Seminars in Cell & Developmental Biology 16 (2005) 683–693

Review

Specification of neural crest cell formation and

migration in mouse embryos
Paul A. Trainor ∗
Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, USA

Available online 25 July 2005

Abstract

Of all the model organisms used to study human development, rodents such as mice most accurately reflect human craniofacial development.
Collective advances in mouse embryology and mouse genetics continue to shape our understanding of neural crest cell development and by
extrapolation the etiology of human congenital head and facial birth defects. The aim of this review is to highlight the considerable progress
being made in our understanding of cranial neural crest cell patterning in mouse embryos.
© 2005 Elsevier Ltd. All rights reserved.

Keywords: Mouse; Neural crest; Craniofacial; Induction; Migration

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683
2. Mouse neural crest cells: formation, migration, differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684
3. Specification of mouse neural crest cell formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685
4. Specification of mouse neural crest cell migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
5. Specification of multipotency versus restricted potency of mouse neural crest cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691

1. Introduction form very early during craniofacial development and they

generate the majority of the cartilage, bone, connective and
The craniofacial complex is anatomically the most sophis- peripheral nerve tissue in the head (Fig. 1). Not only are
ticated part of the body and to function properly it requires neural crest cells crucial to head development but they are
the orchestrated integration of the viscerocranium and also synonymous with vertebrate craniofacial evolution.
neurocranium, the central and peripheral nervous systems, Craniofacial abnormalities are largely attributed to defects
facial muscles, connective tissue, vasculature and dermis. in the formation, migration and differentiation of neural crest
Not surprisingly this integration often goes awry such that cells and the origins of particular congenital syndromes can
craniofacial abnormalities are the most common congenital be traced back specifically to problems in one or more of these
malformation constituting at least a third of all birth defects. phases of neural crest cell development. For example, First
Neural crest cells, are a migratory stem cell population that Arch syndrome broadly describes craniofacial abnormalities
characterized by malformation of the eyes, ears, lower jaw
∗ Tel.: +1 816 926 4414; fax: +1 816 926 2051. and palate. Treacher Collins syndrome and Pierre Robin syn-
E-mail address: pat@stowers-institute.org. drome are two of the more extreme examples falling into this

1084-9521/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcdb.2005.06.007
684 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) 683–693

Fig. 1. Migration and differentiation of neural crest cells. Sox10 in situ hybridization (purple stain) labels the distinct segregated streams of neural crest
cells as they leave the neural tube of mouse embryos at 8.5 dpc (A) and as they condense to contribute to the cranial ganglia at 9.5 dpc (B). Neurofilament
immunohistochemistry highlights the differentiation of neural crest cells in the cranial ganglia into sensory neurons that project axons into the branchial arches
at 10.5 dpc (C). Neural crest cells also give rise to the majority of the cartilage (blue) and bone (red) that constitute the viscerocranium and neurocranium (D,
sagittal view; E, superior view).

category. The clinical abnormalities associated with Treacher
Collins and Pierre Robin syndromes are thought to arise due
to defects in the migration of neural crest cells. In contrast,
craniosynostoses, which are characterized by the premature
fusion of the bony plates in the skull are related to problems
with neural crest cell differentiation [1–3]. Consequently it is
important to understand the distinct mechanisms, which reg-
ulate the formation, migration and differentiation of neural
crest cells.
2. Mouse neural crest cells: formation, migration,
differentiation
Murine neural crest cells are generated transiently along
almost the entire vertebrate axis at the interface between the
surface ectoderm and the neural plate of the embryo, in a
region that is referred to as the neural plate border. During this
induction process, neuroepithelial cells undergo an epithelial
to mesenchymal transformation at which point they delam-
inate and begin to emigrate from the neural tube, a process
that requires significant cytoarchitectural and cell adhesive
changes. The induction of the neural crest cells is typically
assayed by the expression of members of the Snail (Snail
and Slug) zinc-finger transcription factors gene family [4,5],
which play key roles in the epithelial to mesenchymal trans-
formation process by repressing the cell adhesion molecule
E-cadherin [6]. Snail/Slug transcription factors are among the
earliest known markers of neural crest cell formation and the
onset of their expression has been used to study the spatial
and temporal competence of the neural plate to initiate neural
crest induction in response to different signals.
Murine neural crest cell formation and migration com-
mences at approximately the 4–5 somite stage in the region
of the caudal midbrain and rostral hindbrain [7] and proceeds
simultaneously as a wave rostrally towards the forebrain and
caudally towards the tail (Fig. 1). In avian embryos neu-
ral crest cell migration commences after neural tube closure
however this is not the case in mammalian embryos such as
mice where neural crest cell formation and migration com-
mences well before fusion of the bilateral halves of the neural
plate. Typically there is a narrow temporal window during
which neural crest cells are induced to delaminate and emi-
grate from the dorsal neural tube and although this period
varies between species, in mice it typically lasts mice 7–9 h at
each axial level [8]. The neural crest can be subdivided rostro-
caudally into at least four distinct major axial populations;
cranial, cardiac, vagal and trunk, each of which migrates
along unique pathways, contributing to specific cell and tis-
sue types that are characteristic of their axial level of origin.
The cranial neural crest, which is the focus of this
review can be divided into forebrain, midbrain and hindbrain
domains of migrating neural crest cells. Cranial neural crest
cells do not appear to migrate randomly, rather they follow
precise, species and region specific pathways moving subec-
todermally over the surface of the cranial mesoderm [8–10].
 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) 683–693
Cranial neural crest cells typically migrate in discrete seg-
regated streams, the pattern of which is highly conserved
in vertebrate species as disparate as amphibians, teleosts,
avians, marsupials and mammals (reviewed in [11]). Briefly,
forebrain and rostral midbrain neural crest cells colonise the
frontonasal and periocular regions. Caudal midbrain derived
neural crest cells populate the maxillary component of the
first branchial arch [9,10]. The hindbrain is divided into
seven distinct segments known as rhombomeres [12] and
neural crest cells emigrate from each of the rhombomeres,
but predominantly from rhombomeres 2, 4 and 6 in discrete
segregated streams that populate the first, second and third
branchial arches respectively [9,10].
Exquisite fate mapping analyses particularly in avians
using the quail-chick chimera system, have revealed that neu-
ral crest cells derived from each axial region of the cranial
neural plate and in particular from each individual rhom-
bomere generate specific and unique components of the cran-
iofacial complex including the viscero- and neurocraniums
as well as the peripheral nervous system [13–16]. Despite the
generation of lineage fate maps in mice [9,10], the limitations
of mouse embryo culture prevented a long-term differentia-
tion analysis of the extensive fates of murine neural crest cells
although obvious parallels were drawn with avians.
Recently however, genetic recombination experiments
finally permitted the long-term tracing of murine neural crest
cells. Initially P0-cre mice were used in combination with
chicken ␤-actin conditional reporters, which confirmed much
of what was expected in terms of the fates of mammalian neu-
ral crest cells [17]. However, P0-cre mediated recombination
only occurred in subsets of neural crest cells and exhibited
some ectopic expression leaving many questions in mam-
malian neural crest differentiation unanswered.
Shortly thereafter, a more specific neural crest cell pro-
moter from the Wnt1 gene was used in combination with
ROSA26-lacZ conditional reporter mice to indelibly mark
the progeny of the cranial and cardiac neural crest cells dur-
ing embryonic, fetal and post-natal development [18–20].
Murine cranial neural crest cells contribute to the forma-
tion of the condensed dental mesenchyme, dental papilla,
odontoblasts, dentine matrix, pulp, cementum, periodontal
ligaments, chondrocytes in Meckel’s cartilage, mandible, the
articulating disk of the temporomandibular joint and the
branchial arch nerve ganglia [20]. In the neurocranium, the
meninges and frontal bones are derived from neural crest
cells as is the suture mesenchyme, which separates the bony
plates [18]. In the cardiac region, neural crest cells contribute
to the aorticopulmonary septum, conotruncal cushions, and
adult derivatives of the third, fourth and sixth pharyngeal
arch arteries [19]. Not surprisingly, the overall patterns of
migration and long-term differentiation are highly conserved
between mammalian and avian embryos.
Further refinements to lineage and fate determination in
mammalian embryos in terms of using axial level specific
promoters such as those that delineate a single rhombomere
subpopulation of neural crest cells are no doubt in progress.
685
These results are eagerly anticipated as they will substantially
aid comparative analysis of neural crest cell patterning in
diverse organisms and further our understanding of neural
crest cell and craniofacial evolution.
Collectively these analyses performed in mice together
with those from avians, fish and frogs demonstrate that
cardiac, vagal and trunk neural crest cells produce neurons,
glial cells, secretory cells and pigment cells—contributing
to the peripheral nervous system, enteric nervous system,
endocrine system and skin. Cranial neural crest cells however
exhibit an even more surprising diversity of derivatives,
giving rise to pigment cells, nerve ganglia, smooth muscle,
and connective tissue, as well as most of the bone and
cartilage of the head (Fig. 1).
3. Specification of mouse neural crest cell formation
Neural crest cell formation requires contact mediated sig-
nals between the neural plate and paraxial tissues such as
the surface ectoderm and/or the mesoderm [21]. Three key
signaling pathways (BMP, FGF, Wnt) intersect at the neu-
ral plate border, each of which is critical for neural crest cell
induction. Previously, BMP4/7 signaling in the ectoderm and
neuroepithelium in the form of a morphogen gradient was
considered to be the key factor in inducing neural crest cell
formation [5,21–23]. However, more recently, the emphasis
has shifted in favour of Wnt signaling from the surface and
in chick embryos Wnt6 is key, since it is widely expressed
in domains of epithelial to mesenchymal transitions during
embryogenesis [24]. In contrast in zebrafish, Wnt8 signaling
from the surface ectoderm is the main protagonist of neural
crest cell induction [25]. In Xenopus, although Wnt signal-
ing is also critical for neural crest cell induction, the situation
is more complex since FGF signaling from the underlying
mesoderm also independently induces neural crest cell for-
mation [26–28]. The specifics of neural crest cell induction in
these species are discussed in detail elsewhere in this volume,
however, the contact mediated induction is considered to be a
conserved feature of vertebrate neural crest cell formation as
it has been rigorously tested in chick and frog embryos due to
their amenability to tissue manipulation, recombination and
explant culture.
In the case of mammalian embryos, there is currently a
paucity of data linking these different signaling pathways
in neural crest cell induction mechanisms. Part of the prob-
lem is that many of the Wnt, Bmp and Fgf genes described as
playing roles in neural crest cell induction in non-mammalian
embryos also play key roles during gastrulation and/or neural
plate induction. Therefore, many knockout models of these
genes exhibit embryonic lethality prior to the onset of neu-
ral crest cell formation or no phenotype at this specific stage
due to functional redundancy. For example, genetic ablation
of Wnt1 or Wnt3a in mouse embryos failed to demonstrate
a conserved role for Wnt signaling in murine neural crest
cell induction. Wnt1 was shown to be important for mid-
686 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) 683–693
brain patterning while Wnt3a was required for formation of
the paraxial mesoderm [29,30]. However, in the absence of
both Wnt1 and Wnt3a there is a marked deficiency in neu-
ral crest derivatives. Double mutant mice exhibit skeletal
defects and a significant reduction in cranial and spinal sen-
sory neurons as well as melanocytes. [31]. Further evidence
of a role for Wnt signaling in neural crest cell determination
came from targeted inactivation of downstream components
of the Wnt signaling pathway. Inactivation of ␤-catenin, in the
dorsal neural tube of mouse embryos [32,33] or null muta-
tions in APC [34] result in severe defects in cranial neural
crest derivatives including the cranial and dorsal root gan-
glia and the craniofacial skeletal elements. To date there is
no literature available describing a null mutation in Wnt6.
This implies that Wnt signaling in mouse embryos appears
to be more important for the lineage specification of neural
crest cell differentiation rather than neural crest cell induc-
tion. In support of this idea, Wnt/␤-catenin signal activation
in emigrating neural crest stem cells (NCSCs), regulates cell
fate decisions by promoting the formation of sensory neural
cells in vivo at the expense of other neural crest derivatives
[35].
Bmp4 and Bmp7 have been implicated in neural crest cell
induction in avians [22,36], however mutations in Bmp4 or
Bmp7 do not produce obvious deficiencies in murine cranial
neural crest development [37–39]. Functional redundancy
may explain the absence of an effect, however even mouse
embryos mutant for both Bmp7 and Bmp5 also exhibit nor-
mal patterns of neural crest cell formation and migration [40].
Furthermore, Bmpr1a (Alk3) and Bmpr1b (Alk2), which are
expressed in the neural tube of mouse embryos at the time of
neural crest cell induction, do not affect the formation of neu-
ral crest cells when conditionally inactivated [41,42]. They do
however affect the later differentiation of neural crest cells.
In contrast to chick embryos, Bmp4 expression is not
detected in the dorsal neural tube or surface ectoderm of
mouse embryos during the formation of neural crest cells
[43]. Rather Bmp2 is expressed in a small region of the surface
ectoderm that abuts the neural plate suggesting that it may
play a role in murine neural crest cell patterning [43,44]. In
order to disrupt BMP signaling in premigratory and migra-
tory neural crest cells, without the problems of functional
redundancy and early embryonic lethality potentially, Xeno-
pus noggin was expressed under the control of the Hoxa2
promoter in the hindbrain of transgenic mouse embryos [44].
Hoxa2 is expressed in the neural tube and in the second
and third branchial arch mesenchyme. Consequently, noggin
overexpression in the Hoxa2 domain led to a spatially spe-
cific depletion of second and third branchial arch neural crest
derivatives which included components of the cranial gan-
glia, the stapes and styloid process, greater horn and body of
the hyoid bone, thyroid and cricoid cartilages [44]. Underly-
ing the malformation or absence of these skeletal elements
was the observation that the migration of cranial neural crest
cells was nearly completely abolished in the caudal branchial
arch region.
A role for BMP signaling and BMP2 in particular in neural
crest cell formation in mice is further supported by analyses of
Bmp2 null mutant mice which can survive to the 15–17 somite
stage [45]. These embryos exhibit no evidence of migrating
neural crest cells as assayed by the expression of the neural
crest cell marker Crabp1 [44]. We cannot conclusively rule
out that neural crest cells formed but failed to migrate because
markers of neural crest cell formation such as Snail were
not used in this study. However, an absence of neural crest
cell formation seems to be the most likely scenario, which
suggests that roles for the surface ectoderm and also BMP
signaling in neural crest cell formation are conserved even in
mouse embryos.
Interestingly, recent work in avians has reported an inte-
gration between BMP and Wnt signaling with the cell cycle
during avian trunk neural crest cell induction [46]. The over-
expression of noggin in the neural tube inhibits G1/S tran-
sition, which in turn abrogates BMP-induced neural crest
cell delamination. Similarly, interfering with ␤-catenin and
LEF/TCF also inhibits G1/S transition and neural crest cell
formation. Exogenous BMP can stimulate Wnt1 transcrip-
tion, which implies that BMP signaling regulates G1/S tran-
sition and consequently, neural crest cell formation through
the canonical Wnt signaling pathway [46]. It remains to be
seen however if this mechanism also holds true in the mouse
during neural crest cell induction.
Apart from the Bmp2 mutants, currently there is at least
one other clear example of murine neural crest cell induction
failure and that occurs in a restricted domain in the Hoxa1/b1
double null mutant mice [47]. Hoxa1/b1 double mutants lack
all second branchial arch skeletal elements and exhibit strik-
ing external and middle ear malformations. Lineage tracing
in cultured double mutant embryos together with analyses of
neural crest cell markers such as Sox10 revealed a complete
absence of second branchial arch neural crest cells. What
is significant about this particular study was the demonstra-
tion that wild type cells transplanted into the double mutant
embryos could migrate into the second branchial arch. This
suggested that there was no inherent defect in the endogenous
migration pathway but rather that the neural crest cells at this
axial level failed to form [47]. It is unlikely that Hox genes
play a universal role in neural crest cell induction because
they are not expressed throughout the anterior regions of the
head where neural crest cells clearly form. Hox genes are
clearly well recognized for their roles in anterior–posterior
patterning, however new roles are slowly being uncovered
for these genes in dorso-ventral patterning as well [48]. In the
Hoxa1/b1 double mutants it therefore seems likely that the
absence of neural crest cell formation was the secondary con-
sequence of dorso-ventral patterning transformation defects
which respecifies the dorsal neural plate border territory to a
non-neural crest identity.
Overall, there are surprisingly and disappointingly few
mouse mutants that exhibit a clear absence of migrating neu-
ral crest cells. Snail/Slug are widely used as indicators of
neural crest cell formation since they mark the epithelial to
 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) 683–693
mesenchymal transition that neural crest cells undergo dur-
ing their formation and emigration from the neural tube. By
directly repressing E-cadherin, Snail/Slug promotes the for-
mation of neural crest cells and the onset of their migration
from the neural tube [6]. Unfortunately, null mutations in Slug
demonstrate that Slug is not required for neural crest cell gen-
eration [49]. This may possibly be explained by functional
redundancy with Snail, however to date there is no litera-
ture describing a null allele of Snail or a phenotype with a
defect in neural crest cell formation or migration. Although
it is widely believed that the same contact mediated induc-
tive mechanisms underlie murine neural crest cell formation,
interactions between the neural ectoderm and surface ecto-
derm or the neural ectoderm and paraxial mesoderm have yet
to be definitively tested because of the technical difficulty
of manipulating mouse embryos due to their small size and
complex in vitro culture requirements.
4. Specification of mouse neural crest cell migration
One of the key steps in neural crest cell development is
their migration from the neural tube through the extracel-
lular matrix to their final destinations throughout the body
[50]. The formation of neural crest cells is concomitant with
the epithelial to mesenchymal transition of neuroepithelial
cells and their emigration from the neural tube. Therefore,
it is extremely difficult to distinguish between a failure in
neural crest cell induction versus a complete failure of neu-
ral crest cell migration largely because the latter depends on
the former. Currently, there does not appear to be a situa-
tion where neural crest cells are induced to form but fail to
migrate and consequently most of our analyses of the specifi-
cation of neural crest cell migration in mice relate to aberrant
streams of neural crest cells in vivo and in vitro cell culture
assays.
As described above Snail/Slug are key regulators of both
neural crest cell induction and migration. The inhibition
of Slug blocks neural crest migration in Xenopus [51,52].
Similarly, antisense oligonucleotides directed against Slug
in avian embryos also blocks the ability of neuroepithe-
lial cells to emigrate from the neural tube [4]. Conversely,
over-expression of Slug in the chick neural tube leads to an
increased production of migrating neural crest cells [53]. The
caveat of these analyses is that Slug/Snail are expressed in
neural crest cells during their formation and also during their
migration. What is still critically missing from analyses of
neural crest cell determination in mammalian embryos is an
assessment of the function of Slug/Snail during the migration
of neural crest cells completely independently of their for-
mation. To achieve this will require conditional or inducible
inactivation of Slug/Snail in migrating neural crest cells in a
strict spatiotemporally controlled manner.
Neural crest cell delamination and migration from the neu-
ral tube is aided by the down-regulation of cell adhesion
molecules such as NCAM, N-cadherin, and cadherin-6b and
687
the up-regulation of cadherin-7 and cadherin-11 [6,54–57].
A breakdown of the cytoskeletal components in the basement
membrane surrounding the neural tube where the crest cells
emigrate is also required [58].
Hindbrain derived cranial neural crest cells migrate in
distinct segregated streams adjacent to the even numbered
rhombomeres and into the branchial arches in mouse embryos
[10]. Consequently, neural crest free zones exist adjacent to
rhombomeres 3 and 5 (Fig. 2). In contrast to the even num-
bered rhombomeres, we know from lineage tracing in mouse
embryos that generally fewer neural crest cells emigrate from
the odd numbered rhombomeres and also that rather than
migrating laterally these neural crest cells migrate anteriorly
or posteriorly to join the adjacent even rhombomere neu-
ral crest streams [43]. The segregation of these neural crest
cell populations is critical to prevent fusions of the cranial
ganglia and skeletal elements and also to prevent mixing of
neural crest cells with different genetic constitutions, which
as described below are critical for axial neural crest and cran-
iofacial patterning.
The presence of neural crest free zones and the segre-
gation of neural crest cells into discrete streams is not an
intrinsic property of the vertebrate hindbrain or the neural
crest cells. In ErbB4 null mutant mice neural crest cells
from rhombomere 4 acquire the ability to migrate through
the dorsal mesenchyme adjacent to rhombomere 3, which is
normally free of neural crest cells to join the first branchial
arch stream of neural crest cells (Fig. 2) [59]. Consequently,
this leads to fusions of the trigeminal and facio-acoustic
ganglia. This aberrant rostral migration is not autonomous
to the neural crest cells, since wild type neural crest cells
transplanted into Erbb4 mutants also exhibit this aberrant
migration. Conversely, when ErbB4 mutant neural crest cells
are homotopically transplanted into wild type embryos they
emulate the normal endogenous neural crest migratory pat-
tern from rhombomere 4 into the second branchial arch [60].
The aberrant migration of neural crest cells in ErbB4 mutants
is due to as yet unidentified changes in the paraxial mes-
enchyme environment. Since ErbB4 is normally expressed
in rhombomeres 3 and 5 this phenotype reflects defects in
signalling between the hindbrain and the adjacent environ-
ment branchial arches in mutant embryos.
Fusions of the cranial ganglia have also been described
in the neuregulin and Krox20 null mutant mice [61–63],
however, currently no neural crest cell lineage tracing or
gene expression data is available to confirm the suspicion
that neural crest cells also migrate aberrantly in these mice.
Interestingly, neuregulin is a ligand for ErbB4, so it is not
surprising that it too may also play a role in regulating the
segregated pathways of cranial neural crest cell migration.
Coincidentally, Krox20 is expressed in rhombomeres 3 and
5, which overlaps with ErbB4. To date no direct link between
Krox20 and ErbB4 has been established, but given that
Krox20 is one of the earliest determinants of rhombomere
3 identity, it seems plausible (although not yet proven) that
ErbB4 expression is disrupted in the Krox20 mutants which
688 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) 683–693

Fig. 2. Normal and abnormal patterns of neural crest cell migration. Lineage tracing of neural crest cells in mouse embryos with DiI (fluorescent red dots)
demonstrates that rhombomere 2 derived neural crest cells contribute to the first branchial arch (A and B) whereas rhombomere four derived neural crest cells
contribute to the second branchial arch (C and D). Normally cranial neural crest cells migrate as distinct segregated streams into the adjacent branchial arches
(E). Mixing between first and second branchial arch streams is observed dorsally adjacent to the neural tube in ErbB4 mutants (F). Extensive mixing of neural
crest streams is observed in Twist mutants (G). Mixing is also observed in Tbx1 mutants but only ventrally in the vicinity of the first pharyngeal pouch (H).

would account for the same ganglia fusions as observed in
ErbB4 null mutant mice.
Twist mutant embryos also exhibit aberrant neural crest
cell migration such that second branchial arch neural crest
cells stray from their subectodermal route towards the sec-
ond branchial arch and invade the normally neural crest free
mesenchyme adjacent to rhombomere 3 (Fig. 2) [64,65].
Consequently, Twist mutant mice also display fusions of the
trigeminal and facio-acoustic ganglia, similar to the ErbB4
mutants [65]. Interestingly, the expression pattern of ErbB4
is normal in the Twist mutant hindbrain, which implies that
Twist could act downstream of ErbB4 or be part of an addi-
tional important pathway that regulates the segregated migra-
tion pathways of cranial neural crest cells. It remains to be
determined whether the mesodermal or neural expression
domain of Twist regulates the migration patterns of cranial
neural crest cells, but conditional Twist mutants will eventu-
ally answer this question.
Previously it has been proposed that the cranial mesoderm
influences the migration pathways of cranial neural crest cells
in mouse embryos [43] and consequently this implies that
not all craniofacial malformations arise due to defects intrin-
sic to the neural crest. Rather, primary defects in the cranial
mesoderm or other tissues that the neural crest cells contact
could lead to secondary abnormalities in both the migration
and patterning of cranial neural crest cells [43,66]. This idea
appears to be supported by neural crest cell migration anoma-
lies in Tbx1 mutant mice (Fig. 2). In Tbx1 mutant embryos,
patterning of the second and more caudal branchial arches
are disrupted resulting in poor colonisation by neural crest
cells. Consequently, a stream of Hoxa2 expressing neural
crest cells originating from rhombomere four migrate aber-
rantly into the first branchial arch, which results in inner
ear abnormalities [67]. Tbx1 is not expressed in neural crest
cells but rather in the paraxial mesoderm and core regions of
the branchial arches. This suggests that Tbx1 is acting non-
cell autonomously in the branchial region during craniofacial
morphogenesis. This data also provocatively suggests that
craniofacial malformations such as those seen in DiGeorge
syndrome in which Tbx1 mutations are implicated may occur
as a secondary consequence of defects in neural crest cell
migration. The primary defect possibly lies in the mesoderm
or endoderm, which are the main regions of Tbx1 expression.
These results demonstrate that ErbB4, Twist and Tbx1 may
all be acting non-cell autonomously in directing the migration
pathways of cranial neural crest cells and to date surprisingly
few molecules that intrinsically influence the path finding
of murine cranial neural crest cells have been identified.
Evidence obtained primarily from analyses in frog embryos
suggests that bi-directional Eph/ephrin cell signalling plays
an important intrinsic role in keeping the neural crest cell
streams segregated [68]. This is also supported by a recent
study in mouse embryos, where it was recently shown that
ephrinB1 acts cell autonomously in neural crest cells to regu-
late craniofacial development [69]. EphrinB1 deficient mice
exhibit high degree of cleft palate and tympanic ring defects
and the neural crest cells from the post-otic region of the
hindbrain display aberrant migration by invading territories
 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) 683–693
normally devoid of neural crest cells which ultimately led to
nerve fasciculation and branching defects. Each of the studies
detailed above describe subpopulations of neural crest cells
migrating aberrantly but they do not reveal the underlying
mechanism that controls neural crest cell migration along
there normal pathways. This instead appears to be a mor-
phogen regulated event.
BMPs are not only critical for neural crest induction, but
they are also involved in neural crest migration. In mouse
embryos in which Alk2 (Bmpr1b) is conditionally deleted
from neural crest cells, the initial formation and migration
of neural crest cells remains unaffected. However, the later
migration of mutant neural crest cells to the outflow tract of
the heart is dramatically impaired [41]. The effect of BMP
signaling on changes in cell adhesion and the cytoskeletal
components within the neural tube remains to be clearly
established, however one target of BMP signaling is the GTP-
binding protein, rhoB [70]. Ectopic expression of noggin in
the avian neural tube, leads to a reduction in the expression of
rhoB as well as cadherin-6B which prevents neural crest emi-
gration from the dorsal neuroepithelium [71]. Collectively,
these studies support a role for BMP in neural crest migra-
tion from the neural tube through the regulation of rhoB and
cadherins.
Although a role for FGF signaling in murine neural crest
cell formation has not been determined, in vitro migra-
tion assays have revealed that exogenous FGF2 (basic FGF)
and FGF8 exhibit a chemo-attractive activity that influ-
ences the migration of mesencephalic mouse neural crest
cells [72]. Anti-FGF2 and anti-FGF8 neutralising antibod-
ies inhibit the chemotaxic response. In vivo, FGF2 activity is
detected predominantly in target regions such as the mandibu-
lar mesenchyme, which is colonised by mesencephalic neu-
ral crest cells. This characteristic distribution supports the
notion that FGF2 acts as a chemo-attractant in the mouse
embryo that directs mesencephalic neural crest cell migra-
tion. The domains of FGF8 activity are largely restricted to the
branchial arch epithelium suggesting it may not be involved
in guiding neural crest cell migration, however FGF2 activ-
ity can be promoted by FGF8. This has led to the hypothesis
that FGF8 activity in the mandibular arch epithelium is a pre-
requisite for the differential localisation of FGF2 and that in
turn, the distribution of FGF2 is essential for chemotaxis of
mesencephalic neural crest cell migration [72]. In vivo this
is not the case since the conditional inactivation of Fgf8 in
the branchial arch epithelium does not disrupt the migration
of cranial neural crest cells into the branchial arches. Instead
FGF8 activity in the branchial arch surface ectoderm appears
to play an important functional role in neural crest cell sur-
vival [73].
Further evidence for a role of FGF signaling in neural
crest cell migration has come from analyses of the Fgfr1 null
mutant mice. Fgfr1 is expressed in multiple cellular com-
ponents of the branchial arches [74] and recent studies have
shown that perturbation of Fgfr1 function during branchial
arch development results in failure of the neural crest to enter
689
the second arch [75]. The hindbrain derived neural crest
cells migrate toward the second branchial arch, but failing
to enter, they instead accumulate in a region proximal to the
second branchial arch. While this defect in neural crest migra-
tion does not reflect a neural crest cell specific function of
Fgfr1, the second branchial arch exhibited by these mutants
appear to result from abnormal patterning during early devel-
opment of the branchial arch region. The initial generation
of neural crest and the segmentation and patterning of the
hindbrain appear normal in these mutants. Taken together,
this suggests Fgfr1 patterns the branchial arch region cre-
ating permissive environments that enable neural crest cell
migration.
Cranial neural crest cells are also susceptible to retinoic
acid-mediated teratogenesis [76] and in vitro and in vivo
studies have demonstrated that retinoic acid interferes with
neural crest migration [77–81]. Targeted inactivation of the
mouse retinaldehyde dehydrogenase 2 (Raldh2), the enzyme
responsible for early embryonic retinoic acid synthesis, leads
to prenatal death from a lack of aorticopulmonary septation
[82]. Raldh2 null embryos exhibit impaired development of
their posterior (third to sixth) branchial arches. Post-otic neu-
ral crest cells fail to establish segmental migratory pathways
and are misrouted caudally. The disruption of cardiac neural
crest cell migration leads to the absence of outflow tract septa-
tion. Similar defects in vagal neural crest leads to agenesis of
the enteric ganglia, a condition reminiscent of Hirschprung’s
disease in humans. Raldh2 expression is restricted to the pos-
terior most pharyngeal mesoderm, highlighting the potential
importance of the mesoderm during the migration phase of
neural crest cell development. In support of these genetic
studies, treating headfold-stage mouse embryos with a pan-
RAR antagonist in vitro and in vivo results in a complete
absence of the third and fourth branchial arches [83]. Conse-
quently, neural crest cells normally destined for the third and
fourth arches migrate ectopically.
Further roles for retinoic acid in the migration of neural
crest cells have been observed in a stage-dependent manner in
rat embryos [80]. Early-stage retinoic acid treatment induced
an ectopic caudal migration of the anterior hindbrain (rhom-
bomeres (r) 1 and 2) crest cells into the second branchial arch
and acousticofacial ganglion. In contrast, late-stage treatment
did not disturb the segmental migration pattern of hindbrain-
derived crest cells, although it did induce branchial arch
fusions. Anterior hindbrain-derived neural crest cells pop-
ulated the anterior half of the fused arch while neural crest
cells derived from the pre-otic hindbrain (r3 and r4) occupied
its posterior half [80]. Similar treatments of mouse embryos
with retinoic acid in culture result in the ectopic migration of
second arch neural crest cells into the first arch [84], which
interestingly mimics the Tbx1 mutant neural crest cell migra-
tion phenotype.
One of the potential targets of retinoic acid is the c-Jun
N-terminal kinase (JNK) pathway [85]. Cardiac neural crest
migration is inhibited when retinoic acid blocks JNK phos-
phorylation, which is critical for neural crest outgrowth. Fur-
690 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) 683–693
thermore, the use of dominant-negative constructs to perturb
upstream and downstream components of the JNK pathway
also reduce cardiac neural crest outgrowth, implying that JNK
is not only a target for the action of retinoic acid, but is also
critical for the migration of cardiac neural crest cells in vitro.
Neural crest cells express retinoic acid receptors (RARs) and
cellular retinoic acid binding proteins (CRABPs) however
the primary source of retinoic acid comes from the paraxial
mesoderm.
Collectively, these studies demonstrate that appropriate
migration may not be an intrinsic property of neural crest cells
but rather is directed non-cell autonomously in an extremely
intricate fashion that requires complex interactions between
signals from the neuroepithelium, together with the paraxial
mesoderm, ectoderm and endoderm tissues [43,59].
5. Specification of multipotency versus restricted
potency of mouse neural crest cells
One of the many intriguing features of neural crest cell
is their pluripotency. A single neural crest cell will differ-
entiate into any of several distinct cell types depending on
its location within the embryo. However, it is still not certain
whether most individual neural crest cells that leave the neural
tube are pluripotent or whether the majority of the popula-
tion is already restricted to certain fates. Some of the strongest
evidence for pluripotency comes from single neuroepithelial
cell labeling in chick embryos, which revealed that progeny
of a single neural crest cell could become sensory neurons,
melanocytes, adrenomedullary cells and also glia [86,87].
Similar results were obtained from in vitro clonal assays in
mice [88], where it was demonstrated that these cells have
the capacity to self renew and form neurons, glia and smooth
muscle [89]. Consequently they have been termed neural
crest stem cells [90]. In contrast analyses in zebrafish provide
equally strong evidence for restricted lineage determination
since similar labeling experiments revealed that most neural
crest cells differentiate into only one cell type [91,92].
Evidence in mouse embryos suggests that some popu-
lations of neural crest cells are committed at the time of
emigration from the neural tube or very soon after since they
express transcription factors that constrain the cell types they
can produce [93,94]. Using the Cre recombinase system to
permanently mark subsets of neural crest cells that transiently
expressed the transcription factor Ngn2 or Wnt1 revealed that
Ngn2 positive progenitors were four-fold more likely than
Wnt1 positive cells to contribute to sensory and sympathetic
ganglia [95]. Within the dorsal route ganglia however, both
Ngn2 and Wnt1 positive populations were equally likely to
generate neurons or glia. Hence this suggests that very early
in neural crest migration, Ngn2 may mark a sub-population
that is already fate biased. Furthermore, this data also implies
that in the trunk neural crest cells become restricted to sensory
or autonomic lineages before committing to either neuronal
or glial fates.
This issue of multipotency versus lineage restriction has
also been addressed with respect to melanocytes. A sub-
population of cells in the dorsomedial domain of the neu-
ral tube, which expresses the receptor tyrosine kinase, Kit,
migrates exclusively into the developing dermis [96]. Con-
sequently, these cells activate definitive melanocyte lineage
markers indicating they are melanocyte progenitor cells. This
particular sub-population is generated predominantly at the
midbrain–hindbrain junction and at the cervical and lower
trunk levels. Other cells within the dorsal neural tube that are
Kit negative, express p75 and migrate ventrally giving rise
to neurons and glia. Interestingly, the p75 positive cells are
located ventrolateral to the Kit positive cells, suggesting that
there is in vivo neural crest cell lineage segregation within
the mouse neural tube [96]. This poses an important ques-
tion that currently remains unresolved and that is what are
the signaling pathways that establish this lineage segrega-
tion during the period of neural crest cell formation? Is the
interface between FGF, WNT and BMP signaling at the neu-
ral plate border only stimulating the induction of neural crest
cells or do each of these signaling pathways have the capacity
to specify sublineages within the neural crest.
6. Conclusions
Mammalian neural crest cells are a mulitpotent migratory
stem cell population that generates an impressively broad
array of cell and tissue types during craniofacial development.
Currently, BMP2 signaling specifically from the surface ecto-
derm immediately adjacent to the neural plate appears to be
the key player in murine neural crest cell induction. This
is somewhat different to chick, frog and zebrafish embryos
where the emphasis lies with Wnt signaling. Collectively,
BMP, Wnt and FGF signaling all play key roles in regulating
either early or late steps in neural crest cell migration. Surpris-
ingly, these signaling pathways also mediate major aspects
of neural crest cell differentiation. Hence, repeated use of the
same signaling pathways elicits completely different effects
on neural crest cell formation, migration and differentiation.
The studies described in this review highlight the complex-
ity in regulating neural crest cell patterning and the balance
between signals acquired in the neuroepithelium during for-
mation versus signals received from the extrinsic tissue envi-
ronments during migration. This intricate regulation affects
not only the migratory properties of neural crest cells but also
dramatically influences their differentiation and has been cru-
cial to vertebrate neural crest and craniofacial evolution.
Acknowledgements
P.A.T. is supported by research funds from the Stowers
Institute, a Basil O’Connor Research Scholar Award (#5-
FY03-16) from the March of Dimes and grant RO1 DE
016082-01 from the National Institute of Dental and Cranio-
facial Research.
 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) 683–693
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