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Neural Crest: Origin,
Migration and
Differentiation
Introductory article
Article Contents
. Introduction
. Specification of the Neural Crest Lineage and Its
Detachment from the Neural Tube
. Patterns of Neural Crest Cell Migration
Carol A Erickson, University of California, Davis, California, USA
. Control of Lineage Segregation and Differentiation of
the Neural Crest
The neural crest is a population of cells that emigrates from the dorsal neural tube
during early embryogenesis and migrates extensively to give rise to a myriad of cell types.
Patterns of migration are controlled largely by extracellular cues in the environment. Cell
fates are determined both by specification events while the cells are still resident in the
neural tube and by extracellular cues the cells perceive as they migrate.
Introduction
The embryos of vertebrates are distinguished from those of
invertebrates by, among other features, a population of
cells that emigrates from the dorsal surface of the neural
tube, and consequently has been coined ‘the neural crest’.
These cells disperse from the neural tube along stereotyped
pathways and give rise to a remarkable range of
phenotypes, including neurons and glial cells of the
peripheral nervous system, pigment cells of the skin, and
a population of cells in the head collectively called the
‘ectomesenchyme’. The ectomesenchyme produces connective tissues, including cartilage and bones of the face
and jaw, as well as components of the teeth, eye, ear and
heart. Consequently, the neural crest has been a favourite
model system with which to address questions concerning
morphogenesis and cell differentiation.
The neural crest has been studied in many different
organisms. Initially amphibian and chicken embryos were
employed because they are easily observed and experimentally manipulated, and there are a variety of markers with
which to label and identify the neural crest. More recently,
the genetically tractable mouse and zebrafish embryos
have become increasingly popular. Together these model
organisms have allowed us to address questions concerning what specifies the neural crest as a lineage, what
controls the patterns of migration, and what determines
cell fate in this remarkable population of cells.
Specification of the Neural Crest
Lineage and Its Detachment from the
Neural Tube
If a piece of the neural plate is removed from an embryo
and cultured, it will fail to give rise to neural crest cells. If
the neural folds are explanted just before the completion of
. Summary
neurulation, then an abundance of neural crest cells will
materialize. Thus, a question that many laboratories have
considered is when and how the neural crest lineage
segregates from the rest of the neural epithelium. Experimental studies in the chick and the salamander showed that
contact between the neural folds and the contiguous
ectoderm is required to induce the formation of the neural
crest. If this contact is prevented, the neural crest will fail to
form. If a portion of the neural tube that does not give rise
to neural crest cells is placed in contact with the ectoderm,
it will now produce neural crest cells. The most likely
inductive signal emanating from the ectoderm is a member
or members of the bone morphogenetic protein (BMP)
family of signalling molecules. BMP4 and BMP7 are both
produced by the ectoderm at the appropriate time, and
treatment of lateral neural tube with purified BMP in
culture will result in the formation of neural crest cells
(Figure 1). It is not known what downstream molecular
events triggered by BMP determine the neural crest
lineage.
Once the neural crest cells are specified, they migrate
from the neural epithelium as individual cells, a process
known as an epithelial/mesenchymal transformation
(EMT). Cellular changes that are required in order for
neural crest cells to emigrate are: (1) loss of adhesions to the
neural epithelium; and (2) ability to attach to and migrate
on the extracellular matrix in the periphery. There are at
least two models to explain the EMT. In the first, there
could be a downregulation and loss of the cadherin
adhesion molecules that maintain epithelial cell cohesion.
Several different cadherins have been identified in the
neural tube and at least some of these are no longer
expressed by the neural crest cells at the time that they
emigrate. However, there is no direct evidence to show that
neural crest cells are stimulated to migrate if cadherins are
experimentally downregulated. A second possibility is that
neural crest cells are stimulated to migrate owing to an
upregulation of cell–matrix adhesion molecules (integrins)
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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Neural Crest: Origin, Migration and Differentiation
NP
Cranial
Bone and cartilage
Connective tissues
(teeth, eyes, ears)
Sensory neurons
Glial cells
Melanocytes
BMP4/7
N
(a)
Vagal
Enteric neurons
Sensory neurons
Glial cells
Melanocytes
Smooth muscle
Cardiac tissues
NF
N
(b)
NC
Trunk
Sensory neurons
Autonomic neurons
Chromaffin cells
(adrenal medulla)
Glial cells
Melanocytes
S
(c)
Figure 1 Sections through the trunk of (a) a neural plate-stage embryo,
(b) a neurulation-stage embryo, and (c) at the completion of neurulation,
when the neural crest cells are beginning to migrate. At the neural plate
stage, neural crest cells are not yet specified, but under the influence of
BMP4/7 (indicated in blue) produced by the ectoderm, neural crest cells
(indicated in green) are induced to form from the edges of the neural plate
(or neural folds). N, notochord; NC, neural crest cells; NF, neural fold; NP,
neural plate; S, somite.
and a reorganization of the actin cytoskeleton that would
allow these cells to generate sufficient tractional force to
pull away from (actually rupture) their adhesions. There
are several studies that document an upregulation of
integrins at the time of the EMT. In addition, the cells of
the dorsal neural tube express the small G protein, rhoB,
which is required for the neural crest to undergo the EMT
and is known to organize the actin cytoskeleton. Nevertheless, there is no direct imaging of this event to reveal if
this ‘tugging’ hypothesis is correct. Despite the intensity
with which the EMT has been studied, it is still not clear
what mechanism drives this process.
Patterns of Neural Crest Cell Migration
Once neural crest cells detach from the neural tube, they
undergo an extensive migration throughout the embryo
2
Figure 2 Fate map of the neural crest derivatives in a stage-14 chicken
embryo.
(Figure 2). In the trunk (axial level from the neck, posterior),
neural crest cells take two major pathways. The first is
ventral, between the neural tube and somites, and these
cells give rise to the neurons and glial cells of the peripheral
nervous system, including the secretory cells of the adrenal
medulla. Somewhat later (the time depending upon the
species), neural crest cells embark on a second, dorsolateral
pathway between the ectoderm and dorsal surface of the
somites, and these are the crest cells that differentiate into
the pigment cells of the skin.
In the head, the pathways of migration are exceedingly
complex, but as a generality, neural crest migration is
dorsolateral, between the ectoderm and the underlying
mesoderm, with very little ventral migration. The most
distally migrating cells give rise to connective tissues of the
face, jaw, eye, ear and heart, whereas those cells that
remain proximal produce the neurons and glial cells of the
cranial ganglia.
There is a population of neural crest cells between the
cranial and trunk neural crest (at the axial level of somites
1–7), often referred to as the vagal crest, where there
appears to be a transition between these two different
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Neural Crest: Origin, Migration and Differentiation
patterns of migration. At this level, the first pathway of
migration is dorsolateral between the ectoderm and
somites, and these cells give rise to components of
branchial arches 4 and 6, as well as the connective tissues
of the heart and the enteric nervous system of the gut. Next,
vagal neural crest cells migrate ventrally through the
somites to produce elements of the peripheral nervous
system. Finally, the last stage in migration is again
dorsolateral, and these crest cells give rise to the pigment
cells of the skin.
NC
S
N
(a)
Extracellular matrix determines the patterns
of migration
The pathways of neural crest migration are primarily
determined by extracellular components in the environment through which the cells migrate. Control of trunk
neural crest migration is especially well studied.
At the trunk level, neural crest cells migrate ventrally
between the neural tube and somites, until they arrive at the
interface between the myotome and sclerotome. Here they
abruptly turn almost 908 and migrate medially to laterally
through the somite along the undersurface of the myotome
(Figure 3). Eventually, neural crest cells begin to fill up some
of the sclerotome as well. Some neural crest cells migrate as
far as the dorsal aorta and here they coalesce to form the
sympathetic ganglia. Other neural crest cells cease migration close to the dorsal neural tube and these constitute the
dorsal root or sensory ganglia. Another subpopulation
migrates along the ventral root motor fibres and these
differentiate into glial cells. A really startling finding is that
neural crest cells only invade the anterior half of the
somites and avoid the posterior half (Figure 4). This very
early segmental migration is critical in determining the
segmental pattern of the peripheral nervous system.
A major question is what determines the migratory
pathways of the trunk neural crest. Why do they only
migrate through the anterior half of the somite, and why do
they avoid the posterior half and fail to invade other
epithelial tissues that border the path, such as the neural
tube? Apparently there are extracellular matrix molecules
that are permissive for migration and lay out the pathways,
whereas there are others that are inhibitory for migration
and act as impenetrable barriers that border the pathways
and thus constrain migration to particular regions.
Barrier molecules that inhibit cell movement are as
important in determining pathways of neural crest migration as molecules that stimulate motility. Regions in the
trunk that are refractory to neural crest migration include
the dorsolateral path (at least initially), the posterior half of
each somite, and the ventral portion of the anterior
sclerotome. Each of these regions is filled with numerous
molecules that have been demonstrated to inhibit neural
crest migration in culture. These barrier molecules include:
extracellular matrix molecules that bind peanut agglutinin
EC
NC
M
SC
N
(b)
DRG
+ 24 h
+ 24 h
M
VR
SC
(c)
N
SY
Figure 3 Sections through the trunk of a chicken embryo showing the
early (a), mid (b) and late (c) stages of neural crest migration. Initially neural
crest cells (NC) migrate ventrally between the neural tube and somite (a).
Once they reach the somite, they enter at the interface of the myotome (M)
and sclerotome (SC), and migrate laterally across the somite. The cells
localize near the dorsal aorta to form the sympathetic ganglia (SY), align
along the ventral root motor fibres (VR) and differentiate into glial cells, or
coalesce near the dorsal neural tube and constitute the sensory or dorsal
root ganglia (DRG). Twenty-four hours after migration has begun, neural
crest cells begin to invade the dorsolateral path. EC, ectoderm; NC,
notochord.
(PNA), chondroitin sulfate proteoglycans and F-spondin.
In addition, two other ligands that are integral cell
membrane proteins (i.e. not extracellular matrix) and
inhibit cell migration have also been identified in the
posterior somites: ephrin-B1, a ligand for the Eph family of
receptor tyrosine kinases, and collapsin-1, one of the many
members of the collapsin/semaphorin family. In order to
confirm the role of any of these in inhibiting neural crest
migration, their function should be perturbed in vivo. Such
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Neural Crest: Origin, Migration and Differentiation
localized to regions refractory to neural crest migration, it
is likely that there are redundant molecules to assure the
proper patterning of the neural crest.
In addition to mechanisms that prevent neural crest cells
from entering some regions, there must also be molecules
that are permissive for their migration in the anterior
somites, as well as eventually in the dorsolateral path. The
anterior somite contains a variety of molecules that sustain
motility, including laminin, fibronectin, collagen, vitronectin and thrombospondin. However, attempts to perturb their function in vivo using function-blocking
antibodies have not resulted in any disruption in neural
crest migration. The most likely explanation is that neural
crest cells can use any of these molecules for migration and
are therefore functionally redundant. Recently, functionblocking antibodies to the a4b1 integrin substantially
inhibited neural crest migration. Since this integrin is a
receptor of fibronectin and thrombospondin, it is likely
that these are the predominant matrix molecules that
sustain neural crest migration.
Some neural crest derivatives are endowed
with cell-autonomous guidance mechanisms
Figure 4 A stage-16 chicken embryo immunolabelled with the HNK-1
antibody, which identifies neural crest cells, and processed so that the cells
appear brown. Note the segmental migration of the neural crest cells
through the somites in the trunk, and the streams of neural crest cells
migrating into the branchial arches in the head.
studies have been greatly facilitated by the development of
an explant culture system in which pieces of the trunk are
cultured on semipermeable membranes and can be bathed
in medium containing inhibitors of the molecule in
question. Such a system has the additional benefit that
the neural crest cells can be visualized directly as they move
if they are first labelled with DiI. Using this in vitro culture
system, PNA-binding molecules and ephrin ligands have
both been shown to be involved in preventing neural crest
cells from invading the posterior sclerotome. Even more
recently, F-spondin has been implicated as a barrier
molecule because when antibodies that perturb F-spondin
function are injected into the space between the neural tube
and somites, neural crest cells can now invade the posterior
somite. Given the number of proposed barrier molecules
4
The current dogma suggests that neural crest cells are
pluripotent when they detach from the neural tube, migrate
haphazardly into the various pathways when those pathways are permissive for migration, and then differentiate
according to localized cues in the various pathways. There
is considerable evidence for this pluripotentiality (see next
section). However, another possibility is that neural crest
cells become specified (that is, know what they are going to
become) before or early after they detach from the neural
tube, and because of this specification they acquire
migratory properties that allow them to exploit or even
choose a particular migratory route. There is compelling
evidence that this is how melanoblasts become localized in
the skin.
At the trunk level, neural crest cells first migrate
ventrally, and then almost 24 h later they invade the
dorsolateral path, where they will differentiate into
melanocytes. It had been presumed that the delay in
migration was owing to inhibitory molecules in the path
that must be removed in order for neural crest cells to
migrate into the skin. However, when melanoblasts
(melanocyte precursors) are grafted into an early chicken
embryo, they immediately embark on the dorsolateral
path. This result shows that melanoblasts have special
migratory capabilities and further suggests the hypothesis
that, in order to invade the dorsolateral path, neural crest
cells have to be specified as melanoblasts. Molecular
markers of melanoblasts reveal that they are specified
before entering the dorsolateral path, and, furthermore,
that only melanoblasts ever enter the dorsolateral path.
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Neural Crest: Origin, Migration and Differentiation
Therefore, in order to migrate dorsolaterally, neural crest
cells must be specified as melanoblasts.
Melanoblasts are so far the only neural crest subpopulation for which evidence of cell autonomous guidance
properties exists; however, there is tantalizing evidence
that other neural crest phenotypes are specified early,
before entering the appropriate migratory pathway (see
next section). Future work may well reveal that this is, in
fact, a much more common mechanism than was
previously surmised.
Control of Lineage Segregation and
Differentiation of the Neural Crest
When are neural crest lineages specified?
Most evidence, until recently, has generally supported the
view that neural crest cells are multipotent when they
initiate migration, and that their differentiation is controlled by environmental cues that they perceive as they
migrate and after they arrive at their final destination. The
landmark heterotopic grafting experiments of Le Douarin
and Teillet revealed that neural crest cells from one axial
level transplanted to another axial level will differentiate
according to their new position rather than to their origin.
Similarly, when neural crest-derived structures, such as
ganglia, are back-transplanted into the early migratory
pathways, the component cells remigrate to many other
locations and give rise to a variety of neural crest
derivatives in addition to the phenotype of the structure
from which they were derived. All of these studies test the
developmental capability of the neural crest as a population and not as individual neural crest cells.
The developmental potential of individual neural crest
cells has also been investigated using cloning techniques,
both in culture and in the embryo. Sieber-Blum and Cohen
were among the first to culture individual trunk neural
crest cells and then assess how the progeny differentiate. In
many cases, a single clone (all the cells derived by cell
division from the single cell) will contain neurons, glial cells
and pigment cells, showing that the original neural crest
cell was capable of differentiating into all the major classes
of cell types derived from the trunk. Similarly, cloning of
cranial neural crest cells reveals multipotent lineages.
Single-cell labelling techniques have been developed to
mark individual neural crest cells in the embryo in order to
assess their developmental repertoire in situ. Such studies,
which are technically difficult, show that a single labelled
cell gives rise to a clone of cells, and these clonally related
cells sometimes migrate to different neural crest-derived
structures or into various pathways. These results suggest
that they are also differentiating into a variety of cell types,
although the actual phenotype of the clonal derivatives
could not be rigorously tested without the availability of
cell type-specific markers. Nevertheless, these studies
suggest that most neural crest cells are not restricted in
their developmental potential at the time they detach from
the neural tube.
Although most of the previous studies stressed the
multipotentiality of the neural crest, there was also
evidence in these same reports that a large percentage of
the neural crest cells were fate-restricted. In all the cloning
studies, many of the clones gave rise to only one or two
derivatives. Similarly in the back-transplantation studies,
not all neural crest derivatives differentiate. For example,
back-transplanted sympathetic ganglia fail to populate the
sensory ganglion. More recently, with the development of
lineage markers, it has become clear that many neural crest
cells are likely to be fate-restricted quite early in development.
A study by Henion and Weston is the most recent and
best evidence to seriously question a strict view of
multipotentiality of the neural crest. Their approach was
to label individual neural crest cells with DiI as they
detached from the neural tube in culture, and then assess
how the clone of cells derived from single labelled cells
differentiate. In this manner, they could examine the
developmental capability of neural crest cells just as they
emerge from the neural tube, rather than cloning neural
crest cells that are 24–48 h old, as the previous cloning
studies had done. They made several interesting findings.
When they labelled single cells that detach during the first
6 h after explanting the neural tube, and then determined
how the clones differentiate several days later, they found
that 44.5% of the clones contained only one cell type,
showing that neural crest cells are already fate-restricted
(that is, their fate is already specified) at the time they begin
to migrate. The remaining clones were partially restricted,
giving rise to mixed clones of neurons and glial cells, or
clones of glial and pigment cells. A similar study in
zebrafish also revealed that premigratory neural crest cells
are in large part fate-restricted.
Their second major observation was that the first neural
crest cells to detach from the neural tube produce largely
neurons and glial cells, but no pigment cells. Conversely,
neural crest cells that emigrate relatively late in the process
(that is, the last to leave) give rise almost exclusively to
pigment cells. Together, these results suggest that neural
crest cells are specified earlier than previously imagined,
and that the developmental potential of early- versus latemigrating neural crest cells is different.
Studies of head crest also suggest that there is developmental heterogeneity. For example, only cranial neural
crest cells can differentiate into connective tissue, suggesting that this lineage must be specified exclusively in the
head. Similarly, the neural crest cells that contribute to the
heart (the so-called cardiac neural crest) arise from the
postotic level of the neural tube to somite level 6, and when
these crest cells are ablated, neural crest cells from no other
axial level can substitute for them. Finally, when neural
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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Neural Crest: Origin, Migration and Differentiation
crest cells that would normally migrate into branchial
arches 2 or 3 are replaced by premigratory first arch neural
crest cells, the transplanted cells migrate into the second
branchial arch rather than the first (that is, they take the
migratory path appropriate to their new position), but they
differentiate into structures typical of the first arch. How
exactly these cranial lineages acquire their identity is not
known, although hox genes seem to play a role in segmental
identity. All these studies present the intriguing notion that
not all neural crest cells are multipotent.
How does the environment affect neural crest
cell differentiation?
Although the above studies show convincingly that not all
neural crest cells are created equal, there is nevertheless
evidence that some neural crest cells are multipotent (can
give rise to at least two derivatives). Therefore there must
be some cues in the environment to guide their differentiation. Moreover, there is a great deal of phenotypic variety
within the neuron and glial subpopulations. That is, there
are multiple types of neurons and glial cells that are derived
from the neural crest and which come to reside in different
structures. Finally, even though neural crest cells may be
specified early, they still require additional stimuli in the
environment to complete the process of differentiation or
require additional cues to survive. Thus, it is clear that
environmental cues must influence neural crest cell
differentiation as well.
The role of environmental cues in controlling the
development of the sympathoadrenal lineage is particularly well studied. Neural crest cells in this lineage produce
catecholamines as their neurotransmitters and give rise to
the adrenergic neurons of the sympathetic ganglia, the
chromaffin cells of the adrenal gland, and a third cell type
known as SIF cells, which are found in the sympathetic
ganglia, adrenal medulla and in the small paraganglia of
the gut. All of these cells arise from a common precursor,
which is first identified in the primary sympathetic chain
adjacent to the dorsal aorta. There is, at present, no
evidence to suggest that there is a premigratory, faterestricted sympathoadrenal precursor cell, and the earliest
markers of adrenergic neurons do not appear until well
after the cells have reached the dorsal aorta. From this site,
the cells then disperse, in some cases dorsally to form the
definitive sympathetic ganglia, and also ventrally where
they coalesce near the kidney and develop into the
phaeochromocytes of the adrenal gland. Experimental
evidence is consistent with the notion that the first cell type
to differentiate is the neuronal phenotype, and it does so
under the influence of noradrenaline (norepinephrine)
produced by the notochord and BMPs produced around
the dorsal aorta. When some of these neuronal precursors
disperse to the kidney, they are exposed to glucocorticoids,
6
which repress neuronal differentiation and stimulate
differentiation into chromaffin cells.
Neural crest cells also give rise to the neurons and glial
cells of the sensory ganglia. Cloning studies have revealed
that, although there are multipotent neural crest cells that
can differentiate into both sensory neurons and sympathetic neurons, there are also at least two subpopulations of
sensory neurons that are fate-restricted at the time they
leave the neural tube. These latter cells still require growth
factors produced by the dorsal neural tube in order to
complete differentiation. The best-characterized neurotrophic factor is NT-3, and, in its absence, sensory neurons
fail to survive. NT-3 may also act as a chemotactic
molecule, which could attract sensory neuron precursors to
the appropriate site near the dorsal neural tube.
As discussed above, melanoblasts are specified around
the time they leave the neural tube. This specification
appears to be controlled by members of the Wnt family of
signalling molecules, to which they are exposed in the
neural tube. However, Wnt signalling alone is not sufficient
to complete their differentiation. They also require other
growth factors, including endothelin and steel factor
produced by the skin, that appear to act as maintenance
and/or proliferation factors. Mutant mice in which either
of these factors are missing fail to produce melanocytes.
Summary
The neural crest has been a popular subject for studying
morphogenetic movements and cell differentiation, but
critical questions remain unanswered. What are the
molecular and cellular changes that accompany the
EMT? Which lineages of the neural crest are specified
early and acquire cell-autonomous migratory properties
and which are at the mercy of the extracellular environment? And finally, what are the molecular mechanisms that
are responsible for the segregation of the various neural
crest lineages and at what times does this segregation
occur? Answers to these questions will shed light on the
same processes in other developing cells and tissues, and
will reveal the basis for many birth defects that affect neural
crest derivatives.
Further Reading
Erickson CA and Perris R (1993) The role of cell–cell and cell–matrix
interactions in the morphogenesis of the neural crest. Developmental
Biology 159: 60–74.
Henion PD and Weston JA (1997) Timing and pattern of cell fate
restrictions in the neural crest lineage. Development 124: 4351–4359.
Le Douarin NM and Kalcheim C (1999) The Neural Crest. Cambridge:
Cambridge University Press.
Le Douarin NM and Teillet MA (1974) Experimental analysis of the
migration and differentiation of neuroblasts of the autonomic nervous
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Neural Crest: Origin, Migration and Differentiation
system and of neurectodermal mesenchymal derivatives, using a
biological cell marking technique. Developmental Biology 41: 162–184.
Selleck M and Bronner-Fraser M (1996) The genesis of avian neural crest
cells: a classic embryonic induction. Proceedings of the National
Academy of Sciences of the USA 93: 9352–9357.
Sieber-Blum M (1998) Growth factor synergism and antagonism in early
neural crest development. Biochemistry and Cell Biology 76: 1039–
1050.
Sieber-Blum M and Cohen AM (1980) Clonal analysis of quail neural
crest cells: they are pluripotent and differentiate in vitro in the absence
of noncrest cells. Developmental Biology 80: 96–106.
Stemple DL and Anderson DJ (1993) Lineage diversification of the
neural crest: in vitro investigations. Developmental Biology 159: 12–23.
Wehrle-Haller B and Weston JA (1997) Receptor tyrosine kinasedependent neural crest migration in response to differentially localized
growth factors. Bioessays 19: 337–345.
Weston JA (1991) Sequential segregation and fate of developmentally
restricted intermediate cell populations in the neural crest lineage.
Current Topics in Developmental Biology 25: 133–153.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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