 Vangl1 is not expressed in wild‐type trunk NC, nor ectopically expressed in Vangl2Lp/Lp mutants. (A‐C) Whole‐mount in situ hybridisation for Vangl1 in wild‐type (WT; +/+) embryo at E9.5. Transcripts are detected in the ventral half of the neural tube from midbrain to posterior neuropore (* in A indicates probe trapped within the hindgut). This expression pattern is confirmed in transverse sections (B, C; at level indicated by dotted lines in A). (D‐F) Pattern of Vangl1 expression in Vangl2Lp/Lp appears unaltered compared with WT. Marginally more intense cranial staining in Vangl2Lp/Lp (Lp/Lp; D), is likely an artefact due to the widely open neural folds. Transverse sections (E‐F) reveal Vangl1 expression confined to the ventral neuroepithelium at a similar intensity as WT. (G‐I) Erbb3 mRNA expression is confined to migrated NC in the cranio‐facial region (G) and actively migrating NC cells in the trunk (H, I). Vangl1 and Erbb3 expression does not overlap in either genotype. (J‐L) Vangl2 mRNA expression at E9.5 is present at greatest intensity throughout the neuroepithelium, and at lower intensity in mesoderm and gut endoderm (* in J indicates probe trapped within the hindgut). As at E8.5, co‐expression of Vangl1 and Vangl2 occurs only in ventral midline neural tube cells, and Vangl2 does not overlap in expression with Erbb3. Scale bars: 500 µm (A, D, G, J), 200 µm (all sections). 3 Development | Supplementary Material
Figure S4 A
D
B
C
E
Neural crest outgrowth in vitro is equivalent in wild‐type and Vangl2Lp/Lp explants. (A) Method of preparation of neural tube explants for outgrowth culture. After enzymatic digestion, the neural tube was isolated adjacent to the posterior‐most five somites (indicated by white line in left panel). Surrounding surface ectoderm and somitic tissues were removed and explants were plated for culture on fibronectin/ poly‐D lysine‐coated coverslips. (B) Wild‐type (+/+; i) and Vangl2Lp/Lp (Lp/Lp; ii) neural tube explants cultured for 24 and 48 h. Inner yellow dotted lines indicate the area covered by the central mass of neuroepithelial tissue; outer dotted lines indicate the leading edge of the migratory population. (C) Examples of the variable morphology of leading edge cells, as viewed by phase contrast microscopy. Red asterisks: wild‐type and Vangl2Lp/Lp cells which appear polarised. Yellow asterisks: cells which extend protrusions in all directions. (D) Representative Vangl2+/+; Wnt1‐Cre/YFP neural tube explant culture immunostained with anti‐GFP. Higher magnification views of the boxed regions (i and ii) are shown in the lower panels. While the majority of migratory cells are YFP‐positive NC cells (i), other cell types, which do not express YFP, are also present (ii). (E) Representative Vangl2+/+; Wnt1‐Cre/YFP neural tube explant culture immunostained with anti‐GFP and anti‐P75. Most YFP‐positive cells also express the NC marker P75 in vitro. Scale bars: 200 µm (A, B), 50 µm (C), 100 µm (D, E).
4 Development | Supplementary Material