Download neural crest specification: migrating into genomics

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NEURAL CREST SPECIFICATION:
MIGRATING INTO GENOMICS
Laura S. Gammill and Marianne Bronner-Fraser
The bones in your face, the pigment in your skin and the neural circuitry that controls your digestive
tract have one thing in common: they are all derived from neural crest cells. The formation of these
migratory multipotent cells poses an interesting developmental problem, as neural crest cells are
not a distinct cell type until they migrate away from the central nervous system. What defines the
pool of cells with neural crest potential, and why do only some of these cells become migratory?
New genomic approaches in chick, zebrafish and Xenopus might hold the key.
Division of Biology 139-74,
California Institute of
Technology, Pasadena,
California 91125, USA.
e-mails:
[email protected];
[email protected].
doi:10.1038/nrn1219
Neural crest cells are the great explorers of the vertebrate embryo. Although they have the potential to
remain in the central nervous system at the site of their
birth, they strike off on their own and colonize the far
reaches of the embryo. These wandering cells form,
among other things, peripheral neurons, glia, connective
tissue, bone, secretory cells and the outflow tract of the
heart. We are only just beginning to understand how
migratory neural crest cells arise and maintain their
ability to form such diverse cell types.
Many of the properties that make neural crest cells
so interesting to study also make their molecular
characterization a difficult task. As the neural crest is a
vertebrate invention1, it cannot be studied in yeast,
worms or flies, which are the traditional organisms for
rapid, large scale genetic screens. Furthermore, neural
crest cells form during the early stages of embryonic
development, when mouse embryos are small and difficult to manipulate. Historically, the neural crest has
been studied in accessible, externally developing avian
and amphibian embryos. The resulting classical embryological literature provides us with a detailed account of
where neural crest cells arise, which tissues send signals
to cause neural crest formation, and what cell types they
will form1. Until recently, however, this rich descriptive
history of the neural crest has been counterbalanced by
a paucity of molecular information, owing largely to
the scarcity of established molecular techniques in
birds and genomic approaches in vertebrates other
than the mouse. However, with the advent of genome
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sequencing projects in chick, frog and zebrafish, this is
rapidly changing.
In this article, we will provide an overview of early
neural crest development, paying particular attention to
our current understanding of the gene network that
regulates this process. We will then consider how
genomic techniques are changing this view, describing
recent advances and highlighting future possibilities.
Life at the margins
In the earliest phase of neural development, neural
tissue is induced in the ectodermal (outer) layer of the
embryo. As a consequence of neural induction, the
ectoderm becomes divided into three different regions
— the neural ectoderm or neural plate, which will give
rise to the central nervous system; the non-neural
ectoderm, which will form epidermis; and the cells at
the border between neural and non-neural ectoderm,
which for the most part will become the neural crest
(FIG. 1). This border region was first appreciated in 1868
by His, who described chick neural crest as a ‘zwischenstrang’ or ‘in-between strip’, lying between the neural
and non-neural ectoderm2. Neural tissue folds in on
itself to form the neural tube in a process called
neurulation. During neurulation, the neural plate border
cells bend to form the neural folds and eventually
become the dorsal aspect of the neural tube. Depending
on the organism and the axial level, neural crest cells
initiate migration from the closing neural folds or from
the dorsal neural tube.
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Non-neural
ectoderm
Neural plate border
Neuroectoderm
Neural fold
Paraxial
mesoderm
Neural plate
shown that the neural plate border and neural crest cells
form in response to signalling between newly induced
neural tissue and the neighbouring non-neural
ectoderm5,7–10. Signals from the underlying paraxial
mesoderm are also involved in inducing the border
region5,11–15. It is not clear, however, whether these interactions occur coincidentally or sequentially, or whether
they normally serve inductive or maintenance roles in
the embryo.
To complicate matters further, recent evidence
indicates that border induction and neural crest
induction are not necessarily the same process. The
transcription factor Dlx5 is one of the earliest markers of
the neural plate border16–18, and ectopic expression of
Dlx5 in the neural plate results in non-cell-autonomous
induction of neural plate border gene expression without
the induction of neural crest markers18. Furthermore,
when Dlx activity is inhibited in the non-neural ectoderm, neural plate border genes are expressed in their
normal patterns, but are shifted laterally19. Together, these
observations indicate that Dlx proteins act to specify
the border region. Interestingly, Dlx activity is required
for the interaction between neural and non-neural
ectoderm to induce neural crest19. So, Dlx-dependent
induction of an unspecified border region is a requisite
first step for the formation of border cell types, but
neural crest induction requires additional signals.
Secreted factors that induce neural crest…
WNT PROTEINS, BONE MORPHOGENETIC PROTEINS
WNT PROTEINS
A family of highly conserved
secreted signalling molecules,
which are related to the
Drosophila wingless protein and
regulate cell–cell interactions
during embryogenesis. Wnt
proteins bind on the cell surface
to receptors of the Frizzled family.
BONE MORPHOGENETIC
PROTEINS
(BMPs). Multifunctional secreted
proteins of the transforming
growth factor-β superfamily. In
the early embryo, they participate
in dorsoventral patterning.
FIBROBLAST GROWTH FACTORS
(FGFs). Multifunctional factors
that are involved in embryonic
development. More than 20
FGFs and 4 FGF receptors have
been described. Their
coordinated activity controls cell
proliferation, migration, survival
and differentiation. FGFs
regulate growth and
morphogenesis by an early
action on regional patterning,
and a later effect on the growth
of progenitor cells of the
forebrain.
ANAMNIOTES
Vertebrates, such as fish and
amphibians, that do not develop
inside an amnion.
796
Neural
crest cells
Somite
Neural tube
Notochord
Figure 1 | Border induction and neurulation. The neural
plate border (green) is induced by signalling between the
neuroectoderm (purple) and the non-neural ectoderm (blue)
and from the underlying paraxial mesoderm (yellow). During
neurulation, the neural plate borders (neural folds) elevate,
causing the neural plate to roll into a neural tube. Neural crest
cells (green) delaminate from the neural folds or the dorsal
neural tube (shown), depending on the species and axial level.
Although the neural folds are viewed as ‘premigratory’
neural crest, only a subset of these cells will actually
migrate. Cell-marking experiments have shown that
progeny of individual cells within the neural folds can
contribute to the neural tube3–6 and epidermis5, as well
as to the neural crest. Even if cells are marked shortly
before migration initiates, labelled progeny are found in
both the neural tube and neural crest5. So, the neural
crest is not a defined cell population until the cells begin
to migrate. Instead, the earliest events in neural crest
development result in a population of cells in the neural
folds with the potential to become migratory neural
crest cells.
The formation of neural crest precursors at the
neural plate border involves several signalling events.
The neural plate forms first, followed by induction of its
border7. Several groups, using various organisms, have
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(BMPs) and
FIBROBLAST GROWTH FACTORS (FGFs) have all been shown in
various assays to mimic the tissue interactions that
induce neural crest. Another factor, Noelin, regulates the
timing of neural crest production. This topic has
recently been reviewed in detail elsewhere20, and will be
summarized only briefly here.
In birds, BMPs are both necessary21 and sufficient22
to induce neural crest and other dorsal neural tube cell
types from the neural plate, and for several years they
were believed to be the signal from the non-neural ectoderm that mediates the neural/non-neural ectoderm
interaction. However, additional work has shown that
neural crest formation requires BMP signalling only
after the initial induction step, indicating that BMPs
might serve a maintenance role in the induction
process23, or that they signal the emigration of neural
crest cells from the neural tube24. It now seems that the
inducing signal from the non-neural ectoderm is a Wnt
protein. Wnts are both necessary and sufficient for
robust induction of neural crest in isolated neural tissue,
and in birds, Wnt6 is expressed at the correct time
and in the right place in the non-neural ectoderm25.
In support of this idea, components of the Wnt
signalling pathway have been shown to be important
for neural crest formation in several different assays
and organisms26.
However, the role of Wnts and BMPs seems to be
slightly different in Xenopus and zebrafish than in birds.
In ANAMNIOTES, neural-inducing BMP antagonists, such as
Noggin and Chordin, generate a BMP signalling gradient
that specifies dorsoventral pattern in the ectoderm,
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Table 1 | Genes expressed in premigratory neural crest
Gene
Mouse
Chick
Fish
Frog
Ap2
110
112
113
111
Crestin
135
eif4a2
Foxd3
NP
Id2
54
56
X
53
126
Meis1b
108
Msx1
119
Msx2
119
Msxb/c
–
7
–
X
121
–
–
EPI
NC K/O
↓ function
↑ function
X
114,115
111
111
X
109
55
NF
X
122
135
X
109
X
ND
X
NP
53,54
126
X
108
X
X
123
X
X
NP
–
X
c-Myc
103
X
X
Nbx
107
X
X
53–55
NI
X
NI
103
107
107
97
97,98
Notch1
96
97
99
98
X
X
101
Pax3
89
22,90
88
90
X
X
6,94
Pax7
92
91
88
X
X
92
Rhob
138
137
Slug
–
43
–
Snail
43
–
Sox9
68
Sox10
70
69
Twist
133
131
Zic1
82
83,84
81
76,80
X
X
NP
76
Zic2
82
83
81
76,79
X
X
87
76,79
Zic3
82
–
81
75
X
NP
137
44
X
–
46–48
14,41
43
44
X
ND
42
41,42
64
65
X
64,68
65
63
66,67
X
63,72,73
67
130
X
132
66
75
X
X
NP
Zic5
77
X
X
NP
77
Zicr1
78
X
X
NP
78
Numbers indicate the references that contain the expression pattern. –, The gene is not expressed in the neural crest in that organism. X, The gene is expressed in the neural
plate (NP), the neural folds (NF) or the non-neural ectoderm (EPI); NC K/O, mouse or fish mutant phenotype in the neural crest; ↓ function, morpholino or dominant negative
phenotype; ↑ function, overexpression phenotype; ND, the mutant dies too early to determine the phenotype in the neural crest; NP, no phenotype in the neural crest; NI, the
phenotype with regard to the neural crest was not indicated and has not been examined. The numbers in the last three columns indicate the references in which the
phenotypes were reported.
with neural plate border cell types forming at intermediate levels of BMP signalling27. So, in Xenopus, partial
inhibition of BMP signalling together with activation of
Wnt signalling seems to mediate neural crest induction14,28,29. Work in zebrafish also supports a role for
both a BMP gradient30,31 and Wnts32 during neural crest
cell specification.
Also in Xenopus, FGF signalling can induce neural
crest in neuralized ectoderm14,33,34, albeit through a Wnt
intermediary14, and seems to be a component of
the neural crest-inducing signal from the paraxial
mesoderm15. Expression of FGF3, FGF4 and FGF8 has
been observed in the paraxial mesoderm7,15,35–38,
although only FGF8 can induce a subset of neural crest
markers in isolated Xenopus ectoderm without
additional factors15. Finally, Noelin is a secreted glycoprotein that seems to regulate the competence of the
neural folds to give rise to neural crest in avians39.
ZINC FINGER
A protein module in which
cysteine or cysteine–histidine
residues coordinate a zinc ion.
Zinc fingers are often used in
DNA recognition and in
protein–protein interactions.
…and their molecular targets
Less is known about the events downstream of the
signals that induce neural crest. A growing list of
genes (TABLE 1) has been found to be expressed in neural
crest precursors and to be necessary and/or sufficient to
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initiate neural crest development. As one might expect
from lineage analyses3–6, this list includes epidermal,
neural and neural crest markers7. However, as we
will discuss, the relationships between these genes are
not clear.
Neural crest markers. The first category of neural crest
genes is expressed almost exclusively in the neural folds.
These genes are typically used as markers of premigratory
neural crest.
The best understood neural crest marker is the
transcription factor Slug. Slug, and its close relative
Snail, comprise a family of ZINC-FINGER transcriptional
repressors40. Slug and Snail are functionally equivalent41,42 and, depending on the vertebrate, one or the
other is highly specific to the neural crest — in the
premigratory neural crest, chickens express only Slug,
mice and fish express only Snail 43, and Xenopus
expresses both44. Slug expression seems to be a direct
target of neural crest induction, as a functional Slug promoter contains a lymphoid enhancer-binding factor/Tcell factor (LEF/TCF) binding site45, which has been
shown to mediate the transcriptional response to Wnt
signalling in various systems26.
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EPITHELIAL–MESENCHYMAL
TRANSITIONS
(EMT). The transformation of
an epithelial cell into a
mesenchymal cell with
migratory and invasive
properties.
ADHERENS JUNCTION
A cell–cell junction also known
as zonula adherens, which is
characterized by the intracellular
insertion of microfilaments. If
intermediate filaments are
inserted in lieu of
microfilaments, the resulting
junction is referred to as a
desmosome.
TIGHT JUNCTIONS
Belt-like regions of adhesion
between adjacent epithelial or
endothelial cells. Tight junctions
regulate paracellular flux, and
contribute to the maintenance of
cell polarity by stopping
molecules from diffusing within
the plane of the membrane.
HIGH MOBILITY GROUP (HMG)
DOMAIN
A conserved domain that is
present in HMG proteins, which
are non-histone proteins
involved in chromatin structure
and gene regulation.
MORPHOLINO
An antisense oligonucleotide
that acts specifically to block the
initiation of translation.
DORSAL ROOT GANGLIA
The cell bodies of neural crestderived sensory neurons are
collected together in paired
ganglia that lie alongside the
spinal cord. These cell bodies are
surrounded by satellite glial cells,
which share much in common
with the Schwann cells that
ensheath peripheral axons. Very
few synapses have been observed
in these ganglia.
798
Slug/Snail activity is crucial at several stages during
early neural crest development. Overexpression expands
the neural crest-forming region14,41, whereas blocking
Slug and/or Snail function inhibits neural crest specification42,46 and migration46–48. The targets of Slug/Snail
during premigratory neural crest development
are not known. However, Slug and Snail have been
shown to regulate EPITHELIAL–MESENCHYMAL TRANSITIONS
(EMT) in cultured cells through direct repression of
E-cadherin49–51 and claudins/Occludin52, causing the
break-up of ADHERENS and TIGHT JUNCTIONS, respectively.
The ability of Slug/Snail to regulate the junctional
proteins that are expressed in the neural crest has not
been examined, but it is likely that they are also direct
regulators of EMT in the neural crest. Curiously, Slug
RNA is expressed in the neural folds long before migration actually initiates, and not all Slug-expressing cells
will become migratory neural crest cells44. Either a signal
or a newly expressed cofactor activates Slug function in a
subset of premigratory neural crest cells when it is time
to migrate. Indeed, there are probably factors in addition
to Slug/Snail that regulate EMT at trunk levels41.
Foxd3 is another gene whose expression is specific to
neural crest precursors in the ectoderm of all vertebrates53–56. In addition, it is weakly expressed in the paraxial mesoderm. Like Slug, Foxd3 gain-of-function expands
the neural crest field53–55 and loss-of-function ablates
neural crest precursors53,54. Foxd3 is expressed in undifferentiated embryonic stem cells57, and is required for
embryonic stem cell establishment and maintenance58.
On the basis of their homology to linker histones, it has
been postulated that winged-helix transcription factors
like Foxd3 bind to nucleosomes and open compacted
chromatin to potentiate transcription of target genes59.
Indeed, a protein related to Foxd3, FoxA, has been
shown to have such activity60. So, Foxd3 might regulate
the transcriptional accessibility of a collection of genes
that are responsible for the multipotency of neural crest
and other stem cells.
Sox9 and Sox10 are HIGH MOBILITY GROUP (HMG)-DOMAIN
transcriptional activators61,62 that are specific to the
premigratory neural crest and otic placode in frog and
fish63–67. Murine Sox9 is also expressed in premigratory
and migratory neural crest68, whereas Sox10 is initially
expressed just as neural crest migration initiates in
chick69 and mouse69,70. In Xenopus, Sox9 (REF. 65) or Sox10
(REF. 67) MORPHOLINO knockdown inhibits neural crest
specification. However, mouse or fish Sox9 mutants
have no defects in neural crest formation or migration64,68, and in mouse or fish Sox10 mutants, neural
crest cells are specified properly71, but undergo apoptosis
at the start of 63,72 or during72,73 migration. It is not clear if
these differences are due to experimental conditions, or
whether they represent true mechanistic variation
among vertebrates. Sox10 inhibits differentiation and
maintains stem cell potential74, and can synergize with
Pax3 in some neural crest-derived lineages71, although
premigratory neural crest has not been analysed in this
regard. Curiously, Sox10 undergoes nucleocytoplasmic
shuttling, and Sox9 contains identical regulatory
sequences for shuttling61. So, regulating the balance of
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nuclear import and export might provide a mechanism
to regulate the transcriptional activity of these proteins.
Markers for neural crest and neural plate. The next
category of early neural crest genes is more broadly
expressed than Slug, Foxd3 and the Sox genes, but is
sufficient and/or required for aspects of early neural
crest development. In this section, we will consider the
genes that are expressed in the neural crest and neighbouring neural plate, and in the next section, we will
consider those that are expressed in the neural crest and
neighbouring non-neural ectoderm.
The Zic genes (Zic1, Zicr1, Zic2, Zic3 and Zic5) are a
family of zinc-finger transcription factors that are
expressed in the neural crest and neural plate/dorsal
spinal cord75–83, although Zic5 is the most specific to
neural crest precursors77. The onset of Zic gene expression occurs early in the neural ectoderm, and this is
likely to be an early response to neural inducers75,78.
Overexpression of any Zic gene induces both neural and
neural crest-marker gene expression75–79, and inhibits
differentiation while promoting proliferation79,84,85. A
role in regulating proliferation and/or differentiation is
confirmed in Zic1 and Zic2 mutant mice, which exhibit
decreased proliferation84,86 and impaired differentiation of
the dorsal neural plate87 and cerebellum86, although these
mice do not have obvious neural crest defects except for
a reduction in the size of the DORSAL ROOT GANGLIA in Zic2–/–
mice87. It is not clear how the neural crest-specification
activity that is observed in Xenopus overexpression
assays fits with the mouse mutant phenotypes, but gene
redundancy could mask these effects in mice.
Pax3 (REFS 22,88–90) and Pax7 (REFS 88,91,92) are
transcriptional activators93 that are expressed in both the
neural crest and neural plate, with Pax3 being expressed
earlier than Pax7 in mice92. Mice that are mutant for
Pax3 or Pax7 display defects in various neural crest
derivatives92,94, and Pax3 mutants exhibit a decrease or
loss of migratory neural crest cells caudal to the otic
vesicle6. However, the requirement for Pax3 in the early
neural crest is non-cell-autonomous: neural crest cells
migrate from Pax3–/– neural tubes that have been transplanted to chick hosts6 or in vitro substrates95, and
Pax3–/– migratory neural crest cells are observed in wildtype/Pax3–/– chimeric mice89. Foxd3 is not expressed in
caudal regions of Pax3 mutant mice55, correlating with
the failure to form neural crest in these regions6.
However, Foxd3 expression has not been examined
in wild-type/Pax3–/– chimaeras. As migratory neural
crest is observed in chimaeras, it would be interesting
to see if a wild-type environment rescues Foxd3
expression in Pax3–/– premigratory and migratory
neural crest, or if neural crest cells are migrating without
expressing Foxd3.
Notch1 is broadly expressed in the neural plate,
although its expression is elevated in neural crest96–99.
When the Notch receptor binds one of its ligands such
as Delta, the intracellular domain is cleaved and translocates into the nucleus to activate transcription100.
Overexpressing an activated Notch ablates neural
crest markers and results in a loss of neural crest
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E-BOX
The conserved nucleotide
sequence CANNTG that is
recognized and bound by basic
helix–loop–helix and other
proteins.
HOMEOBOX
A sequence of about 180 base
pairs that encodes a DNAbinding protein sequence known
as the homeodomain. The 60amino-acid homeodomain
comprises three α-helices.
DOMINANT-NEGATIVE
A mutant molecule that
interferes with and inhibits the
activation of normal molecules.
BASIC HELIX–LOOP–HELIX
(bHLH). A structural motif
present in many transcription
factors that is characterized by
two α-helices separated by a
loop. The helices mediate
dimerization, and the adjacent
basic region is required for DNA
binding.
SOMITES
Paired blocks of mesoderm cells
along the vertebrate body axis
that form during early vertebrate
development and differentiate
into dermal skin, bone and
muscle.
RETROELEMENTS
Segments of genetic material
that transpose around the
genome using an RNA
intermediate.
derivatives97,98, yet Delta/Notch signalling is required for
neural crest formation97,101. So, finely tuned levels
of Delta/Notch signalling and/or exquisite temporal
regulation of Notch activity are needed in the neural
crest97. In zebrafish at least, Notch promotes neural
crest formation by inhibiting neurogenesis through
repression of Neurogenin-1 (Ngn1)101.
The proto-oncogene c-Myc, which stimulates proliferation and prevents differentiation102, was recently
shown to be expressed in neural crest precursors and
anterior neural plate in Xenopus, with expression in the
neural crest temporally preceding that of Slug103. Loss
of c-Myc activity inhibits neural crest-marker gene
expression and results in the loss of various neural crest
derivatives. In tissue culture, c-Myc is a target of Wnt
signalling104,105 and its expression in the neural crest
depends on Wnt signals103. c-Myc activates signal-dependent target gene expression through E-BOX binding and
histone H4 acetylation106, so like Foxd3, it might regulate
the transcriptional accessibility of a cohort of genes that
are necessary for early neural crest development.
Another recently identified neural crest marker with a
neural plate component is the NK-1 HOMEOBOX gene Nbx.
When overexpressed in Xenopus, this transcriptional
repressor expands neural crest at the expense of neural
plate, whereas a DOMINANT-NEGATIVE construct inhibits
neural crest formation107. Meis1b, a cofactor that
regulates the transcriptional activity of Hox proteins, also
seems to be sufficient to promote the formation of
neural crest — overexpression induces anterior neural
and neural crest markers in Xenopus108. Finally, the RNAhelicase translation initiation factor eif4a2 is expressed
in the neural plate and its border in Xenopus, and is
sufficient to induce neural and neural crest markers109.
Markers for neural crest and non-neural ectoderm. The
transcription factor Ap2 is an example of a neural crest
gene that is initially expressed throughout the neural
plate border and non-neural ectoderm, and that is later
enhanced in the neural folds in all vertebrates110–113.
Mice that are mutant for Ap2 show defects in neural
crest derivatives114,115, and in Xenopus, Ap2 is necessary
and sufficient to promote Slug and Sox9 gene
expression111. Ap2 is also required for epidermal development116. Expression of Ap2 in neural crest depends on
Wnt signalling111, indicating that it is a target of inducing
signals from the non-neural ectoderm. Ap2 fosters
proliferation by repressing genes that promote terminal
differentiation117. Ap2 and c-Myc interact to regulate
gene expression; for example, they activate or repress
transcription of E-cadherin depending on the relative
expression levels of the two different c-Myc isoforms118.
Msx1 and Msx2 are homeobox transcriptional
repressors119 that are also expressed in the neural folds
and, at lower levels, in neighbouring non-neural
ectoderm, with Msx2 being more restricted in its expression pattern119,120 than Msx1 (REFS 7,119,121). Zebrafish
Msx genes are not orthologous to Msx genes in other
vertebrates, although Msxb and Msxc also show expression at the borders of the neural plate122. Msx1 mutant
mice exhibit a loss of neural crest derivatives in the face,
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although the severity of the phenotype is probably
diminished by redundancy in Msx1 and Msx2 expression
and function123. Msx1 and Msx2 inhibit differentiation
without promoting proliferation124. Like Ap2, Msx1 has
also been implicated in epidermal induction, and is
induced by BMP121 and Wnt104 signalling.
Miscellaneous neural crest genes. Id2, a helix–loop–helix
(HLH) protein that negatively regulates basic HLH
(BHLH) proteins125, is expressed in rostral but not caudal
neural folds126, although we know little about the mechanisms that determine rostral/caudal differences in
the neural crest. Id2 has been implicated in neural
crest formation from the non-neural ectoderm126, and
in cultured cells it is a target of BMP127 and Wnt
signalling104, and of Pax3 (REF. 128) and Myc activity129.
Like many other neural crest factors, Id genes stimulate
proliferation and inhibit differentiation125.
Twist is a bHLH protein that is expressed in the
neural crest, SOMITES and lateral plate mesoderm130–132.
Twist is an early neural crest marker in Xenopus130,
whereas in the mouse, Twist is expressed in migrating
neural crest cells in the head133. Mouse mutants show
that Twist is required for neural crest migration and
differentiation, but not for neural crest specification132.
Twist is a direct target of Wnt signalling in murine
mammary epithelial cells134.
In zebrafish, a molecule called Crestin, which is a
member of a multi-copy family of RETROELEMENTS, is a
very specific marker of neural crest135. In addition, large
scale genetic screens have generated numerous mutant
fish lines with neural crest defects, although the genes
responsible have not been identified136.
Finally, Rhob is a target of BMP signalling that is
expressed in the dorsal neural tube137 and migrating
neural crest137,138. Rho activity is not required for neural
crest specification, but is necessary for the delamination
of neural crest cells137. Although Rho GTPases are
typically viewed as regulators of the actin cytoskeleton,
they have been implicated in many other processes,
including transcription and cell cycle progression139.
A minimal ‘network’?
As the list of genes expressed by premigratory neural
crest cells grows, there is an increasing need to define
the interrelationships between these genes (FIG. 2).
Overexpression analyses have not been altogether helpful
in this regard. For example, in Xenopus animal cap
assays, Foxd3 can activate Slug and Zicr1 expression, and
Zicr1 can induce Foxd3 and Slug expression53. Which
comes first, Foxd3 or Zicr1? To add to the confusion,
investigators tend to look at the same markers — Slug,
Sox9 and Foxd3. In many cases, including Foxd3 overexpression53, neural tissue is induced along with neural
crest. How do we know that neural crest is not induced
as a secondary consequence of the formation of ectopic
neural tissue, which itself induces neural crest?
Furthermore, overexpression assays have mostly been
performed in Xenopus, in which all neural crest genes
seem to turn on other neural crest genes. How can we
find order in this chaos?
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Neural crest
induction
Wnt
FGF
BMP
Premigratory
neural crest
Slug/Snail
Foxd3
Sox9, Sox10
Zic genes
Pax3, Pax7
c-Myc, Notch1
Ap2, Nbx
Msx2
Dlx5
Msx1
Border
induction
Neural plate
border
Neural crest
emigration
Rhob
N-cadherin
Figure 2 | The current status of a neural crest gene
regulatory network. The neural crest (yellow) and neural plate
border (orange) are induced as separate events regulated by
overlapping secreted factors. A complex set of genes are
expressed in the neural folds (green and blue) as a
consequence of these inductions. This developmental
programme leads to changes in gene expression (purple) that
result in the emigration of neural crest cells from the neural
tube. However, the interrelationships between neural crest
genes are only just beginning to be elucidated.
As a starting point, it is possible to identify trends in
our gene list (TABLE 1). In terms of function, many neural
crest genes have been shown to stimulate proliferation
and prevent differentiation (Zic genes79,84,85, Pax3 (REF.
140), c-Myc102, Ap2 (REF. 117), Msx1 and Msx2 (REF. 124), Id2
(REF. 125), Notch1 (REF. 101) and Twist 134) or maintain stem
cell potential (Foxd3 (REF. 58) and Sox10 (REF. 74)), both of
which are key characteristics of the neural crest lineage.
The list includes several transcriptional repressors
(Slug/Snail40, Zic1 (REF. 85), Nbx107, Msx1 and Msx2
(REF. 119) and Id2 (REF. 125)), as well as transcriptional
activators (Sox9 and Sox10 (REFS 61,62), Pax3 (REF. 93),
c-Myc102, Ap2 (REF. 141) and Notch1 (REF. 100)), indicating
that the formation of neural crest cells requires repression as well as activation of new gene expression.
Interestingly, with the exception of Pax3, all of the
transcriptional activators have been shown to bind to
CREB-binding protein (CBP)/p300 (REFS 142–146), as
does β-catenin, a downstream effector of Wnt
signalling147,148. Furthermore, CBP/p300 is a target
of Wnt signalling104. CBP and p300 are closely related
transcriptional co-activators that connect sequencespecific transcription factors to the general transcriptional machinery149. They are histone acetyltransferases
(HATs) and, along with Foxd3 and c-Myc, might have
roles in regulating the chromatin structure of neural
crest target genes. In another twist, the HAT activity of
p300 is inhibited by Twist150. CBP/p300 also regulate the
cell cycle in a complex containing Mdm2 (REF. 149),
which is preferentially expressed in the neural folds
and migrating neural crest151. Finally, Cited2 is a protein
that interacts with Ap2 and CBP/p300 to activate
transcription, and Cited2 mutant mice have various
neural crest defects152.
Another interesting correlation in the premigratory
neural crest gene list is the direct regulation of E-cadherin
expression. Expression of the cell adhesion molecule
E-cadherin characterizes most epithelial cells, although
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| OCTOBER 2003 | VOLUME 4
premigratory neural crest expresses N-cadherin and
cadherin-6b in lieu of E-cadherin153. During EMT,
including the transition from a tumour to a metastatic
cancer cell, E-cadherin is downregulated154. Likewise,
N-cadherin and cadherin-6b expression are downregulated during EMT at the onset of neural crest
migration153. E-cadherin and N-cadherin are functionally
equivalent155, although the mechanisms that regulate their
expression have not been compared. As Slug/Snail49–51,
Ap2 and c-Myc118 directly regulate E-cadherin expression,
it is possible that they also modulate N-cadherin and/
or cadherin-6b expression to initiate EMT at the start
of neural crest migration.
Although they provide a framework for formulating
future experiments, these correlations do not describe
the molecular mechanism by which migratory neural
crest cells are generated from the neural folds. How
can we distill this complex array of information into a
meaningful regulatory network?
Filling in the gaps
One way to create a clearer picture of early neural crest
development is to take a comprehensive approach.
Genomic-level screens could potentially identify the full
collection of genes that are involved in early neural crest
development. These genes can then be assembled into
functional networks. Researchers have been using
microarrays to achieve these goals for several years
now156. The advantage of using this approach for neural
crest development is that increasingly sophisticated
bioinformatic tools are constantly being generated to
identify relationships and infer pathway models from
array data156. What is truly exciting about the chick and
Xenopus genomic era, however, is the possibility of
combining powerful array technologies with the ability
to do experimental embryology. Equally enticing is the
intersection of vertebrate genetics, transgenics and
genomics in zebrafish.
As we alluded to earlier, documenting the complete
gene expression profile of a premigratory neural crest
cell is not a straightforward endeavour. The availability
of genomic tools for studying neural crest is not the only
obstacle. The neural folds contain a heterogeneous
population of cells, and neural crest precursors within
this population are multipotent, with the ability to give
rise to neural crest, neural tube and epidermis3–6. So, it is
not possible to simply purify cells from the neural folds
and characterize gene expression in those cells.
One study recently circumvented this problem to
identify a collection of genes that are expressed in neural
crest precursors at a single time-point following neural
crest induction157. First, by co-culturing pieces of avian
non-neural ectoderm and neural plate, neural crest precursors were induced in vitro5,10. Then, genes that were
expressed as a consequence of neural crest induction
were enriched in the cDNA population by subtracting
cDNA from non-neural ectoderm and neural
plate that had been cultured in isolation157. Because
chick microarrays were not available, the subtracted
cDNA was used to screen ‘macroarrays’ containing a
cDNA library synthesized from 4 to 12 somite embryos,
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REVIEWS
which was arrayed and spotted on nylon membranes.
Only clones of interest were sequenced on the macroarray, circumventing the need for the databases of
existing sequence information that are required for
microarray production.
The results of this screen have provided new markers
and regulators of neural crest development. For example,
the cDNA with the most specific expression pattern,
provisionally called precrest-1, has no homology to any
sequences in the database157. This gene is expressed at the
borders of the neural plate as soon as they are apparent,
earlier than Slug or Foxd3, and is probably a direct target
of early neural crest-inducing signals. Neuropilin-2a1, a
receptor for semaphorins and vascular endothelial
growth factor158, is also expressed in head and trunk
neural folds at very early stages in neural crest development, implying that this signalling pathway is involved
in neural crest cell specification and migration157.
The macroarray screen also emphasized the importance
of proliferation, chromatin remodelling, nucleocytoplasmic export, post-translational regulation and the
Rho pathway for the generation of migratory neural
crest cells.
Defining a neural crest gene regulatory network
EXPRESSED SEQUENCE TAGS
(ESTs). Short (200–500 base
pairs) DNA sequences that
represent the sequences
expressed in an organism under
a given condition. They are
generated from the 3′- and 5′ends of randomly selected cDNA
clones. The purpose of EST
sequencing is to scan for all the
protein-coding genes, and to
provide a tag for each gene on
the genome.
ELECTROPORATION
The transient generation of
pores in a cell membrane by
exposing the cell to a high field
strength electrical pulse. This
allows the entry of large
molecules, such as DNA
constructs, into the cell.
The macroarray screen did not, however, identify the
full complement of genes that are expressed at different
steps in early neural crest development, nor did it organize the neural crest gene list into a cascade or network.
To achieve these goals, a combination of approaches will
be required.
First, we can adopt a comparative strategy for the
molecular analysis of neural crest formation, taking
advantage of the microarrays and other genomic
resources that are beginning to be developed for all
vertebrates. Just as Slug and Snail gene expression patterns differ among vertebrates43, there are likely to be
variations in the molecular cascade of genes that specify
the neural crest. By comparing and contrasting vertebrate neural crest development, we can define the conserved events. The potential for mechanistic variation is
best exemplified by comparing neural induction in frog
and chick, where differences in the importance and
timing of the various factors that are involved help us to
understand the entire process more fully159. The chicken
genome project is well underway160, and chick EXPRESSED
SEQUENCE TAG (EST) databases are rapidly being generated161, so commercially produced microarrays of chick
ESTs are on the horizon. For future avian genomic
screens, it will probably be faster to screen avian
microarrays by differential hybridization, rather than
screening macroarrays with subtracted probe157.
Furthermore, there have been important advances
recently in the toolbox that is available for chick experimentation. For example, ELECTROPORATION allows ectopic
expression of DNA, including morpholino antisense
oligonucleotides and RNA-interference constructs for
loss-of-function analyses162. Microarrays that have
recently been developed in Xenopus163,164 and
zebrafish165,166 could be screened in a similar manner.
Many of the neural crest regulatory molecules that have
NATURE REVIEWS | NEUROSCIENCE
been defined in the past few years were initially identified and characterized in Xenopus, owing to the ease of
ectopic molecular manipulation in this organism.
Furthermore, regulatory pathways can be tested in
zebrafish and Xenopus using transgenics and/or genetic
approaches.
Second, neural crest can be induced in various ways,
and the subsequent changes in gene expression can be
documented using microarrays. Although we do not
know which events most closely mimic neural crest
induction in vivo, the use of different neural crest-inducing strategies should nevertheless allow the identification
of the most complete neural crest gene repertoire that is
possible. For example, neural crest can be induced by
co-culturing neural and non-neural tissue, by treating
competent neural tissue with Wnt or BMP, or by forcing
the expression of a key transcription factor (FIG. 3). In
addition, one could compare gene expression profiles
between wild type and mutant zebrafish or mouse
embryos with neural crest defects (FIG. 3). Data from
each successive screen can be assembled in the form of a
gene expression profile database for every gene in the
catalogue, giving an indication of how each neural crest
gene responds to different conditions, and allowing
coordinately regulated genes to be recognized. These
experiments will not only identify target genes, but will
also determine whether there are qualitative differences in
the neural crest induced by different treatments and transcription factors. Compiling these expression data and
comparing them with in vivo expression patterns might
help us to clarify what factors are required and when.
Third, the response to tissue interactions and treatments that induce neural crest could be documented
over time. If the activity of an inducing factor is temporally regulated through the use of inducible forms of
proteins167, or if tissue is harvested at regular time intervals, immediate early responses (direct targets) can be
characterized along with downstream changes in gene
expression. Furthermore, when array data is collected as
a function of time, co-regulated genes can be identified
using cluster analysis, in which genes are organized into
groups with similar expression profiles156. These
relationships can then be used to recognize regulatory
interactions.
The combination of tissue-specific and temporal
microarray data is already being applied to developmental problems. This approach was elegantly used in
Drosophila to define the networks of signalling pathways that regulate the response to ecdysone during
metamorphosis168. With regard to the neural crest, the
transcriptional response to Wnt104 and Pax3 (REF. 128)
has been documented in tissue culture cells using
human cDNA and oligonucleotide arrays, respectively.
By performing a time course of Wnt3a treatment in
embryonic carcinoma cells, it was possible to identify
genes that are likely to be direct targets, based on the
presence of TCF binding sites in almost all of the target
gene promoters104. Several neural crest genes were identified, including c-Myc, Id2, Msx1 and Msx2, confirming
the utility of this approach. The transcriptional
response to Pax3 was slightly more complex, as the cells
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REVIEWS
Chick intermediate neural plate
Electroporated
(For example, Slug,
Foxd3 and Sox9)
+/– inducing factor
(For example, Wnt
and BMP)
Array hybridization and analysis
+/– inducing tissue
(For example,
non-neural ectoderm)
Xenopus animal cap
Microinjected
(For example, Slug,
Foxd3 and Sox9)
+/– inducing factor
(For example, Wnt
and BMP inhibition)
+/– inducing tissue
(For example,
paraxial mesoderm
and neural plate)
Zebrafish
Neural crest mutant
versus wild type
Cy3/Cy5labelled RNA
1. Create a gene expression profile database of all
genes upregulated by neural crest-inducing treatments
2. Identify groups of genes with similar expression
profiles to predict direct targets and co-regulated genes
3. Test interrelationships with additional microarray
experiments
Morpholino injected
(For example, Snail,
Foxd3 and Sox9)
Figure 3 | Integrating embryology and genomics to define a neural crest gene regulatory network. Examples of various
protocols for inducing neural crest in chick and Xenopus. Microarray hybridization can be used to compare gene expression in
control tissue and induced neural crest tissue; for example, in chick intermediate neural plate that has been electroporated with a
control construct or with a transcription factor that initiates the neural crest programme. Gene expression in zebrafish neural crest
mutants can also be compared. The resulting gene expression profiles can then be compiled, analysed and tested further.
that were used were stably transfected with Pax3 (REF. 128).
However, many of the genes that were upregulated in
this screen contained putative Pax3 binding sites. These
screens emphasize the need to perform time courses to
characterize both direct targets and the subsequent
downstream effects.
Once a cohort of neural crest genes has been identified, an early neural crest-specific microarray can be
generated. This reagent would facilitate future experiments to define the neural crest gene regulatory network,
as only the genes that are involved in the process under
investigation would be included. This type of reagent has
been created to study neural crest-derived melanocyte
differentiation by selecting genes with particular expression profiles from mouse EST databases169. A neural
crest-specific microarray is not an essential requirement,
however, as it is possible to analyse only those clones that
are of interest on a global microarray.
To determine how different factors are interrelated,
the effect of one gene or condition on the expression of
all neural crest genes could be tested. Gain- and loss-offunction techniques should allow the identification
of downstream targets, as well as potential regulatory
interactions. Microarray screens of Drosophila and
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| OCTOBER 2003 | VOLUME 4
Caenorhabditis elegans mutant embryos have identified
target genes in an analogous manner170,171. The most
comprehensive example of a gene regulatory network to
be synthesized from this type of information is the network that underlies endomesoderm specification in the
sea urchin172. This complex network of transcription
factors, signalling pathways and their targets has been
compiled by experimentally testing interconnections by
loss-of-function approaches.
Finally, to definitively characterize regulatory interactions, it will be necessary to dissect elements in the
regulatory regions of key neural crest gene targets.
Promoter constructs can be assayed quite effectively by
electroporation into a tissue of interest in chicken
embryos85,173. Meanwhile, cis regulatory elements can be
assessed using transient and stable transgenic approaches
in frogs174 and fish175. In addition, the chick160, Xenopus176,
and zebrafish177 genome projects will place the genomic
sequence of all genes and their flanking regions into the
public domain. This will allow defined enhancer
elements to be annotated and putative regulatory regions
to be computationally identified as shared sequence
motifs in coordinately regulated genes168,178. Complete
genomic sequences will also permit the construction of
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REVIEWS
intergenic sequence microarrays, which can be used
to identify protein binding sites and reveal direct
regulatory interactions on a genome scale179,180.
Conclusions
To date, neural crest development has been best studied
in chick and frog due to their accessibility and ease of
manipulation. Although differences exist between
species, the assumption is that the most crucial mechanisms must be conserved across vertebrates, including
mammals. In general, mouse null mutations have
shown that neural crest genes have largely homologous
roles in the chick, fish and frog (for example, transcription factor Ap2 (REFS 111,114,115), Sox10 (REFS 63,67,71–73)
and the Zic gene family75–79,84–87), although interesting
switches in gene usage occur between paralogues
(for example, Slug/Snail43). The ultimate goal is to
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Acknowledgements
The authors would like to thank S. Fraser, M. García-Castro, V. Lee, Y.
Marahrens and L. Ziemer for critical comments on the manuscript,
and the Bronner-Fraser lab for insightful discussions. L.S.G.
is supported by a K22 Career Transition Award from the NIH.
Work in M.B.F.’s lab is supported, in part, by grants from NIH
and NASA.
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
Ap2 | BMPs | β-catenin | cadherin-6 | CBP | Cited2 | claudins |
Crestin | Delta | Dlx5 | E-cadherin | FGFs | Foxa | Foxd3 | Id2 |
Mdm2 | Msx1 | Msx2 | Msxb | Msxc | c-Myc | N-cadherin | Ngn1 |
Noelin | Notch1 | Occludin | p300 | Pax3 | Pax7 | Rhob | Slug |
Snail | Sox9 | Sox10 | Twist | Wnts | Zic1 | Zic2 | Zic3 | Zic5
Xenbase: http://www.xenbase.org/
nbx
TIGR Gallus gallus Gene Index:
http://www.tigr.org/tdb/tgi/gggi/
Chordin | Noggin
TIGR Xenopus laevis Gene Index:
http://www.tigr.org/tdb/tgi/xgi/
eif4a2 | Meis1b | Zicr1 |
Access to this interactive links box is free online.
VOLUME 4 | OCTOBER 2003 | 8 0 5