Wnt Signaling in Neural Crest Ontogenesis and Oncogenesis

Abstract

Neural crest (NC) cells are a temporary population of multipotent stem cells that generate a diverse array of cell types, including craniofacial bone and cartilage, smooth muscle cells, melanocytes, and peripheral neurons and glia during embryonic development. Defective neural crest development can cause severe and common structural birth defects, such as craniofacial anomalies and congenital heart disease. In the early vertebrate embryos, NC cells emerge from the dorsal edge of the neural tube during neurulation and then migrate extensively throughout the anterior-posterior body axis to generate numerous derivatives. Wnt signaling plays essential roles in embryonic development and cancer. This review summarizes current understanding of Wnt signaling in NC cell induction, delamination, migration, multipotency, and fate determination, as well as in NC-derived cancers.

Full text

cells
Review
Wnt Signaling in Neural Crest Ontogenesis
and Oncogenesis
Yu Ji 1,2,3,*, Hongyan Hao 3,4, Kurt Reynolds 1,2,3 , Moira McMahon 2,5
and Chengji J. Zhou 1,2,3,*
1Department of Biochemistry and Molecular Medicine & Comprehensive Cancer Center, University of
California at Davis, School of Medicine, Sacramento, CA 95817, USA; ksreynolds@ucdavis.edu
2Institute for Pediatric Regenerative Medicine, UC Davis School of Medicine and Shriners Hospitals for
Children, Sacramento, CA 95817, USA; mmcmahon2000@berkeley.edu
3
Graduate Program of Biochemistry, Molecular, Cellular and Developmental Biology, University of California,
Davis, CA 95616, USA; hyhao@ucdavis.edu
4Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA
5College of Letters & Science, University of California, Berkeley, CA 94720, USA
*Correspondence: yvji@ucdavis.edu (Y.J.); cjzhou@ucdavis.edu (C.J.Z.); Tel.: +1-916-452-2268 (C.J.Z.)
Received: 30 August 2019; Accepted: 25 September 2019; Published: 29 September 2019


Abstract:
Neural crest (NC) cells are a temporary population of multipotent stem cells that generate a
diverse array of cell types, including craniofacial bone and cartilage, smooth muscle cells, melanocytes,
and peripheral neurons and glia during embryonic development. Defective neural crest development
can cause severe and common structural birth defects, such as craniofacial anomalies and congenital
heart disease. In the early vertebrate embryos, NC cells emerge from the dorsal edge of the neural
tube during neurulation and then migrate extensively throughout the anterior-posterior body axis
to generate numerous derivatives. Wnt signaling plays essential roles in embryonic development
and cancer. This review summarizes current understanding of Wnt signaling in NC cell induction,
delamination, migration, multipotency, and fate determination, as well as in NC-derived cancers.
Keywords: Wnt; neural crest stem cells; neural crest-derived cancer
1. Introduction
Neural crest (NC) cells are a unique group of cells originated from the dorsal margins of the neural
tube during early vertebrate development [
1
,
2
]. They migrate extensively into most tissue/organs
and generate numerous differentiated cell types. Consequently, the NC has long been designated
the “fourth germ layer” despite originating from the ectoderm. NC development begins during
gastrulation, induced at the junction between the neural plate and non-neural ectoderm [
3
]. After
induction, NC cells delaminate from the neuroepithelium by undergoing epithelial-mesenchymal
transition (EMT) and migrate long distances in distinct streams according to their positions along the
anterior-posterior axis within the embryo [
4
,
5
]. Subsequently, NC cells differentiate into various cell
types to contribute to different organs in a region-specific manner, including the peripheral nervous
system, melanocytes, the adrenal medulla, smooth muscle, as well as various skeletal, connective,
adipose, and endocrine cell subtypes [6,7].
NC cells originate from four major segments of the neural tube to form cranial (or cephalic), vagal,
trunk, and sacral neural crest cells [
8
] (Figure 1). Distinct populations of cranial NC cells originate from
the diencephalon, midbrain, and hindbrain [
9
] and have the capability to differentiate into craniofacial
bone, cartilage, and connective tissue [
10
]. The rostral cranial NC cells generate the frontonasal
skeleton, while the posterior cranial NC cells fulfill the pharyngeal arches in vertebrates (also known
Cells 2019,8, 1173; doi:10.3390/cells8101173 www.mdpi.com/journal/cells

Cells 2019,8, 1173 2 of 40
as branchial (gill) arches in fish and amphibians), to generate middle ear, bone and cartilage of the jaw
and neck [
11
]. Cranial NC cells also make essential contributions to the membranous bones of the skull
vault [
11
]. The vagal NC, which arises from somites 1–7, has been depicted as a crossbreed between
the cranial and trunk NC [
12
]. The vagal NC forms various cell types in thymus, thyroid, parathyroid,
and heart; and also forms ganglia in lung, pancreas, and the gut [
13
]. Cardiac NC cells arise from the
dorsal neural tube between the otic vesicle and the third somite in the vagal crest segment [
13
,
14
].
These cells are required in avians to trigger restructuring of the developing cardiac outflow tract [15].
In mammals, NC-derived cells occupy conotruncal cushions and the aorticopulmonary septum during
overt septation of the outflow tract and envelop both the thymus and thyroid as these organs form [
16
].
Trunk NC cells arise in the posterior part of the embryo and migrate along three distinct pathways:
a dorsolateral pathway between the ectoderm and the somites, a ventro-lateral pathway between
somites, and a ventro-medial pathway between the neural tube and the posterior sclerotome [
17
].
These cells generate pigment cells of the skin, the peripheral nervous system, and secretory cells of the
endocrine system [18].
Cells 2019, 8, x 2 of 39
(also known as branchial (gill) arches in fish and amphibians), to generate middle ear, bone and
cartilage of the jaw and neck [11]. Cranial NC cells also make essential contributions to the
membranous bones of the skull vault [11]. The vagal NC, which arises from somites 1–7, has been
depicted as a crossbreed between the cranial and trunk NC [12]. The vagal NC forms various cell
types in thymus, thyroid, parathyroid, and heart; and also forms ganglia in lung, pancreas, and the
gut [13]. Cardiac NC cells arise from the dorsal neural tube between the otic vesicle and the third
somite in the vagal crest segment [13,14]. These cells are required in avians to trigger restructuring of
the developing cardiac outflow tract [15]. In mammals, NC-derived cells occupy conotruncal
cushions and the aorticopulmonary septum during overt septation of the outflow tract and envelop
both the thymus and thyroid as these organs form [16]. Trunk NC cells arise in the posterior part of
the embryo and migrate along three distinct pathways: a dorsolateral pathway between the ectoderm
and the somites, a ventro-lateral pathway between somites, and a ventro-medial pathway between
the neural tube and the posterior sclerotome [17]. These cells generate pigment cells of the skin, the
peripheral nervous system, and secretory cells of the endocrine system [18].
Figure 1. Anatomically distinct neural crest cell populations and their major derivates. Schematic
lateral view of a mouse embryo at embryonic day 9.5 shows the cranial (green), vagal (azure), trunk
(purple), and sacral (ruby) neural crest cells and their major derivatives. Vagal segment includes
cardiac neural crest (indigo).
Environmental cues or cell-autonomous factors can affect appropriate NC cell differentiation,
causing cell cycle dysregulation and ectopic tissue formation [19]. Defects in NC cell development
are related with numerous serious diseases, many of which mainly affect children [19]. These
abnormalities, named neurocristopathies [20], are one of the commonest birth defects in newborns,
including congenital heart defects, craniofacial malformations, and familial dysautonomia [19,21,22].
Hirschsprung disease is caused by the failing of vagal NC cell migration to the colon, leading to the
absence of enteric ganglia required for peristaltic bowel movement [23,24]. Treacher Collins
syndrome is an uncommon human congenital disorder which presents with craniofacial
abnormalities due to excessive apoptosis within a pool of cranial NC cells which migrate to the first
and second branchial arches [21]. DiGeorge syndrome is a congenital disease which is also caused by
Figure 1.
Anatomically distinct neural crest cell populations and their major derivates. Schematic
lateral view of a mouse embryo at embryonic day 9.5 shows the cranial (green), vagal (azure), trunk
(purple), and sacral (ruby) neural crest cells and their major derivatives. Vagal segment includes cardiac
neural crest (indigo).
Environmental cues or cell-autonomous factors can affect appropriate NC cell differentiation,
causing cell cycle dysregulation and ectopic tissue formation [
19
]. Defects in NC cell development
are related with numerous serious diseases, many of which mainly affect children [
19
]. These
abnormalities, named neurocristopathies [
20
], are one of the commonest birth defects in newborns,
including congenital heart defects, craniofacial malformations, and familial dysautonomia [
19
,
21
,
22
].
Hirschsprung disease is caused by the failing of vagal NC cell migration to the colon, leading to the
absence of enteric ganglia required for peristaltic bowel movement [
23
,
24
]. Treacher Collins syndrome
is an uncommon human congenital disorder which presents with craniofacial abnormalities due to
excessive apoptosis within a pool of cranial NC cells which migrate to the first and second branchial

Cells 2019,8, 1173 3 of 40
arches [
21
]. DiGeorge syndrome is a congenital disease which is also caused by abnormal migration
of NC cells into the pharyngeal arches. DiGeorge patients usually present with immunodeficiency,
cardiac defects, learning disabilities, psychiatric impairment, and craniofacial malformations [
25
].
Waardenburg syndrome is an autosomal dominant disorder that often includes hypopigmented patches
of skin. In many cases, patients have mutations in PAX3,SNAI2, or SOX10, which are all involved in NC
induction and specification [
2
]. CHARGE syndrome is induced by mutations in helicase DNA-binding
protein 7 (CHD7) gene, which is critical for maintenance of NC multipotency and migration [
26
,
27
].
Patients with CHARGE syndrome have malformation in NC-derived tissues, such as heart defects and
coloboma [28].
Niche signals acting prior to and after migration of NC cells are known to be involved in NC
development [
8
]. Strong evidence shows that Wingless-type MMTV integration site family member
(Wnt) signaling is involved in various stages of NC development [
29
–
31
]. Exogenous Wnt activation is
sufficient to induce human NC cells from pluripotent stem cells [
32
]. Wnt signaling is both necessary
and sufficient for inducing NC cells in Xenopus, chicks, and mice [
33
–
36
]. Furthermore, loss- and
gain-of-function experiments reveal that non-canonical Wnt signaling plays an essential role in NC
cell migration by regulating actin polymerization [
37
,
38
]. In contrast, canonical Wnt signaling may
control NC cell lineage differentiation [
37
,
39
]. In mice, conditional knockout of intracellular Wnt
signal transducer
β
-catenin (Ctnnb1) in pre-migratory NC cells suppresses both sensory neuron and
melanocyte formation [
40
]. In this review, we summarize current understanding of Wnt signaling in
NC development based on published data from the mouse, chick, quail, frog, and zebrafish models,
and in NC-derived cancers.
2. Wnt Signaling Pathways
2.1. β-Catenin-Dependent Canonical Wnt Signaling Pathway
Canonical Wnt/
β
-catenin signaling plays vital roles in development and disease [
41
]. In this
pathway, Wnt proteins bind to two distinctly different types of cell surface receptors, Frizzled (Fzd) and
low-density lipoprotein (LDL) receptor-related protein 6 (Lrp6) or Lrp5 [
41
]. Fzd family proteins are
seven-transmembrane proteins with an extracellular cysteine-rich domain (CRD) [
42
], which interacts
with both the N-terminal domains (D1) and C-terminal domains (D2) of Wnt proteins [
43
,
44
]. Lrp5 and
Lrp6 are single-span transmembrane co-receptors that bind to the D2 domain of Wnt proteins [
44
–
47
].
Therefore, the canonical Wnt proteins have the ability to bridge these different types of receptors
Fzd and Lrp5/Lrp6 [
44
,
45
]. The binding of Wnt proteins causes a conformational change of the
receptor complex, resulting in phosphorylation of the cytoplasmic domain of Lrp5/Lrp6 by several
kinases, such as glycogen synthase kinase 3 (GSK3), allowing the recruitment of the scaffold protein
Axin [
41
]. This leads to the inhibition or saturation of
β
-catenin degradation [
48
]. Therefore, the
newly synthesized
β
-catenin can be accumulated and translocated to the nucleus [
49
,
50
]. Within the
nucleus,
β
-catenin binds to a T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor
family member to regulate the expression of genes involved in cell proliferation, differentiation, and
apoptosis [
51
]. Without Wnts, the cytoplasmic
β
-catenin is continually degraded by the destruction
complex [
48
]. In this complex, Axin acts as a scaffold and interacts with
β
-catenin, the tumor suppressor
protein adenomatous polyposis coli (APC), and two constitutively active kinases, GSK3, and Casein
kinase 1 (CK1) [
52
]. There,
β
-catenin is phosphorylated by GSK3, causing its ubiquitination and
degradation [41].
2.2. β-Catenin-Independent Non-Canonical Wnt Signaling Pathways
Among the
β
-catenin-independent non-canonical Wnt signaling pathways is planar cell
polarity (PCP) signaling, which provides positional information to mediate asymmetric cytoskeletal
organization [
53
]. Therefore, Wnt/PCP is essential for tissue patterning and morphogenesis as well
as polarized cell migration [
53
,
54
]. Binding of Wnt proteins to Fzd receptors can activate and

Cells 2019,8, 1173 4 of 40
recruit Dishevelled (Dvl or Dsh) to the cell membrane where it forms a complex with Dvl-associated
activator of morphogenesis 1 (Daam1) [
55
]. This complex activates Rho GTPases which leads to the
subsequent activation of Rho-associated kinase (Rock) [
56
]. This contributes to asymmetric cytoskeletal
organization and polarized cell migration [
57
]. Another type of non-canonical Wnt signaling is the
Wnt-cGMP/Ca
2+
signaling pathway, which regulates intracellular Ca
2+
flux and levels [
58
]. In this
pathway, the binding of Wnt proteins to Fzd leads to the production of inositol 1,4,5-triphosphate (IP3)
and diacylglycerol (DAG) [
58
,
59
]. IP3 leads to the releasing of Ca
2+
from the endoplasmic reticulum
(ER). DAG is activated by high concentration of calcium released from the ER, which activates protein
kinase C (PKC) [
60
]. Ca
2+
also activates calcium/calmodulin-dependent protein kinase II (CaMKII) [
59
].
Both CaMKII and PKC activate various transcription factors, such as NFκB [61].
3. Wnt Signaling in NPB Formation and NC Induction
NC induction is a multistep process regulated by a complex gene regulatory network [
62
]. It is
initiated during gastrulation and continues through neural tube closure [
63
]. The vertebrate ectoderm
can be separated into three major subdivisions at the end of gastrulation: the non-neural ectoderm,
the neural plate, and the neural plate border (NPB) [
64
] (Figure 2). The non-neural ectoderm will
develop into the epidermis, and the neural plate will give rise to the central nervous system [
65
]. In
most experimental vertebrate species, the NPB begets the NC as well as the pre-placodal ectoderm [
66
].
The induction of NC can be distinguished into three steps. First, inductive signals drive the expression
of a set of transcription factors that define the NPB, known as NPB specifiers. Subsequently, the NPB
specifiers and inductive signals work together to stimulate another set of transcription factors more
restricted to the NC, which are known as NC specifiers [
62
,
63
,
66
]. Finally, inhibitory interactions
between the neural plate, the non-neural ectoderm, the anterior neural fold, and the NC acutely
define the boundaries between these territories [
35
,
67
,
68
]. NPB specifiers, such as Zic1,Gbx2,Msx1,
Pax3/7,Gata2/3,Foxi1/2,Dlx5/6, and Hairy2, are expressed in the early gastrula [
62
–
64
,
66
,
69
–
72
]. NC
specifiers include Snai2,Foxd3,Sox8/9/10,Ets1,cMyc, and Twist [
62
,
71
,
73
,
74
]. Multiple signals regulate
the expression of NPB and NC specifiers, including Wnt, Bmp, Fgf, and Notch [
62
]. Since the formation
of NC occurs at the border between the neural plate and non-neural ectoderm, and ventrally adjacent
to mesoderm, these tissues have been suggested as the source of NC induction signals [
3
]. Here,
we summarize the involvement of Wnt signaling (in the order of canonical then non-canonical for
classified Wnt signaling molecules) in NPB formation and NC induction ( Figures 2and 3; Table 1).
The general description is based on findings on multiple vertebrates, and the species/region-specific
findings are clearly noted in the tables and as much as in the text.

Cells 2019,8, 1173 5 of 40
Table 1.
Experimental findings of Wnt signaling molecules, modulators, and effectors in vertebrate neural crest induction and specification. (cKO, conditional
knockout; GOF, gain of function; KO, knockout; LOF, loss of function; MO; morpholino).
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
ADAM13
upregulates canonical
Wnt signaling by cleaving
class B Ephrins
MO LOF cranial Xenopus
defective NC induction
(Snai2, Sox9, Foxd3) and head
cartilage
[75]
ADAM19
upregulates canonical
Wnt signaling by
stabilizing ADAM13
MO LOF cranial Xenopus
defective NC induction
(Snai2, Sox9, Foxd3) and head
cartilage
[76]
Aldh1a2 delay Wnt3a and Wnt8a
expression KO LOF trunk mouse
diminished NPB specification
(Msx1, Pax3) [77]
Apoc1 downstream effector of
canonical Wnt signaling MO LOF cranial Xenopus
defective NPB induction
(Msx1, Pax3, Zic1), eyes and
head deformation
[78]
Awp1 stabilizes Ctnnb1 MO LOF cranial Xenopus
defective NPB induction
(Msx1, Pax3), pigmentation
and craniofacial cartilage
[79]
Axud1 downstream effector of
canonical Wnt signaling
MO, dominant-negative
construct LOF trunk chick defective NC inductions
(Foxd3, Sox9, Sox10, Ets1) [80]
Cdh11
competitive binding with
Ctnnb1 to repress
Wnt/Ctnnb1 signaling
MO LOF cranial Xenopus
increased Wnt/Ctnnb1
signaling and NC induction
(Sox10, Ap2)
[81]
Ctnnb1 coactivator for Tcf/Lef1
transcription factor RNA injection, MO GOF;
LOF cranial Xenopus
expanded (GOF) or
diminished (LOF) NC
induction (Snai2, Twist)
[82,83]
Daam1
mediator of non-canonical
Wnt signaling and actin
polymerization
MO, mutations LOF cranial Xenopus defective NC induction
(Twist, Sox8, Snai2, Sox10) [38]
Dkk1 antagonist of canonical
Wnt signaling
blocking antibody,
Dkk1-null mouse LOF cranial Xenopus,
mouse
NC generated in the anterior
neural fold, expanded cranial
cartilages
[68]
Dkk2
positive regulator of
canonical Wnt signaling
independent of Gsk3b
MO LOF cranial Xenopus
defective NC induction (Snai2,
Twist1, Sox10), reduced
craniofacial cartilages
[34]

Cells 2019,8, 1173 6 of 40
Table 1. Cont.
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
Dvl (Dsh)
canonical and
non-canonical Wnt
signaling
mutants (dd1, dd2) LOF cranial Xenopus repressed NC induction
(Snai2) [37]
PCP signaling PCP mutants: ∆N-Dsh,
Dsh-DEP+
GOF,
LOF cranial Xenopus
expanded (GOF) or decreased
(LOF) NC induction (Foxd3,
Sox8, Snai2)
[84]
Fgf8a induce Wnt8 in the
paraxial mesoderm MO, RNA injection LOF,
GOF cranial Xenopus
defective (LOF) NPB
induction (Pax3), induced
(GOF) NC in anterior neural
plate
[83]
Fzd3 receptor for Wnt1 RNA injection, MO GOF,
LOF cranial Xenopus
Induced (GOF) or diminished
(LOF) NC induction (Snai,
Twist)
[85]
Fzd7 receptor in canonical
Wnt/Ctnnb1 signaling MO LOF cranial Xenopus inhibited NC induction, loss
of pigment cells [86]
Gbx2 direct target of canonical
Wnt signaling MO, RNA injection LOF,
GOF cranial Xenopus
diminished (LOF) or rescued
(GOF) NPB specifiers (Pax3,
Msx1)
[87]
Gsk3b phosphorylation and
degradation of Ctnnb1 RNA Injections GOF cranial Xenopus increased NC induction
(Krox20, Ap2, Snai2) [88]
Hes3 inhibition of Wnt/Ctnnb1
signaling expression constructs GOF cranial,
trunk Xenopus
blocked NC specifiers (Snai2,
Sox10), supernumerary
pigment cells
[35]
kctd15 attenuate canonical
Wnt/Ctnnb1 signaling MO, mRNA injection LOF cranial zebrafish
defective NC induction
(Sox10, Foxd3), increased
pigmentation, loss of jaw
elements
[89]
expression constructs GOF cranial,
trunk zebrafish
increased NC induction
(Foxd3, Sox10), loss of
pigmentation, small head
[89]
Krm2
promotes Lrp6-mediated
Wnt signaling in the
absence of Dkks
MO, RNA microinjection LOF,
GOF cranial Xenopus
diminished (LOF) NC
markers, ectopic NC-derived
structures (GOF)
[90]

Cells 2019,8, 1173 7 of 40
Table 1. Cont.
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
Lrp6 Wnt co-receptor normal and mutant RNA
injection
GOF,
LOF cranial Xenopus
induced (GOF) or diminished
(LOF) NC specifier Snai2 [47,90]
Mark2 (Par-1) bind to Dvl and regulated
by Wnt5a/Wnt11 MO, mRNA injection LOF cranial Xenopus
repressed (LOF) or enhanced
NC specification (Sox8, Foxd3,
Snai2)
[84]
Rap2 stabilizes Lrp6 through
TNIK kinase MO, siRNA LOF cranial Xenopus
abrogate ectopic expression of
NC markers Snai, Foxd3 [91]
retinoic acid regulate Wnt1 and Wnt3a
expression vitamin A deficiency LOF trunk quail defective NPB (Pax7) and NC
induction (Snai2 and Sox9) [92]
Rgs2
regulates
Wnt-Ppard-Sox10
signaling cascade
MO, dominant negative
rgs2 construct LOF cranial zebrafish
increased NC induction
(Sox10, Snail1b), reduced
cranial cartilage formation
[93]
RhoV downstream effector of
canonical Wnt signaling MO LOF cranial Xenopus
defective NC induction (Sox9,
Sox10, Snai), abnormal
craniofacial skeletons
[94]
Ror2
co-receptor in
non-canonical Wnt/PCP
signaling
MO LOF cranial Xenopus
defective NPB induction
(Gbx2, Zic1, Msx1, Msx2),
decreased BMP signaling at
NPB
[84,95]
Skip
a potential scaffold in
Ctnnb1/Tcf transcriptional
regulation
MO, siRNA LOF cranial Xenopus
defective NC induction
(Snai2, Sox3, Foxd3), loss of
pigment cells
[96]
Sp5
downstream effector of
canonical Wnt and Fgf
pathways
MO LOF cranial Xenopus
defective NPB induction
(Msx1, Pax3); defects in
craniofacial cartilage and
pigmentation
[97]
Tcf7l1 transcription factor cKO by AP2α-Cre LOF cranial mouse
anteriorly expanded NC
specifiers (Foxd3, Sox9, Sox10,
Pax3); exencephaly
[67]
inhibitory mutants LOF cranial Xenopus defective NPB induction
(Msx1) [98]

Cells 2019,8, 1173 8 of 40
Table 1. Cont.
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
THVGR
(hormone-inducible
Tcf7l1)
GOF cranial Xenopus increased NC induction
(Snai2, Twist) [82]
Wnt1
ligand, canonical pathway
dominant negative Wnt1 LOF trunk chick repressed NC induction
(Snai2) [99]
Wnt1 expressing cells GOF trunk chick inhibited NC induction [100]
Wnt1/Wnt3a
ligand, canonical pathway
RNA and DNA Injections GOF cranial Xenopus increased NC induction
(Krox20, Ap2, Snai2) [88]
Wnt1 and/or Wnt3a
knockouts LOF cranial,
trunk mouse
defective NPB induction
(Pax3), cranial skeletons,
cranial ganglia
[101]
Wnt5a ligand, non-canonical
pathway
MO, dominant negative,
RNA injection
LOF,
GOF cranial Xenopus
defective (LOF) or enhanced
(GOF) NC specification (Pax3,
Foxd3, Sox8)
[84]
Wnt6
ligand, upstream of
Dvl-Rho-JNK in PCP
signaling
Wnt6 cell implantation,
siRNA,
GOF,
LOF trunk chick
induced (GOF) or diminished
(LOF) NC induction [100]
Wnt7b ligand RNA microinjection GOF cranial Xenopus increased NC induction
(Snai2, Twist) [102]
Wnt8
ligand, canonical pathway,
downstream of Fgf8a MO, mRNA injection LOF,
GOF cranial Xenopus
defective (LOF) NPB
induction (Pax3, Sox8),
rescued (GOF) NC in
Fgf8a-deficient embryos
[83]
dominant negative, RNA
microinjection
LOF,
GOF cranial Xenopus defective (LOF) or increased
(GOF) NC induction (Snai2) [103]
Wnt11 ligand, non-canonical
pathway
MO, dominant negative,
RNA injection LOF cranial Xenopus
defective (LOF) or enhanced
(GOF) NC specification (Pax3,
Foxd3, Sox8)
[84]
Wnt11r ligand, non-canonical
pathway MO LOF cranial Xenopus defective NC specification
(Foxd3, Sox8) [84]

Cells 2019,8, 1173 9 of 40
Cells 2019, 8, x 1 of 39
Figure 2. Wnt signaling regulates neural plate border (NPB) induction by regulating NPB specifiers.
(A) NPB induction begins during gastrulation and is regulated by both canonical (orange) and non-
canonical (blue) Wnts secreted from NPB and paraxial mesoderm. (B) Key components and possible
interactions between Wnt signaling and NPB specifiers. NNE, non-neural ectoderm; NP, neural plate.
3.1. Wnt Signaling in NPB Formation
The canonical ligand-encoding genes Wnt1 and Wnt3a are expressed in the NPB (Figure 2A) and
dorsal neural tube of mice [105]. In the absence of both Wnt1 and Wnt3a, NPB formation is disrupted,
resulting in a defect in neural crest derivatives [102]. Wnt3a expression is modulated by retinoic acid
(RA) signaling in mice [78]. Ablation of mouse Raldh2 (Aldh1a2), a RA biosynthetic enzyme [106],
delays Wnt3a expression in the dorsal neural tube and decreases the expression of NPB specifiers,
such as Msx1 and Pax3 [78]. Unlike Wnt1 and Wnt3a, Wnt8 is expressed in the paraxial mesoderm
Figure 2.
Wnt signaling regulates neural plate border (NPB) induction by regulating NPB specifiers.
(
A
) NPB induction begins during gastrulation and is regulated by both canonical (orange) and
non-canonical (blue) Wnts secreted from NPB and paraxial mesoderm. (
B
) Key components and
possible interactions between Wnt signaling and NPB specifiers. NNE, non-neural ectoderm; NP,
neural plate.
3.1. Wnt Signaling in NPB Formation
The canonical ligand-encoding genes Wnt1 and Wnt3a are expressed in the NPB (Figure 2A) and
dorsal neural tube of mice [
104
]. In the absence of both Wnt1 and Wnt3a, NPB formation is disrupted,
resulting in a defect in neural crest derivatives [
101
]. Wnt3a expression is modulated by retinoic acid
(RA) signaling in mice [
77
]. Ablation of mouse Raldh2 (Aldh1a2), a RA biosynthetic enzyme [
105
],
delays Wnt3a expression in the dorsal neural tube and decreases the expression of NPB specifiers,
such as Msx1 and Pax3 [
77
]. Unlike Wnt1 and Wnt3a,Wnt8 is expressed in the paraxial mesoderm

Cells 2019,8, 1173 10 of 40
(Figure 2A). In Xenopus, Fgf8 induces the expression of Wnt8 in the paraxial mesoderm and regulates
the formation of NPB [
83
] (Figure 2B). Tcf7l1 is a transcription factor activated by Wnt/
β
-catenin
signaling. Inhibiting Tcf7l1’s ability to bind with
β
-catenin blocks NPB formation (Msx1) and NC
induction (Snai2,Sox9) in Xenopus [
98
]. The transcription factor Gbx2 is a direct target of Wnt/
β
-catenin
signaling, and impaired Tcf7l1 function negatively affects Gbx2 activation in Xenopus [
87
]. During early
NC formation, Gbx2 interacts with the neural fold gene Zic1 and upregulates the expression of NPB
specifiers Pax3 and Msx1. In the absence of Gbx2, Zic1 drives the expression of pre-placodal genes, but
Gbx2 activation inhibits pre-placodal fate and induces NC cells [87].
In Xenopus, the canonical Wnt/
β
-catenin signaling could induce the expression of Apoc1 that
belongs to the apolipoprotein family and binds lipids to form lipoprotein particles and function in lipid
transport [
106
]. Depletion of Apoc1 protein resulted in defective formation of a neural plate border
(Msx1,Pax3,Zic1) and loss of neural crest cells (c-Myc,Sox9,Snai2,Twist1,Id3) [
78
]. The transcription
factor Sp5 has also been shown to be a direct target of Wnt signaling in mice [
107
]. In Xenopus,Sp5 is
induced by Fgf8a or Wnt8 signals to promote NC formation [
97
]. Sp5 has been shown to regulate the
expression of NPB specifiers Msx1 and Pax3 and alters Zic1 expression to promote NC fate during
gastrulation [
97
]. Awp1 is a lipid-activated kinase which associates with the serine/threonine polarity
kinase Par1 [
108
]. In Xenopus, Awp1 mediates NPB formation (Msx1,Pax3) and NC induction (Sox10,
Snai2) by modulating the stability of β-catenin and regulating Wnt signaling [79].
Non-canonical Wnt signaling also plays an important role in NPB formation mainly based on
findings in Xenopus. Gain- and loss-of-function experiments showed that Wnt5a and Wnt11 are
required for the formation of NPB (Pax3,Sox8) and NC (Foxd3,Snail2,Twist) through Dvl and Ror2
in Xenopus [
84
]. Par1 plays an important role in neural crest induction (Sox8,Foxd3, and Snai2).
Wnt5/Wnt11 signaling induce the dissociation of Par1 from the cell cortex, upregulating its enzymatic
activity, which then regulates the expression of Pax3 [
84
]. Ror2 is a major regulator of non-canonical
Wnt signaling [
109
]. In Xenopus, Wnt5a-Ror2 signaling upregulates Papc or Pcns which then upregulates
Bmp ligand Gdf6, thus activating Bmp signaling (pSmad1/5/8) in the dorsolateral marginal zone [
95
].
Moreover, Ror2 regulates cell polarity in the neuroectoderm and shapes the NPB during early neurula
stages. Ror2 loss-of-function causes reduced expression of neural plate border specifiers (Gbx2,Msx1,
Msx2, Zic1) and neural crest marker genes (Twist,Snail2, c-Myc,Tfap2a) [95].
3.2. Wnt Signaling in NC Induction and Specification
Canonical Wnt signaling is well-known to regulate NC induction, and Wnt1, Wnt3a, Wnt7b, Wnt8,
Fzd3, and Lrp6 have all been shown to be required for vertebrate NC specification [
47
,
85
,
88
,
102
,
103
,
110
]
(Figure 3). In Xenopus, the Wnt receptor Fzd7 is required to induce NC markers (Sox10,Sox9,Snail,
Twist,Foxd3). Fzd7 can induce neural crest through binding with different Wnts, including Wnt1,
Wnt7b, and Wnt8. Fzd7 may activate both canonical and non-canonical Wnt signaling pathways. Fzd7
knockdown can be rescued by overexpressing
β
-catenin, suggesting that Fzd7 regulates neural crest
specification through the canonical Wnt pathway in Xenopus [
86
]. Canonical Wnt signaling via Wnt1
regulates the expression of RhoV in NC cells, which is required for the induction of NC in Xenopus [
94
].
RhoV has been shown to regulate Pak1 [
111
], which can phosphorylate and activate Snai1 [
112
].
Therefore, RhoV may act as mediator of canonical Wnt signaling in NC development [
113
]. Axud1
is a transcription factor which acts downstream of Wnt/
β
-catenin signaling during NC induction in
chicks [
114
]. Axud1 knockdown inhibits the expression of NC specifiers (Sox9,Sox10, and Ets1), but
not the NPB gene Pax7 [
80
]. Axud1 directly interacts with Pax7 and Msx1 to form a transcriptional
complex. This complex can bind to the Foxd3 NC1 enhancer to regulate Foxd3 expression [80].

Cells 2019,8, 1173 11 of 40
Cells 2019, 8, x 3 of 39
Figure 3. Wnt signaling induces neural crest cells by regulating NC specifiers. (A) NC is induced from
the neural plate border at the end of gastrulation (zebrafish and Xenopus) or the beginning of
neurulation (chick and mouse). This process is regulated by both canonical (orange) and non-canonical
(blue) Wnts secreted from NPB, adjacent NNE, and paraxial mesoderm. (B) Key components and
possible interactions between Wnt signaling and NC specifiers. NC, neural crest; NNE, non-neural
ectoderm; NT, neural tube.
The canonical Wnt mediator β-catenin affects NC survival in mice. Knockout of β-catenin by a
Wnt1-driven Cre recombinase causes increased apoptosis in pre-migratory NC cells, suggesting
canonical Wnt signaling is needed for the expansion of NC progenitors in mice [116]. However, Wnt1-
Cre-mediated reporter activity is first detected about 0.5–1 days after NC induction begins [117].
Figure 3.
Wnt signaling induces neural crest cells by regulating NC specifiers. (
A
) NC is induced
from the neural plate border at the end of gastrulation (zebrafish and Xenopus) or the beginning of
neurulation (chick and mouse). This process is regulated by both canonical (orange) and non-canonical
(blue) Wnts secreted from NPB, adjacent NNE, and paraxial mesoderm. (
B
) Key components and
possible interactions between Wnt signaling and NC specifiers. NC, neural crest; NNE, non-neural
ectoderm; NT, neural tube.
The canonical Wnt mediator
β
-catenin affects NC survival in mice. Knockout of
β
-catenin by
a Wnt1-driven Cre recombinase causes increased apoptosis in pre-migratory NC cells, suggesting
canonical Wnt signaling is needed for the expansion of NC progenitors in mice [
115
]. However,

Cells 2019,8, 1173 12 of 40
Wnt1-Cre-mediated reporter activity is first detected about 0.5–1 days after NC induction begins [
116
].
Therefore, these results may not be able to reject the role of canonical Wnt signaling in NC induction
in mice.
Dickkopf-related protein 2 (Dkk2) acts as either an antagonist or activator of Wnt signaling
by binding to the Wnt co-receptor Lrp6 [
117
–
119
]. A recent study in Xenopus shows that Dkk2 is
required for neural crest induction [
34
]. Blocking Dkk2 does not affect the expression of NPB specifiers
(Pax3,Sox8, and Snai1) but blocks the formation of neural crest cells by causing a reduction of neural
crest specifiers (Snai2,Twist1, and Sox10) [
34
]. In Dkk2-depleted embryos, overexpression of Lrp6 or
β
-catenin could rescue neural crest formation; however, inhibition of GSK-3
β
could not, indicating
that Dkk2 also activates Wnt/
β
-catenin signaling independently of GSK-3
β
[
34
]. Moreover, Xenopus
Wnt8 induces NC genes in animal cap explants. Dkk2 knockdown significantly blocks the induction of
Snail2 by Wnt8, implying two independent mechanisms by either Wnt8 or Dkk2 to activate
β
-catenin
and induce NC formation [34].
The canonical Wnt signaling is also known to be required for anterior-posterior patterning. It has
been proposed that NC specification by Wnts is an indirect effect of posteriorization activity rather than
a direct effect [
120
,
121
]. In Xenopus, canonical Wnt signaling induces posterior neural tissue through
activation of Fgf signaling [
122
]. Therefore, the posteriorizing activity of Wnt signaling can be blocked
by inhibiting Fgf signaling [
82
]. In contrast, studies in Xenopus show that Wnt proteins could induce
NC specifiers (Snai2,Twist) even if its posteriorizing activity is inhibited, suggesting that NC induction
is a direct consequence of Wnt signaling [82].
Although a requirement for canonical Wnt signaling in NC induction has been thoroughly
demonstrated, the role of non-canonical Wnt signaling in this process remains understudied. In
chicks, Wnt6 is expressed in the ectoderm and controls the induction of NC through Dvl-mediated
non-canonical Wnt signaling [
99
,
100
]. Surprisingly, Schmidt et al. showed that canonical Wnt signaling
(Wnt1) inhibited neural crest formation in the chicken embryo [
100
]. These results may indicate
evolutionary changes in NC induction during vertebrate evolution. The noncanonical Wnt11 pathway
activates the formin family protein Daam1 during NC induction in Xenopus [
38
]. The ability of Daam1
to induce NC formation requires its FH2 domain, which binds to G-actin, suggesting that Daam1
induces NC through actin polymerization in Xenopus [
38
]. However, the role of actin remodeling in
NC specification has yet to be defined.
3.3. Crosstalk of Wnt Signaling with Upstream Modulators in NC Induction
Many signals control NC induction by regulating canonical Wnt signaling. In zebrafish, regulator
of G protein signaling 2 (Rgs2) has been shown to negatively regulate Wnt signaling (Wnt1,Wnt8a) [
93
].
Disruption of Rgs2 expression in zebrafish caused upregulation of many Wnt target genes (Nfatc2a/b,
Nfatc3a/b,Nfatc4,Tcf7l2,Axin2,Tcf7,Ppar
δ
(Pparda/b),Ccnd1,Myca/b, and Tp53) and induced neural
crest specifiers (Sox10,snail1b) [
93
]. Among these genes, the expression of Pparda was upregulated at
the neural crest progenitor stage. Ppar
δ
could bind to the promoter of Sox10 directly [
93
]. Therefore,
Rgs2-Wnt1/8a–Pparδ–Sox10 signaling mediates neural crest development in zebrafish [93].
Adams are multi-domain transmembrane proteins involved in many developmental processes.
Studies have shown that knockdown of two paralogous disintegrin proteases, Adam13 and Adam19
in Xenopus embryos, inhibits Wnt signaling and the expression of NC specifiers (Snai2,Sox9,Foxd3),
but does not affect NPB specifiers (Pax3,Zic1,Msx1) [
76
]. Moreover, ephrin B1(EfnB1) and ephrin B2
(EfnB2) have been determined as substrates for Adam13 [
75
]. EfnB1 and EfnB2 are cell-surface ligands
for EphB receptor tyrosine kinases which act as antagonists of Wnt signaling [
75
,
123
]. Through cleaving
EphB2, Adam13 permits Wnt signaling and induces NC cell formation (Snai2) [
75
]. Unlike Adam13,
the ability of Adam19 to induce NC specification does not depend on its protease activity [
76
]. Using
immunocytochemistry and immunoprecipitation, the authors further show that Adam19 interacts
with Adam13 in the ER and protects Adam13 from ubiquitin-proteasome-mediated degradation in
Xenopus [75,76].

Cells 2019,8, 1173 13 of 40
Cadherin-11 (Cdh11) binds
β
-catenin at cell-cell adhesion complexes. During NC induction in
Xenopus, Cdh11 competes with Wnts for the cytoplasmic
β
-catenin [
81
]. Depletion of cadherin-11
results in increased levels of
β
-catenin in the nucleus, and therefore activates canonical Wnt signaling
and increases NC marker gene expression (Sox10,AP2) [81,124].
Kremen 2 (Krm2) is a transmembrane receptor for Wnt antagonists Dkk proteins, and its expression
is regulated by canonical Wnts (Wnt3a and Wnt8) in Xenopus [
125
]. Krm2 is required for NC induction
in Xenopus, and this function is independent from that of Dkk. Krm2 regulates NC induction by direct
binding to Lrp6 to promote Wnt signaling in Xenopus [90].
Rap2 belongs to the Ras GTPase family. Studies in Xenopus showed that Rap2 physically
binds with Lrp6 and stabilizes it from proteasome and/or lysosome-dependent degradation [
91
].
TRAF2/Nck-interacting kinase (TNIK) is a downstream effector of Rap2 that controls the stability of
Lrp6 [
91
]. Rap2/TNIK kinase pathway plays a critical role in Wnt signaling (Wnt8)-mediated NC
induction in Xenopus [
91
]. Depletion of Rap2 could inhibit NC formation (Snai1, Snai2, and Foxd3) [
91
].
Skip is a transcriptional co-regulator that plays an important role in NC induction in Xenopus [
126
].
Both under- and overexpression of Skip inhibits Wnt/
β
-catenin signaling, therefore blocking NC
induction (Snai2,Sox3, and engrailed2) in Xenopus [
126
]. Upon overexpression, Skip forms a complex
with Lef1 and Hdac1 to repress Wnt target gene expression [
126
]. However, Skip also interacts with
β
-catenin and acts as a scaffold in
β
-catenin/TCF-mediated transcriptional regulation [
126
]. These
results suggest that Skip expression level needs to be properly modified during NC induction in
Xenopus [96].
Finally, inhibiting Wnt signaling in surrounding tissues can sharpen the boundaries between
NC and their neighboring territories. In zebrafish, the potassium channel tetramerization domain
containing 15 (Kctd15) inhibits NC induction (Sox10,Foxd3,Dlx3b,Sox9b,Tfap2a,Snai1b) by antagonizing
Wnt3a signaling and inhibiting the transcription factor Tfap2a, thereby restricting lateral expansion
of the neural crest beyond its domain [
89
,
127
–
129
]. Hes3 is a member of the Hes family of basic
helix-loop-helix transcriptional repressors and is expressed at the boundary of the neural plate [
35
].
Overexpression of Hes3 blocks Snai2 and Sox10 induction by Wnt8 in Xenopus, suggesting that Hes3
establishes the NP/NC boundary by blocking the mesoderm-derived Wnt signals [
35
]. Tcf7l1 is a
transcriptional repressor of canonical Wnt target genes [
130
]. In mice, Tcf7l1 is expressed in the anterior
neural fold region during neurulation and is required for forebrain development [
67
]. Conditional
inactivation of Tcf7l1 using AP2
α
-Cre results in anterior expansion of NC cells, suggesting Tcf7l1
defines the anterior boundary between NC and forebrain by inhibiting canonical Wnt signaling [
67
].
In both mice and Xenopus, the Wnt antagonist Dkk1 is secreted by the prechordal mesoderm to inhibit
NC formation and prevents the anterior neural fold from transforming into NC [68].
4. Wnt Signaling and Crosstalk with Other Signaling Pathways in NC Delamination and EMT
After induction and specification, NC cells emigrate from the dorsal neural tube by undergoing
EMT. Although all NC cells undergo EMT and become migratory, differences between delamination
of cranial NC cells and trunk NC cells exist [
131
]. For example, cranial NC cells delaminate from
the neural tube together in mice and Xenopus [
132
–
134
]. However, trunk NC cells undergo EMT
individually. Moreover, in chicks, the delamination and EMT of cranial NC cells are irrespective of
the cell cycle, but almost all trunk NC cells delaminate in S phase [
135
]. Despite these axial level
differences, Wnt signaling participates in the regulation of delamination for both cranial and trunk NC
cells [
136
,
137
] (Figure 4; Table 2). In order to emigrate from the neural tube, NC cells need to firstly,
have an appropriate substratum for migration; secondly, lose intercellular adhesion; and thirdly, obtain
migratory ability [
138
]. Wnt interacts with Bmp, Fgf, retinoic acid (RA), and Yes-associated-protein
(YAP) signaling in response to the extracellular microenvironment [
92
,
139
]. If the microenvironment is
suited for NC migration, such as the segmental plate mesoderm, canonical Wnt signaling induces G1/S
transition and prepares NC cells for EMT in the chicken embryo [
92
,
137
,
139
]. Several essential signaling
molecules have been identified which regulate NC intercellular adhesion and motility, including the

Cells 2019,8, 1173 14 of 40
Rho family of small GTPases [
140
], cadherins [
141
], and the non-canonical Wnt/planar cell polarity
(PCP) signaling [
37
]. The role of Wnt/PCP signaling in controlling polarized NC cells during migration
will be discussed later. Here, we will focus on Wnt interactions with different signaling pathways to
regulate NC delamination.
Cells 2019, 8, x 1 of 39
Figure 4. Bmp/Wnt signaling regulates trunk neural crest cell delamination. Undefined factors from
somites inhibit Noggin (purple) expression anteriorly, creating a gradient Bmp (green) activity with a
high level in anterior and low level in posterior of the dorsal neural tube. Bmp4, Yap, and RA signaling
induce canonical Wnt1 expression, leading to Cyclin D1 transcription and G1/S transition to promote
NC cell emigration. However, at the segmental plate mesoderm at the posterior region, Fgf signaling
maintains high levels of Noggin that inhibits Bmp activity. Low Bmp activity blocks Wnt signaling,
which prevents NC cell delamination from the caudal neural tube.
4.1. Wnt Signaling and Crosstalk in Trunk NC Delamination and EMT
Canonical Wnt signaling is required for the delamination of trunk NC cells, and its disruption
blocks NC emigration in chicks [138,147]. Rabconnectin-3a (Rbc3a) is a v-ATPase-interacting protein
expressed in pre-migratory NC cells of zebrafish and regulates Wnt signaling by controlling
intracellular trafficking of Fzd7, while its depletion disrupts Wnt signaling and blocks NC cell
emigration in zebrafish [148]. In chicken embryos, the signals from developing somites inhibit Noggin
expression in the dorsal neural tube, resulting in high Bmp activity [138]. Bmp4, in turn, up-regulates
Figure 4.
Bmp/Wnt signaling regulates trunk neural crest cell delamination. Undefined factors from
somites inhibit Noggin (purple) expression anteriorly, creating a gradient Bmp (green) activity with a
high level in anterior and low level in posterior of the dorsal neural tube. Bmp4, Yap, and RA signaling
induce canonical Wnt1 expression, leading to Cyclin D1 transcription and G1/S transition to promote
NC cell emigration. However, at the segmental plate mesoderm at the posterior region, Fgf signaling
maintains high levels of Noggin that inhibits Bmp activity. Low Bmp activity blocks Wnt signaling,
which prevents NC cell delamination from the caudal neural tube.

Cells 2019,8, 1173 15 of 40
Table 2.
Experimental findings of Wnt signaling molecules, modulators, and effectors in vertebrate neural crest delamination and migration. (cKO, conditional
knockout; EMT, epithelial-mesenchymal transition; GOF, gain of function; KO, knockout; LOF, loss of function; MO; morpholino).
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
ADAM13 regulated by Gsk3 and
Plk MO LOF cranial Xenopus inhibited NC migration [142]
Bmp4 stimulate Wnt1
expression BMP4-coated microbeads GOF trunk chick, quail promoted G1/S transition
and NC delamination [137]
Cdh2
cleaved product CTF2
induces Ctnnb1
expression
expression vectors GOF trunk quail enhanced NC delamination [143]
KO LOF cardiac mouse elevated NC proliferation
and reduced NC migration [144]
Cnn2
downstream PCP
signaling, actin
dynamics
MO LOF cranial chick, Xenopus
inhibited NC migration and
reduced cartilage [145]
Ctnnb1 coactivator for Tcf/Lef1
transcription factor overexpression GOF trunk chick
rescued NC delamination in
Noggin-treated neural tubes
[143]
Dact1
repress Ctnnb1 as the
transcriptional
coactivator
MO, expression vectors LOF, GOF cranial Xenopus blocked (LOF) or enhanced
(GOF) NC delamination [146]
Dact2
repress Ctnnb1 as the
transcriptional
coactivator
RNAi, expression vectors LOF, GOF truck chick blocked (LOF) or enhanced
(GOF) NC delamination [146]
Dmxl2 (Rbc3a)
regulate Fzd7
endocytosis and
enhance Wnt signaling
MO LOF
cranial, trunk
zebrafish
defective NC migration,
cardiac edema, reduced
melanocytes
[147]
Draxin
repress Wnt signaling
via Lrp5, modulate
laminin
MO, CRISPR; expression
vectors LOF, GOF cranial chick
premature NC delamination
(LOF), inhibited EMT (GOF)
[136,148]
Dvl (Dsh) PCP signaling PCP mutants (Dsh-DN,
Dsh-DEP+)LOF cranial Xenopus repressed NC migration [37]
Efhc1 (Efhc1b) downregulate Wnt8a MO LOF cranial Xenopus upregulated Wnt signaling
and defective NC migration
[149]

Cells 2019,8, 1173 16 of 40
Table 2. Cont.
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
Fgf8/4 inhibit Wnt1 expression Fgf8/4-soaked beads GOF trunk chick
repressed NC emigration or
delamination [92]
Fgfr1 inhibit Wnt1 expression dominant-negative,
inhibitor LOF trunk chick premature NC emigration [92]
Gsk3 phosphorylation and
degradation of Ctnnb1 Gsk3 inhibitors LiCl, BIO Wnt GOF
trunk, cranial
chick, Xenopus inhibited NC delamination
and migration [150,151]
Lef1 transcription factor,
canonical pathway inducible Lef1-GR GOF cranial Xenopus repressed NC migration [151]
Lrp5 co-receptor MO, CRISPR LOF cranial zebrafish
defective NC migration,
cranial skeleton
malformation
[152]
Musk
downstream of
non-canonical
Wnt11r-Dsh signaling
transgenic fish, KO mouse
LOF trunk zebrafish, mouse defective segmental NC
migration [153]
Noggin inhibit Wnt1 expression CHO-Noggin cells LOF trunk Chick inhibited G1/S transition
and NC delamination [143]
Ovo1
Wnt target, regulate
intracellular trafficking
of Cdh2
MO LOF cranial zebrafish
defective migration of
NC-derived pigment
precursors
[154]
Pes1 (Pescadillo) downstream of
Wnt4/Fzd3 MO LOF cranial Xenopus
increased apoptosis;
defective NC migration, eye
and craniofacial cartilage
[155]
Ptk7
co-receptor, interact
with Ror2 in Wnt/PCP
signaling
MO LOF cranial Xenopus defective NC migration [156]
required for
Fzd7-mediated Dsh
localization
MO LOF cranial Xenopus defective NC migration [157]
Rara regulate Wnt1/Wnt3a
expression
dominant negative/active
RA receptors LOF, GOF trunk chick
diminished (LOF) or
enhanced (GOF) NC
emigration
[92]

Cells 2019,8, 1173 17 of 40
Table 2. Cont.
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
RhoA, RhoB regulated by Wnt6 [100]dominant-negative,
inhibitor, activator LOF, GOF trunk chick
enhanced (LOF) or
repressed (GOF) NC
EMT/delamination
[158]
RhoU activated by Wnt1 [159]mutant construct, MO,
RNA injection LOF, GOF cranial chick, Xenopus
blocked (LOF) or impaired
NC migration, reduced
cartilages
[160]
Ror2
co-receptor, interact
with Ptk in Wnt/PCP
signaling
expression vector GOF cranial Xenopus rescued migration defect in
Ptk7-deficient NC cells [156]
Sfrp (Fz4-v1) secreted splice variant
of fz4 receptor MO, mRNA injection LOF, GOF
cranial, trunk
Xenopus
defective NC migration
(LOF), altered Wnt
signaling
[161]
Sox9 phosphorylated by
Wnt1 and Bmp signaling
MO; point mutations LOF trunk chick failed to initiate NC
delamination [162]
Syn4
(Syndecan4)
interact with
Wnt5/Dvl/PCP signaling
MO LOF trunk Xenopus, zebrafish
diminished NC migration,
reduced cartilage and
melanocytes
[163]
Tcf7l1 (Tcf3) transcription factor,
canonical pathway
inducible Tcf3-VP16-GR,
Tcf3∆C-GR GOF, LOF cranial Xenopus impaired NC migration [151]
Vgll3 induce Wnt5a and
Wnt8b expression MO, mRNA injection LOF, GOF cranial Xenopus
impaired NC migration,
trigeminal and profundal
placodes
[164]
Wnt1 ligand, canonical
pathway Wnt1 producing cells GOF trunk chick inhibited NC delamination
and migration [150]
Wnt1 DNA
electroporation GOF trunk chick enhanced NC delamination [143]

Cells 2019,8, 1173 18 of 40
Table 2. Cont.
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
Wnt3a ligand, canonical
pathway
Wnt3a cells, melanoma
cells GOF trunk chick, human cell
line
enhanced EMT and NC
migration [165]
Wnt5 ligand, PCP signaling MO LOF trunk Xenopus defective NC migration [163]
Wnt11 ligand, PCP signaling expression vectors LOF, GOF cranial Xenopus inhibited NC migration [37]
Wnt11r ligand MO, mRNA injection LOF, GOF cranial Xenopus repressed or rescued NC
migrating [166]
Yap (Yap1)
bidirectional crosstalk
with Wnt and Bmp
signaling
expression vectors,
mutants, siRNA GOF, LOF trunk chick, quail
stimulated (GOF) or
inhibited (LOF) NC EMT
and emigration
[139]

Cells 2019,8, 1173 19 of 40
4.1. Wnt Signaling and Crosstalk in Trunk NC Delamination and EMT
Canonical Wnt signaling is required for the delamination of trunk NC cells, and its disruption
blocks NC emigration in chicks [
137
,
146
]. Rabconnectin-3a (Rbc3a) is a v-ATPase-interacting protein
expressed in pre-migratory NC cells of zebrafish and regulates Wnt signaling by controlling intracellular
trafficking of Fzd7, while its depletion disrupts Wnt signaling and blocks NC cell emigration in
zebrafish [
147
]. In chicken embryos, the signals from developing somites inhibit Noggin expression in
the dorsal neural tube, resulting in high Bmp activity [
137
]. Bmp4, in turn, up-regulates its downstream
target gene, Wnt1 in chicks [
137
]. Through the canonical Wnt signaling pathway, Wnt1 positively
regulates transcription of cyclin D1 and G1/S transition of NC cells in chicks [
137
]. Since most trunk
NC cells delaminate during the S phase in chicks, Wnt-mediated Bmp-dependent G1/S transition is
required for NC cell delamination at the rostral segmental plate [
135
]. However, the segmental plate
mesoderm expresses a high level of Noggin, which inhibits Bmp activity and expression of Wnt1 and
cyclin D1, blocking the emigration of NC cells from the caudal neural tube [
137
]. In chicks, Bmp/Wnt
signaling also induces Sox9 phosphorylation. Phosphorylated Sox9 can be SUMOylated
in vivo
to
facilitate interaction with Snai2 to trigger NC delamination [162].
N-cadherin belongs to the Ca
2+
-dependent cell adhesion family. It contains an intracellular
β
-catenin-binding domain, a transmembrane, and five extracellular cadherin-binding domains [
167
,
168
].
In quails, overexpression of N-cadherin inhibits NC delamination by reducing G1/S transition [
143
].
The cleaved
β
-catenin-binding cytoplasmic tail of N-cadherin, C-terminal fragment 2 (CTF2), could
induce
β
-catenin and cyclin D1 transcription and therefore stimulate NC delamination [
143
]. Bmp4
regulates the cleavage of N-cadherin through Adam10. Adam10 cleaves N-cadherin into CTF1, which
will be further cleaved by γ-secretase to form soluble CTF2 [143].
Rho signaling has also been shown to regulate NC delamination through Rock activity. In
chicken embryos, RhoA and RhoB are expressed in the dorsal neural tube at stages corresponding
to the delamination of NC cells [
158
]. Rho/Rock signaling is regulated by Bmp/Noggin and active
at the membrane of NC cells before they undergo EMT [
158
]. The Rho/Rock signaling stabilizes
N-cadherin at the cell membrane, inhibiting NC EMT. Upon delamination, Rho/Rock activities are
downregulated, causing a loss of stress fibers and decreased N-cadherin-mediated adhesion [
158
].
Altogether, these studies suggest that Bmp/Wnt signaling regulates NC delamination through four
separate yet converging pathways.
Other than Bmp, Wnt also interacts with YAP, Fgf, and RA signaling to regulate NC EMT. Fgf
signaling blocks NC cell emigration by inhibiting the expression of Wnt1 in premature chicken NC
cells [
92
]. Fgf signaling also maintains the expression of Noggin in the caudal neural tube, whereas
RA signaling induces the expression of Wnt1 in the dorsal neural tube and controls the initiation
of NC cell emigration. Therefore, Fgf and RA signaling play opposing roles to control the timing
of NC EMT [
92
]. The Hippo signaling pathway controls many aspects of developmental processes,
such as cell proliferation and survival. YAP, the main effector of Hippo signaling, interacts with a
transcriptional co-activator called TAZ to regulate gene expression in response to specific molecular
and mechanical signals from the microenvironment [
169
]. In chicken embryos, YAP regulates Bmp
and Wnt signaling and stimulates G1/S transition, survival, and delamination of pre-migratory NC
cells [139].
Although canonical Wnt signaling has been suggested to be required for NC delamination, other
studies show that emigration of neural crest cells needs transient Wnt inhibition. Tracing the expression
of a Wnt-responsive reporter in chicken embryos show that pre-migratory NC cells exhibit endogenous
canonical Wnt activity. However, this activity will be transiently inhibited during chicken trunk NC
and Xenopus cranial NC delamination [
146
]. Activation of the small intracellular scaffold proteins
Dact1/2 is required for the inhibition of Wnt/
β
-catenin signaling for NC delamination [
146
]. Dact1/2
blocks canonical Wnt signaling by binding to
β
-catenin and accumulating it in nuclear bodies to
prevent it. Therefore, Dact1/2 prevents
β
-catenin from interaction with TCF transcriptional co-activator
and inhibits Wnt signaling [
146
]. Dact2 is expressed in the trunk NC in chicken embryos, whereas

Cells 2019,8, 1173 20 of 40
pre-migratory NC expresses Dact1 in Xenopus embryos [
170
]. A chicken neural tube explant assay
showed that knockout of Dact1/2does not influence the motility of NC cells, but forces their release
from the dorsal neural tube. These results seem conflicting but may indicate that Wnt signaling plays
complicated roles during NC EMT. For example, NC cells must lose intercellular adhesion with neural
epithelium and increase motility to delaminate, and Wnt signaling regulates cadherins involved in this
adhesion [140].
In chicken NC, Wnt3a increases expression of cadherins Cdh7 and Cdh11, which further accumulate
at cell-cell interfaces [
141
]. In zebrafish, Rbc3a-deficient NC cells also display increased Cdh11 expression
levels during EMT [
147
]. Overexpression of Cdh11 prevents NC migration, suggesting a requirement
for Wnt inhibition for NC cells to lose intercellular adhesion and undergo EMT [
124
]. Additionally,
as discussed before, Rho/Rock activity is repressed during EMT. However, activation of Rho by Wnt
signaling has been suggested during Xenopus gastrulation [
171
]. Therefore, the role of canonical
Wnt/β-catenin signaling in trunk NC delamination remains unclear.
4.2. Wnt Signaling and Crosstalk in Cranial NC Delamination and EMT
Unlike trunk NC, canonical Wnt signaling is repressed during delamination of cranial NC.
Draxin, which is temporarily expressed in pre-migratory cranial NC cells before EMT, interacts with
Lrp extracellularly, and inhibits canonical Wnt signaling in chicken embryos [
136
]. Draxin activity
causes a decrease in Snai2 expression and an increase in Cad6b expression to block the emigration of
pre-migratory cranial NC cells. However, when cranial NC cells become migratory during EMT, Draxin
is downregulated and canonical Wnt signaling is activated, which represses Cad6b, and cranial NC
can delaminate from the neural tube. Therefore, Draxin controls the timing of cranial NC EMT [
136
].
In 1982, Newgreen and Gibbins suggested that physical barriers, such as the basal lamina, must be
lost for NC cells to emigrate from the neural tube, but it was not experimentally confirmed for many
years [
138
]. A recent study in chicken embryos finally found that during cranial NC EMT, the basement
membrane protein laminin continually undergoes remodeling [
148
]. When pre-migratory NC cells are
induced and localized at the dorsal neural tube, the basement membrane will form a space between
the non-neural ectoderm and neural tube, which is termed “Regression” [
148
]. As cranial NC cells
begin to delaminate, the basement membrane expands at the junction of the neural tube and ectoderm
to encapsulate NC and creates a physical barrier that blocks NC delamination. This stage is called
“Expansion” [
148
]. Finally, the lateral laminin barrier disappears and a laminin-lined “channel” forms,
and cranial NC cells complete EMT and migrate through the “channel.” Draxin is involved in the
channel formation [
148
]. Draxin knockdown inhibits basement membrane remodeling during the
regression stage, and ectopic introduction of Draxin inhibits the dissolution of the lateral laminin
barrier [
148
]. Snai2 has been shown to rescue the formation defects of laminin channel from Draxin
overexpression, suggesting that Wnt/
β
-catenin signaling is required for the formation of laminin
channels and NC EMT at least in chicks [148].
5. Wnt Signaling in NC Migration
After induction and delamination, NC cells migrate long distances to contribute to the development
of various tissues [
172
]. A common feature of all migrating NC cells is that they organize into discrete
streams [
54
,
173
]. During migration, NC cells are influenced by a large number of activating and
inhibitory signals [
131
,
173
]. Therefore, the direction of NC migration is regulated by many different
signals present in the local environment, including chemotactic signaling, cell-cell interactions, and the
extracellular matrix [131,172–174].
NC cells need to be polarized to migrate directionally (Figure 5). Protrusions, such as filopodia
and lamellipodia, are formed at the leading edge of migrating cells, while a retraction region is usually
formed at the trailing edge [
54
]. Rho family GTPases have been shown to regulate this directional
polarity by controlling the polymerization of actin at the leading edge [
57
]. Rac proteins regulate
actin polymerization by activating actin nucleating proteins, such as the Arp2/3 complex [
175
]. Rho

Cells 2019,8, 1173 21 of 40
proteins activate Rock (Rho-associated kinase) and induce stress fibers [
57
]. During NC cell migration,
environmental signals control the polarized localization of Rho GTPases and regulate the direction
of NC cell migration. However, chemoattractants have been shown to be unable to establish the
directionality of NC cell movement due to lack of
in vivo
chemoattractant gradients through the long
migratory routes [
172
,
176
]. Therefore, movement directionality is more likely controlled by local signals.
Cell-cell interactions such as contact inhibition of locomotion (CIL) have been shown to directly affect
the localization of Rho GTPases and NC cell polarity [
54
,
172
]. CIL is a process by which a cell paralyzes
protrusions in response to a collision with another cell to cease migration in that direction [
177
]. NC
cells exhibit CIL both
in vitro
and
in vivo
, and physical contact between two NC cells inhibits their
protrusions, leading to movement in opposite directions after collision [
54
,
172
,
173
,
176
]. At high density,
only cells adjacent to a free region may migrate away from the cluster. Moreover,
in vivo
, CIL can
cause the directional migration of NC cells since not all areas are available for migration [
54
,
172
,
173
].
Non-canonical Wnt signaling has been proposed to link CIL with the asymmetric distribution of Rho
GTPases during NC migration [54].
Cells 2019, 8, x 4 of 39
the cluster. Moreover, in vivo, CIL can cause the directional migration of NC cells since not all areas
are available for migration [54,173,174]. Non-canonical Wnt signaling has been proposed to link CIL
with the asymmetric distribution of Rho GTPases during NC migration [54].
Figure 5. The role of Wnt signaling in CIL (contact inhibition of locomotion)-mediated directional
migration of neural crest cells. Cell-cell interaction between NC cells localizes and activates Dsh (Dvl)
at the cell membrane of the contact point, activating the small GTPase RhoA. The activation of RhoA
is at least partly regulated by non-canonical Wnt signaling. RhoA inhibits Rac activity at the trailing
edge of the cell, restricting a maximal Rac activation at the leading edge (green). Rac stimulates
branched actin polymerization and drives the directed migration of NC cells.
5.1. Non-Canonical Wnt Signaling in NC Migration
Many studies have revealed the role of the non-canonical Wnt planar cell polarity (PCP)
signaling pathway as the primary source of signals that direct NC cell migration (Figure 5; Table 2).
Different factors involved in PCP signaling localize at the cell contact points during CIL. One example
is the translocation of Dvl from cytoplasm to membrane during NC migration [179]. Different
mechanisms control the localization of Dvl. In Xenopus, two non-canonical Wnt ligands, Wnt11 and
Wnt11r, are required for cranial NC migration as extracellular signals. Before migration, Wnt11r, a
Xenopus homolog of the mammalian Wnt11 gene, is expressed medially while Wnt11 is expressed
lateral to the first migrating NC cells, which express the Wnt receptor Fzd7 [37,167]. Disruption of
Wnt11/Fzd7 signaling causes NC cells to generate fewer cell protrusions and lamellipodia at the
Figure 5.
The role of Wnt signaling in CIL (contact inhibition of locomotion)-mediated directional
migration of neural crest cells. Cell-cell interaction between NC cells localizes and activates Dsh (Dvl)
at the cell membrane of the contact point, activating the small GTPase RhoA. The activation of RhoA is
at least partly regulated by non-canonical Wnt signaling. RhoA inhibits Rac activity at the trailing edge
of the cell, restricting a maximal Rac activation at the leading edge (green). Rac stimulates branched
actin polymerization and drives the directed migration of NC cells.

Cells 2019,8, 1173 22 of 40
5.1. Non-Canonical Wnt Signaling in NC Migration
Many studies have revealed the role of the non-canonical Wnt planar cell polarity (PCP) signaling
pathway as the primary source of signals that direct NC cell migration (Figure 5; Table 2). Different
factors involved in PCP signaling localize at the cell contact points during CIL. One example is the
translocation of Dvl from cytoplasm to membrane during NC migration [
178
]. Different mechanisms
control the localization of Dvl. In Xenopus, two non-canonical Wnt ligands, Wnt11 and Wnt11r,
are required for cranial NC migration as extracellular signals. Before migration, Wnt11r, a Xenopus
homolog of the mammalian Wnt11 gene, is expressed medially while Wnt11 is expressed lateral to
the first migrating NC cells, which express the Wnt receptor Fzd7 [
37
,
166
]. Disruption of Wnt11/Fzd7
signaling causes NC cells to generate fewer cell protrusions and lamellipodia at the leading edge during
migration, suggesting Wnt11 controls NC migration through the PCP signaling pathway [
37
]. On the
other hand, blocking Wnt11r causes cells to lose contact-mediated inhibition, suggesting that Wnt11r
may act as a repellent signal that causes cranial NC cells to move away from the neural plate [
166
].
Moreover, Wnt11 or Wnt11r, Fzd7, and Dvl also accumulate at the contact site when migrating NC cells
collide, suggesting a significant role of Wnt/PCP signaling in CIL and NC migration directionality [
179
].
Another protein that is essential for recruiting Dvl to the cell membrane is protein tyrosine kinase
7 (Ptk7). Ptk7 is a transmembrane pseudokinase which regulates the Wnt/PCP signaling pathway [
180
].
Xenopus Ptk7 is expressed in migratory NC cells and interacts with Ror2 through its extracellular
domain [
156
]. Knockdown of Ptk7 inhibits the motility of NC cells and results in a rounded rather
than a disperse shape
in vitro
, while the kinase activity of Ror2 can rescue the Ptk7 loss of function
phenotype [
156
]. These results suggest that the Ptk7/Ror2 complex regulates non-canonical Wnt
signaling and NC migration. In Xenopus, Ptk7 forms a complex with Fzd7 and Dvl, suggesting Ptk7
is also required for Fzd7-mediated recruitment of Dvl to the cell membrane. Knockdown of Ptk7
inhibits cranial NC migration, suggesting Ptk7 intersects with the PCP signaling pathway through Dvl
localization to regulate NC mobility [
157
]. The accumulation of Dvl at the cell membrane leads to the
localized activation of RhoA in Xenopus [
179
]. Activated RhoA antagonizes Rac, leading the retraction
of protrusions at cell-cell contacts at the trailing edge of a migrating NC cell [57,175,179].
After cell-cell contacts cause the localized activation of Rho GTPases, Wnt/PCP signaling (Wnt11)
activates the actin-binding protein calponin 2 (Cnn2) in both chicks and Xenopus [
145
]. In pre-migratory
NC cells, Cnn2 is phosphorylated by RhoA/Rock signaling, leading to its degradation. However, in
the early migratory NC cells, RhoA activity restricts Cnn2 to the leading edge of migrating NC cells,
which causes the formation of a polarized cortical actin network [145].
Non-canonical Wnt signaling also activates the atypical RhoU GTPase during cranial NC cell
migration [
159
,
160
]. The level of RhoU activity is essential for NC cells to form polarity and generate
adhesive structures [
160
]. Explants from RhoU-depleted Xenopus embryos show a rounded phenotype
and reduced adhesion to the substrate [160]. Overall, these experiments suggest that RhoU regulates
the direction of cranial NC migration in Xenopus. Further studies show that RhoU controls cranial NC
migration by regulating polarized cell adhesion [
160
]. Many effectors have been identified for RhoU
such as PAKs (P21 activated kinases), including Pak1 and Pak2, which are known to participate in cell
adhesion and motility [
160
,
181
]. PAK activates Rac1 and induces the formation of lamellipodia [
175
,
182
].
Therefore, RhoU regulates NC migration through the Pak1–Rac1 signaling pathway. The non-canonical
Wnt4 also induces the expression of Pescadillo (Pes1) in Xenopus [
155
]. Pescadillo is a nuclear protein
involved in ribosomal biogenesis and gene transcription [
183
,
184
]. Pescadillo knockdown blocks
cranial NC migration and also triggers p53-mediated apoptosis, suggesting dual roles of Pescadillo in
NC migration and survival [155].
Moreover, non-canonical Wnt signaling modulates the extracellular matrix to regulate the direction
of NC migration in Xenopus and zebrafish. In migrating NC cells, the proteoglycan Syndecan-4 (Syn4)
is activated by fibronectin. Activated Syn4 directly inhibits Rac activity [
163
]. Syn4 also interacts with
the Wnt/PCP pathway through Dvl to inhibit Rac at the trailing edge of migrating NC cells, resulting
in the formation of cell protrusions such as filopodia and lamellipodia at the leading edge [
163
]. Since

Cells 2019,8, 1173 23 of 40
persistent migration depends on the orientation of cell protrusions, Wnt/PCP and Syn4 signaling
control the direction of NC migration.
Trunk NC cells migrate along three different pathways: a dorsolateral pathway [
185
,
186
],
a ventro-lateral pathway [
187
], and a ventro-medial pathway [
185
,
186
]. In the ventro-lateral
pathway and the ventro-medial pathway, the continuous migration sheet of NC cells rearranges
into narrow, restricted streams. In zebrafish, the initiation of this segmentation is triggered by
Nrp2/Sema3F signaling [
153
]. However, maintenance of the segmental migration is regulated by
Wnt/PCP signaling [
153
]. The zebrafish Wnt11r binds to the surface of the adaxial muscle cells at
the central portion of the somite. Wnt11r-Dvl signaling activates the expression of a muscle-specific
receptor kinase (MuSK). MuSK signaling possibly modifies the extracellular matrix at the central
region of the somite [
153
]. High-resolution imaging finds that NC cells interact with the extracellular
microenvironment using their filopodia and guide the direction of their migration. Therefore,
Wnt11r-MuSK signaling keeps NC cells into restricted streams in zebrafish embryos [153].
5.2. Canonical Wnt Signaling in NC Migration
The role of canonical Wnt signaling in controlling NC migration is less well understood. In
Xenopus, canonical Wnt activity is decreased in migrating NC cells, while its activation inhibits NC
migration [
151
]. Moreover, inhibition of Efhc1, a ciliary component, upregulates Wnt8, leading to
defective NC migration in Xenopus [
149
]. However,
in vitro
studies show that although
β
-catenin is
localized at the intercellular contact regions and associates with N-cadherin in most migrating NC
cells, early migrating cells have
β
-catenin in their nuclei, suggesting that canonical Wnt signaling may
be only transiently required in early migrating NC cells [
150
]. Recent studies in Xenopus also show that
canonical Wnt signaling is activated in the cranial NC cells shortly after delamination, which drives
the re-expression of Snai2 in early migratory NC cells [
188
]. In zebrafish, Lrp5 is required for cranial
NC migration. Knockdown of Lrp5 leads to disrupted migration of cranial NC cells in the branchial
stream [
152
]. In chicks, Wnt3a enhances NC migration along the distal medial pathway [
165
].
In vitro
studies show that Wnt3a induces the expression of Cdh11 [
141
]. Cdh11 is cleaved by Adam9 and
Adam13 to generate a shed extracellular fragment of Cdh11 (EC1-3). EC1-3 promotes Xenopus cranial
NC migration through an unknown mechanism [
189
,
190
]. Interestingly, other than cleavage of Cdh11,
Adam13 also regulates cranial NC cell migration by controlling the expression of many genes through
its cytoplasmic domain (C13), such as the protease calpain 8 [
142
,
191
]. Gsk3 and Polo-like kinase (Plk)
regulate the activity of Adam13 by successive phosphorylation of its C13 domain at two sites (first at
S752 and S768 by Gsk3, second by Plk at T833). The phosphorylation of Adam13 is critical for cranial
NC migration [
142
]. In Xenopus,Fzd4 mRNA can be alternatively spliced to generate a secreted Frizzled
related protein (Sfrp) Fz4-v1 [
192
]. Fz4-v1 regulates canonical Wnt signaling in a non-autonomous
manner [
161
]. Knocking down Fz4-v1 blocks NC cell migration [
161
]. The vestigial-like 3 (Vgll3)
protein is expressed at hindbrain rhombomere 2 to activate the expression of Wnt5a and Wnt8b in
Xenopus [
164
]. Depletion of Vgll3 causes a down-regulation of myosin X that is essential for cell-cell
adhesion [
193
]. These results suggest that Vgll3 regulates NC migration through modification of their
adhesion properties [164].
Canonical Wnt signaling is also involved in establishing transient cell-cell contacts during NC
migration. This process possibly is regulated by localizing N-cadherin to filopodial tips of NC cells,
which has been shown to be regulated by canonical Wnt signaling in zebrafish [
154
]. Wnt signaling
activates the expression of Ovo1, a transcription factor gene [
194
]. Ovo1 inhibits the expression of
several genes involved in intracellular trafficking, such as rab12,rab11fip2,rab3c, and sec6, which
maintain the balance of cytoplasm and membrane-localized N-cadherin in zebrafish [
154
]. In mice,
N-cadherin and
β
-catenin colocalize with the gap junction protein, connexin 43 or
α
1 connexin (Cx43
α
1)
in migrating cardiac NC cells [
144
]. Further studies show that Wnt1 and N-cadherin regulate gap
junction channels in NC cells, suggesting that cadherin-based adherents junction controls NC migration
by modulating gap junction communication [
144
]. An Armadillo protein, p120 catenin (p120ctn),

Cells 2019,8, 1173 24 of 40
links gap junctions to the actin cytoskeleton [
195
]. N-cadherin and Cx43
α
1 interact with p120ctn and
regulate cell motility through the Rho GTPases in mice [144].
6. Wnt in NC Multipotency and Fate Determination
The fact that the NC cells can generate many cell and tissue types makes them represent a
multipotent stem cell population. Several studies have been performed
in vivo
to address the
developmental potential of individual NC cells by retrovirus-mediated gene transfer or dye injection
to label cell lineages [
196
–
198
]. These studies showed that at least some NC cells generate different
cell types
in vivo
, including smooth muscle cells, neurons, glia, and melanocytes [
198
,
199
]. Cultured
NC cells from mouse neural tube explants derived prior to NC cell migration demonstrated their
multipotency
in vitro
[
200
]. After delamination from the neural tube, NC cells can differentiate into
various derivatives. The multipotency of chicken pre-migratory NC cells has also been demonstrated
using a clonal culture system [
201
]. A few studies proposed that pre-migratory NC cells are
fate-determined before delamination in chicks [
202
,
203
]. A recent study employed advanced genetic
fate mapping approaches to convincingly demonstrate that both pre-migratory and migratory trunk
NC cells are multipotent in mice [204].
6.1. Wnt Signaling and Crosstalk in Maintaining NC Multipotency
In addition to demonstrating the stem cell nature of pre-migratory and migratory NC cells,
in vitro
studies have also revealed environmental signals regulating multipotential maintenance in NC
cells [
19
]. Combinatorial canonical Wnt and Bmp proteins sustain persistent expression of the NC
cell markers p75 and Sox10, suppress sensory neurogenesis and maintain multipotency in early NC
stem cells [
205
] (Table 3). Further studies showed that Bmp/Wnt treatment induces the expression
of a chromatin remodeler, chromodomain helicase DNA-binding protein 7 (Chd7), which maintains
the multipotency of NC cells [
27
]. In mice, Chd7 is expressed in the undifferentiated migratory trunk
neural crest cells, the dorsal root ganglia (DRG) and sciatic nerve, which normally contain neural crest
stem cells [
206
–
208
]. Therefore, Bmp/Wnt signaling can maintain multipotency in cultured NC cells
and increase the number of multipotent cells in DRG
in vivo
[
27
]. A more recent study showed that a
high concentration of canonical Wnt ligand at the dorsal neural tube directly induces the expression of
Lin28a in pre-migratory chicken NC cells [
209
]. Lin28a is an RNA-binding protein that binds to let7
pre-microRNA and blocks its maturation into let7 miRNA [
210
]. Therefore, Lin28a protects neural crest
specifiers from inhibition by let7 and maintains the multipotency of pre-migratory NC [
209
]. As NC
cells migrate away from the dorsal neural tube, expression of Lin28a decreases, leading to increased
let7 activation, resulting in a loss of stem cell identity [209].

Cells 2019,8, 1173 25 of 40
Table 3.
Experimental findings of Wnt signaling molecules, modulators, and effectors in vertebrate neural crest proliferation and differentiation. (ca, conditional active;
cKO, conditional knockout; GOF, gain of function; KO, knockout; LOF, loss of function; MO; morpholino).
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
Axin2 scaffold protein for
Ctnnb1 degradation KO LOF cranial mouse
enhanced osteogenic potential
and regeneration of NC-derived
frontal bone
[211]
Bmp2 crosstalk with
Wnt/Ctnnb1 signaling protein to NC culture GOF truck mouse
suppressed sensory neurogenesis
of early NC stem cells [205]
repress Wnt antagonists
Dkk1 and Sost cKO by Wnt1-Cre LOF tooth mouse
early tooth mineralization defects
[212]
Bmp4 repress Wnt antagonists
Dkk2 and Sfrp2 cKO by Wnt1-Cre LOF tooth mouse bud-stage arrest of the
mandibular molar tooth germs [213]
Chd7 chromatin remodeler,
activated by Wnt/Bmp
siRNA, DN, WT
expression LOF, GOF trunk,
DRG mouse
inhibited (LOF) or maintained
(GOF) undifferentiated state
(Sox10, p75) of NCSC
[27]
Ctnnb1 coactivator for Tcf/Lef1
transcription factor mRNA injection GOF cranial zebrafish promoted pigment and cartilage
fates from NC cells [214]
ca by Wnt1-Cre in
premigratory NC cells GOF trunk mouse
suppressed melanocyte (Dct,
Mitf) differentiation from
premigratory NC cells
[39]
ca by Sox10-Cre in
migratory NC cells GOF trunk mouse
ectopic melanocytes, inhibited
other lineages from migratory
NC cells
[39]
cKO by Wnt1-cre LOF trunk mouse lack melanocytes and dorsal root
ganglia [40]
ca, cKO by Wnt1-Cre GOF, LOF trunk,
cranial mouse
promoted (GOF) or blocked
(LOF) sensory neurogenesis of
NC stem cells
[215]
cKO by PdgfraCreErt2,
Dermo1Cre LOF cranial mouse forebrain meningeal hypoplasia
derived from NC cells [216]
Wnt1-Cre LOF cranial mouse
defective maintenance of Pitx2
expression in NC cells and
abnormal eyes
[217]

Cells 2019,8, 1173 26 of 40
Table 3. Cont.
Molecule Role in Wnt Signaling Experimental Approach Function Region Species Phenotype Reference
Wnt1-cre LOF cranial mouse
affected NC survival and
differentiation; failure of
craniofacial development
[115]
let-7 miRNA repressed by
Wnt/Lin28a
electroporation of let-7
mimic GOF trunk chick
down-regulation of NC
multipotency, promoted
differentiation
[209]
Lin28a activated by Wnt siRNA; MO; CRISPR LOF trunk chick suppressed NC multipotency
(Sox10, Foxd3) [209]
Msx1 repress Dkk2 and Sfrp2,
interact Bmp4 KO LOF tooth mouse bud-stage arrest of the
mandibular molar tooth germs [213]
Osr2 upregulate Dkk2 and
Sfrp2 expression KO LOF tooth mouse supernumerary teeth [213,218]
Prmt1 inhibit Wnt, Bmp and
other signaling cKO by Wnt1-Cre LOF cranial mouse
decreased mesenchymal
proliferation, cleft palate,
craniofacial anomalies
[219]
Tcf7l1 (Tcf3) transcription factor of
Wnt/Ctnnb1 signaling mutant mRNA injection LOF cranial zebrafish promoted neural fates, repressed
pigment cells [214]
Wnt1 ligand,
Ctnnb1-dependent
Wnt1-expressing
fibroblasts GOF trunk mouse promoted sensory neurogenesis
of early NC stem cells [215]
Wnt1 and
Bmp2 ligands Wnt1 cell and Bmp2
protein GOF truck mouse
repressed neurogenesis and
maintained multipotency of NC
stem cells
[205]
Wnt3a and
Bmp2 ligands proteins to NC culture GOF trunk,
DRG mouse maintained multipotency (Sox10
or p75) of NC stem cells [27]
Wnt
inhibitors stabilize or elevate Axin XAV939, IWR1 LOF cranial Xenopus repressed NC differentiation and
defective cartilage [188]

Cells 2019,8, 1173 27 of 40
6.2. Wnt Signaling in NC Fate Specification and Differentiation
Wnt signaling is also involved in the fate specification and differentiation of NC cells [
7
] (Table 3).
In zebrafish, activation of canonical Wnt signaling in pre-migratory NC cells promotes pigment cell
formation, but inhibition of Wnt signaling promotes neuronal fates [
214
]. However, in mice, activation
of Wnt/
β
-catenin signaling in pre-migratory NC cells promotes a sensory neuronal fate at the expense of
all other NC derivatives [
215
]. Genetic ablation of
β
-catenin in pre-migratory NC cells using Wnt1-cre
suppresses the formation of both melanocytes and dorsal root ganglia [
40
]. These contradictory results
may be due to taxa-specific differentiation mechanisms, but nevertheless suggest that the role of
canonical Wnt signaling in NC lineage specification needs to be addressed further. A recent study
in mice shows that induced activation of
β
-catenin in migratory NC cells using Sox10-cre promotes
the formation of melanoblasts and melanocytes, and the repression of other lineages [
39
]. However,
β
-catenin activation at later stages, such as glial progenitors or melanoblasts, does not produce similar
effects [
39
]. These results imply a narrow time window in which canonical Wnt signaling controls
migratory NC fate determination. It also suggests that NC cells maintain multipotency both before
and shortly after delaminating from the dorsal neural tube.
Wnt signaling also affects NC differentiation at late stages in mice. For example, disruption
of Axin2 enhances canonical Wnt signaling, increasing osteogenic potential and regeneration of
NC-derived frontal bone in adult mice [
211
]. Conditional knockout of Bmp2 in mouse NC cells
increases the expression of Dkk1 in epithelium and reduces Wnt activity, leading to tooth mineralization
defects [
212
]. NC-secreted Bmp4 genetically interacts with Msx1, repressing Osr2-dependent expression
of Wnt antagonists Dkk2 and Sfrp2 in mouse tooth organogenesis [
213
,
218
]. Cranial NC cells generate
cranial mesenchyme, which further develops into the forebrain meningeal progenitors [
220
]. Studies
in mice show that canonical Wnt signaling is required to maintain the meningeal mesenchymal
progenitors [
216
]. In ocular NC cells, canonical Wnt signaling maintains the expression of Pitx2,
which is required for eye development [
217
]. Protein arginine methyltransferase 1 (Prmt1) negatively
regulates canonical Wnt signaling [
221
–
224
]. Disruption of Prmt1 in NC cells causes cleft palate and
craniofacial defects [219].
7. Wnt Signaling in NC-Derived Cancers
NC derivatives, including melanocytes and glial cells, can transform into cancers such as
melanoma, neuroblastoma, and glioblastoma [
225
]. Indeed, WNT signaling and NC regulators are
tightly associated with these types of cancers. Melanoma that develops from the pigment-producing
melanocytes is the severest type of skin cancers. Among all skin cancers, melanoma is the most
malignant and causes about 80% of all skin cancer deaths [
226
]. The development of melanoma often
involves mutations in BRAF, CDKN2A, CCND1, INK4A, and MAPK signaling pathway components,
many of which are also important for the development of neural crest-derived melanocytes [
227
].
Studies using melanoma cell lines discovered mutations in
β
-catenin, suggesting that canonical
Wnt signaling may play an important role in this type of cancers [
228
]. Other studies have also
detected
β
-catenin accumulation in the cytoplasm and nucleus of melanoma cells [
229
,
230
]. Moreover,
expression of WNT proteins, including WNT2, 4, 5A, 7B, and 10B, have been found in melanoma cells
or their local environment [
231
]. However, the sources and spatiotemporal expression patterns of
these WNTs as well as the precise role of canonical signaling in melanoma remains unclear [
232
]. For
example, melanomas with high
β
-catenin levels are correlated with a lower proliferative index [
233
],
suggesting that
β
-catenin may inhibit melanoma. However, other studies showed that high
β
-catenin
level correlates with enhanced melanoma metastasis [234].
Neuroblastoma is the commonest type of cancers in infants with the median age of 17 months at
diagnosis, reflecting the embryonic origin of the disease [
235
–
237
]. It develops from the NC-derived
sympathoadrenal system [
238
,
239
]. WNT signaling is implicated in onset and progression of
neuroblastoma [
240
]. The oncogene MYCN, which is important for NC development, has been found
to be upregulated in neuroblastoma cell lines or patient samples [
241
,
242
]. Xenograft experiments

Cells 2019,8, 1173 28 of 40
showed that the expression level of WNT5A is higher in human IGR-N-91 neuroblastoma cells than
control grafted cells [
243
]. This is the first evidence for WNT signaling playing a role in human primary
neuroblastoma. High WNT5A levels are associated with low-risk neuroblastoma [
243
]. Further
studies showed that the core WNT/PCP signaling components PRICKLE1 and VANGL2 directly
inhibit canonical WNT signaling in neuroblastoma cells [
244
]. High expression level of PRICKLE1 and
VANGL2 correlates with low risk of neuroblastoma [
244
]. In contrast, transcriptome analyses show that
high expression of WNT3A or WNT5A correlates with longer survival, while high WNT3 expression
level indicates higher risk of neuroblastoma [
245
]. These seemingly conflicting results suggest that the
role of WNT signaling in neuroblastoma needs to be further addressed.
WNT signaling is also implicated in tumors of NC-derived endocrine cells, which include
pheochromocytoma and paraganglioma [
239
,
246
]. The WNT signaling components
β
-CATENIN,DVL3,
and GSK3 are overexpressed in the MAML3 fusion-positive subtype of these tumors and CSDE1 somatic
mutation. WNT4,WNT5A, and WNT11 are also overexpressed after truncated MAML3 activation [
246
].
These results suggest that overactivation of both canonical and non-canonical WNT signaling pathways
may play important roles in these tumors.
Glioblastomas form from astrocytes and can occur in either the brain or spinal cord [
247
]. Key
components of the WNT signaling pathway have been shown to be altered in glioblastoma. For example,
CTNNB1, LEF1, TCF7L2, MYC, and CCND1 are not expressed, while APC and GSK3 are downregulated
in glioblastoma [
248
]. Overexpression of WNT5A increases proliferation of glioblastoma cells
in vitro
,
while knockdown inhibits proliferation and tumorigenicity [
249
]. Notably, the NC specifier Sox10 has
been found to be upregulated in human low-grade gliomas and a mouse model of glioma [250].
NC and cancer cells possess similarities in several aspects [
251
,
252
]. During development, NC
cells undergo EMT, emigrate from the dorsal neural tube, migrate through the embryo. This process is
similar to the early stages of metastasis, when cancer cells disseminate from the original location [
227
].
At molecular and cellular level, the similarity of the development of NC cells and metastasis of cancer
cells is more significant. Many signaling pathways, such as the Wnt signaling pathway, are shared by
these two processes. For instance, WNT signaling also plays crucial roles in maintenance of cancer stem
cells and metastasis [
253
,
254
], which is comparable to the roles of Wnt signaling in NC stemness and
migration. Thus, NC cells represent an excellent model to study how developmental processes can be
re-activated and usurped by cancer cells [
255
]. Studying the changes in cell-cell junctions, cell polarity,
signaling, transcription factors, and the role of Wnt signaling at each step during NC development
may provide insightful mechanisms of how cancer processes and hopefully trigger novel ideas for
cancer treatments.
8. Conclusions
Since William His identified NC in 1868 [
73
], great advances have been made in understanding NC
development for one and a half centuries. NC cells represent a group of migratory, multipotent stem cell
population which are unique to vertebrate evolution. Therefore, NC cells serve as an excellent model for
biologists to study the molecular and cellular mechanism of various developmental and evolutionary
processes like morphogenetic induction, cell motility, and fate specification. Environmental cues
and transcription factors have been shown to control the induction, delamination, migration, and
differentiation of NC cells. Wnt signaling is a key driver for most NC developmental processes.
Mutations in Wnt signaling components are involved in many diseases and cancers. However,
important questions remain for the role of Wnt signaling in NC development and disease. For
instance, does Wnt signaling play a role in NC evolution? Does Wnt signaling play a conserved role
in human NC development? What are the roles of epigenetic factors, such as non-coding RNAs and
DNA/RNA/protein methylation in Wnt signaling regulation of NC development? Whether and how
environmental factors modulate Wnt signaling and thus may influence NC development or cause
NC-derived disease and cancer? It is no doubt that studying Wnt signaling in NC development can
lead to a better understanding of diseases, which hopefully will translate into practical therapies.

Cells 2019,8, 1173 29 of 40
Author Contributions:
Conceptualization, Y.J. and C.J.Z.; literature analysis, Y.J.; writing—original draft
preparation, Y.J.; writing—review and editing, H.H., K.R., M.M. and C.J.Z.; writing—revision, Y.J., K.R. and C.J.Z.;
visualization, Y.J., H.H. and C.J.Z.; supervision, C.J.Z.; funding acquisition, C.J.Z.
Funding:
This work is supported by grants from the NIH (R01DE026737, R01NS102261, and R01DE0221696 to
C.J.Z.) and the Shriners Hospitals for Children (85105 and 86600 to C.J.Z.).
Acknowledgments:
We are grateful to Chelsey Lee for proofreading and to the rest of Zhou lab members for their
supports during the manuscript preparation. We apologize to colleagues whose important work we were unable
to cite due to space constraints or inadvertently overlooking.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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