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Neural crest cells are the great explorers of the verte-brate embryo. Although they have the potential toremain in the central nervous system at the site of theirbirth, they strike off on their own and colonize the farreaches of the embryo. These wandering cells form,among other things, peripheral neurons, glia, connectivetissue, bone, secretory cells and the outflow tract of theheart. We are only just beginning to understand howmigratory neural crest cells arise and maintain theirability to form such diverse cell types.

Many of the properties that make neural crest cellsso interesting to study also make their molecular characterization a difficult task. As the neural crest is avertebrate invention1, it cannot be studied in yeast,worms or flies, which are the traditional organisms forrapid, large scale genetic screens. Furthermore, neuralcrest cells form during the early stages of embryonicdevelopment, when mouse embryos are small and diffi-cult to manipulate. Historically, the neural crest hasbeen studied in accessible, externally developing avianand amphibian embryos. The resulting classical embry-ological literature provides us with a detailed account ofwhere neural crest cells arise, which tissues send signalsto cause neural crest formation, and what cell types theywill form1. Until recently, however, this rich descriptivehistory of the neural crest has been counterbalanced bya 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

sequencing projects in chick, frog and zebrafish, this israpidly changing.

In this article, we will provide an overview of earlyneural crest development, paying particular attention toour current understanding of the gene network thatregulates this process. We will then consider howgenomic techniques are changing this view, describingrecent advances and highlighting future possibilities.

Life at the marginsIn the earliest phase of neural development, neural tissue is induced in the ectodermal (outer) layer of theembryo. As a consequence of neural induction, theectoderm becomes divided into three different regions— the neural ectoderm or neural plate, which will giverise to the central nervous system; the non-neural ectoderm, which will form epidermis; and the cells atthe 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 1868by His, who described chick neural crest as a ‘zwischen-strang’ or ‘in-between strip’, lying between the neuraland non-neural ectoderm2. Neural tissue folds in onitself to form the neural tube in a process called neurulation. During neurulation, the neural plate bordercells bend to form the neural folds and eventuallybecome the dorsal aspect of the neural tube. Dependingon the organism and the axial level, neural crest cellsinitiate migration from the closing neural folds or fromthe dorsal neural tube.

NEURAL CREST SPECIFICATION:MIGRATING INTO GENOMICSLaura S. Gammill and Marianne Bronner-Fraser

The bones in your face, the pigment in your skin and the neural circuitry that controls your digestivetract have one thing in common: they are all derived from neural crest cells. The formation of thesemigratory multipotent cells poses an interesting developmental problem, as neural crest cells arenot a distinct cell type until they migrate away from the central nervous system. What defines thepool 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 ofTechnology, Pasadena,California 91125, USA.e-mails:lgammill@caltech.edu;mbronner@caltech.edu.doi:10.1038/nrn1219

WNT PROTEINS

A family of highly conservedsecreted signalling molecules,which are related to theDrosophila wingless protein andregulate cell–cell interactionsduring embryogenesis.Wntproteins bind on the cell surfaceto receptors of the Frizzled family.

BONE MORPHOGENETIC

PROTEINS

(BMPs). Multifunctional secretedproteins of the transforminggrowth factor-β superfamily. Inthe early embryo, they participatein dorsoventral patterning.

FIBROBLAST GROWTH FACTORS

(FGFs). Multifunctional factorsthat are involved in embryonicdevelopment. More than 20FGFs and 4 FGF receptors havebeen described. Theircoordinated activity controls cellproliferation, migration, survivaland differentiation. FGFsregulate growth andmorphogenesis by an earlyaction on regional patterning,and a later effect on the growthof progenitor cells of theforebrain.

ANAMNIOTES

Vertebrates, such as fish andamphibians, that do not developinside an amnion.

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shown that the neural plate border and neural crest cellsform in response to signalling between newly inducedneural tissue and the neighbouring non-neural ectoderm5,7–10. Signals from the underlying paraxialmesoderm are also involved in inducing the borderregion5,11–15. It is not clear, however, whether these inter-actions occur coincidentally or sequentially, or whetherthey normally serve inductive or maintenance roles inthe embryo.

To complicate matters further, recent evidence indicates that border induction and neural crest induction are not necessarily the same process. Thetranscription factor Dlx5 is one of the earliest markers ofthe neural plate border16–18, and ectopic expression ofDlx5 in the neural plate results in non-cell-autonomousinduction of neural plate border gene expression withoutthe induction of neural crest markers18. Furthermore,when Dlx activity is inhibited in the non-neural ecto-derm, neural plate border genes are expressed in theirnormal patterns, but are shifted laterally19. Together, theseobservations indicate that Dlx proteins act to specifythe border region. Interestingly, Dlx activity is requiredfor the interaction between neural and non-neural ectoderm to induce neural crest19. So, Dlx-dependentinduction of an unspecified border region is a requisitefirst step for the formation of border cell types, butneural crest induction requires additional signals.

Secreted factors that induce neural crest…WNT PROTEINS, BONE MORPHOGENETIC PROTEINS (BMPs) andFIBROBLAST GROWTH FACTORS (FGFs) have all been shown invarious assays to mimic the tissue interactions thatinduce neural crest. Another factor, Noelin, regulates thetiming of neural crest production. This topic hasrecently been reviewed in detail elsewhere20, and will besummarized only briefly here.

In birds, BMPs are both necessary21 and sufficient22

to induce neural crest and other dorsal neural tube celltypes from the neural plate, and for several years theywere believed to be the signal from the non-neural ecto-derm that mediates the neural/non-neural ectoderminteraction. However, additional work has shown thatneural crest formation requires BMP signalling onlyafter the initial induction step, indicating that BMPsmight serve a maintenance role in the inductionprocess23, or that they signal the emigration of neuralcrest cells from the neural tube24. It now seems that theinducing signal from the non-neural ectoderm is a Wntprotein. Wnts are both necessary and sufficient forrobust 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 beslightly different in Xenopus and zebrafish than in birds.In ANAMNIOTES, neural-inducing BMP antagonists, such asNoggin and Chordin, generate a BMP signalling gradientthat specifies dorsoventral pattern in the ectoderm,

Although the neural folds are viewed as ‘premigratory’neural crest, only a subset of these cells will actuallymigrate. Cell-marking experiments have shown thatprogeny of individual cells within the neural folds cancontribute to the neural tube3–6 and epidermis5, as wellas to the neural crest. Even if cells are marked shortlybefore migration initiates, labelled progeny are found inboth the neural tube and neural crest5. So, the neuralcrest is not a defined cell population until the cells beginto migrate. Instead, the earliest events in neural crestdevelopment result in a population of cells in the neuralfolds with the potential to become migratory neuralcrest cells.

The formation of neural crest precursors at theneural plate border involves several signalling events.The neural plate forms first, followed by induction of itsborder7. Several groups, using various organisms, have

Neural plate borderNeuroectoderm

Neural fold

Neuralcrest cells

Neural tubeNotochord

Non-neuralectoderm

Paraxialmesoderm

Somite

Neural plate

Figure 1 | Border induction and neurulation. The neuralplate border (green) is induced by signalling between theneuroectoderm (purple) and the non-neural ectoderm (blue)and from the underlying paraxial mesoderm (yellow). Duringneurulation, the neural plate borders (neural folds) elevate,causing the neural plate to roll into a neural tube. Neural crestcells (green) delaminate from the neural folds or the dorsalneural tube (shown), depending on the species and axial level.

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initiate neural crest development. As one might expectfrom lineage analyses3–6, this list includes epidermal,neural and neural crest markers7. However, as we will discuss, the relationships between these genes arenot clear.

Neural crest markers. The first category of neural crestgenes is expressed almost exclusively in the neural folds.These genes are typically used as markers of premigratoryneural crest.

The best understood neural crest marker is the transcription factor Slug. Slug, and its close relativeSnail, comprise a family of ZINC-FINGER transcriptionalrepressors40. Slug and Snail are functionally equiva-lent41,42 and, depending on the vertebrate, one or theother is highly specific to the neural crest — in the premigratory neural crest, chickens express only Slug,mice and fish express only Snail43, and Xenopusexpresses both44. Slug expression seems to be a directtarget of neural crest induction, as a functional Slug pro-moter contains a lymphoid enhancer-binding factor/T-cell factor (LEF/TCF) binding site45, which has beenshown to mediate the transcriptional response to Wntsignalling in various systems26.

with neural plate border cell types forming at inter-mediate levels of BMP signalling27. So, in Xenopus, partialinhibition of BMP signalling together with activation ofWnt signalling seems to mediate neural crest induc-tion14,28,29. Work in zebrafish also supports a role forboth a BMP gradient30,31 and Wnts32 during neural crestcell specification.

Also in Xenopus, FGF signalling can induce neuralcrest in neuralized ectoderm14,33,34, albeit through a Wntintermediary14, and seems to be a component ofthe neural crest-inducing signal from the paraxial mesoderm15. Expression of FGF3, FGF4 and FGF8 hasbeen observed in the paraxial mesoderm7,15,35–38,although only FGF8 can induce a subset of neural crestmarkers in isolated Xenopus ectoderm without additional factors15. Finally, Noelin is a secreted glyco-protein that seems to regulate the competence of theneural folds to give rise to neural crest in avians39.

…and their molecular targetsLess is known about the events downstream of the signals that induce neural crest. A growing list ofgenes (TABLE 1) has been found to be expressed in neuralcrest precursors and to be necessary and/or sufficient to

ZINC FINGER

A protein module in whichcysteine or cysteine–histidineresidues coordinate a zinc ion.Zinc fingers are often used inDNA recognition and inprotein–protein interactions.

Table 1 | Genes expressed in premigratory neural crest

Gene Mouse Chick Fish Frog NP NF EPI NC K/O ↓↓ function ↑↑ function

Ap2 110 112 113 111 X X 114,115 111 111

Crestin 135 X 135

eif4a2 109 X X 109

Foxd3 55 54 56 53 X ND 53,54 53–55

Id2 126 X NP 126

Meis1b 108 X X 108

Msx1 119 7 – 121 X X 123 NI

Msx2 119 – X X NP

Msxb/c – – 122 – X X

c-Myc 103 X X NI 103

Nbx 107 X X 107 107

Notch1 96 97 99 98 X X 101 97 97,98

Pax3 89 22,90 88 90 X X 6,94

Pax7 92 91 88 X X 92

Rhob 138 137 X NP 137

Slug – 43 – 44 X – 46–48 14,41

Snail 43 – 43 44 X ND 42 41,42

Sox9 68 64 65 X 64,68 65

Sox10 70 69 63 66,67 X 63,72,73 67 66

Twist 133 131 130 X 132

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 X NP 75

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 neuralplate (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 negativephenotype; ↑ 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, thephenotype 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 thephenotypes were reported.

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nuclear import and export might provide a mechanismto regulate the transcriptional activity of these proteins.

Markers for neural crest and neural plate. The next category of early neural crest genes is more broadlyexpressed than Slug, Foxd3 and the Sox genes, but is sufficient and/or required for aspects of early neuralcrest development. In this section, we will consider thegenes that are expressed in the neural crest and neigh-bouring neural plate, and in the next section, we willconsider those that are expressed in the neural crest andneighbouring non-neural ectoderm.

The Zic genes (Zic1, Zicr1, Zic2, Zic3 and Zic5) are afamily of zinc-finger transcription factors that areexpressed in the neural crest and neural plate/dorsalspinal cord75–83, although Zic5 is the most specific toneural crest precursors77. The onset of Zic gene expres-sion occurs early in the neural ectoderm, and this islikely to be an early response to neural inducers75,78.Overexpression of any Zic gene induces both neural andneural crest-marker gene expression75–79, and inhibitsdifferentiation while promoting proliferation79,84,85. Arole in regulating proliferation and/or differentiation isconfirmed in Zic1 and Zic2 mutant mice, which exhibitdecreased proliferation84,86 and impaired differentiation ofthe dorsal neural plate87 and cerebellum86, although thesemice 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-specificationactivity that is observed in Xenopus overexpressionassays fits with the mouse mutant phenotypes, but generedundancy 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 theneural crest and neural plate, with Pax3 being expressedearlier than Pax7 in mice92. Mice that are mutant forPax3 or Pax7 display defects in various neural crestderivatives92,94, and Pax3 mutants exhibit a decrease orloss of migratory neural crest cells caudal to the oticvesicle6. However, the requirement for Pax3 in the earlyneural crest is non-cell-autonomous: neural crest cellsmigrate from Pax3–/– neural tubes that have been trans-planted to chick hosts6 or in vitro substrates95, andPax3–/– migratory neural crest cells are observed in wild-type/Pax3–/– chimeric mice89. Foxd3 is not expressed incaudal regions of Pax3 mutant mice55, correlating withthe failure to form neural crest in these regions6.However, Foxd3 expression has not been examined in wild-type/Pax3–/– chimaeras. As migratory neuralcrest is observed in chimaeras, it would be interesting to see if a wild-type environment rescues Foxd3expression in Pax3–/– premigratory and migratoryneural crest, or if neural crest cells are migrating withoutexpressing 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 suchas Delta, the intracellular domain is cleaved and translo-cates into the nucleus to activate transcription100.Overexpressing an activated Notch ablates neural crest markers and results in a loss of neural crest

Slug/Snail activity is crucial at several stages duringearly neural crest development. Overexpression expandsthe neural crest-forming region14,41, whereas blockingSlug and/or Snail function inhibits neural crest specifi-cation42,46 and migration46–48. The targets of Slug/Snailduring premigratory neural crest development are not known. However, Slug and Snail have beenshown to regulate EPITHELIAL–MESENCHYMAL TRANSITIONS

(EMT) in cultured cells through direct repression ofE-cadherin49–51 and claudins/Occludin52, causing thebreak-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 notbeen examined, but it is likely that they are also directregulators of EMT in the neural crest. Curiously, SlugRNA is expressed in the neural folds long before migra-tion actually initiates, and not all Slug-expressing cellswill become migratory neural crest cells44. Either a signalor a newly expressed cofactor activates Slug function in asubset of premigratory neural crest cells when it is timeto migrate. Indeed, there are probably factors in additionto Slug/Snail that regulate EMT at trunk levels41.

Foxd3 is another gene whose expression is specific toneural crest precursors in the ectoderm of all verte-brates53–56. In addition, it is weakly expressed in the parax-ial mesoderm. Like Slug, Foxd3 gain-of-function expandsthe neural crest field53–55 and loss-of-function ablatesneural crest precursors53,54. Foxd3 is expressed in undiffer-entiated embryonic stem cells57, and is required forembryonic stem cell establishment and maintenance58.On the basis of their homology to linker histones, it hasbeen postulated that winged-helix transcription factorslike Foxd3 bind to nucleosomes and open compacted chromatin to potentiate transcription of target genes59.Indeed, a protein related to Foxd3, FoxA, has beenshown to have such activity60. So, Foxd3 might regulatethe transcriptional accessibility of a collection of genesthat are responsible for the multipotency of neural crestand 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 andfish63–67. Murine Sox9 is also expressed in premigratoryand migratory neural crest68, whereas Sox10 is initiallyexpressed just as neural crest migration initiates inchick69 and mouse69,70. In Xenopus, Sox9 (REF. 65) or Sox10(REF. 67) MORPHOLINO knockdown inhibits neural crestspecification. However, mouse or fish Sox9 mutantshave no defects in neural crest formation or migra-tion64,68, and in mouse or fish Sox10 mutants, neuralcrest cells are specified properly71, but undergo apoptosisat the start of63,72 or during72,73 migration. It is not clear ifthese differences are due to experimental conditions, orwhether they represent true mechanistic variationamong vertebrates. Sox10 inhibits differentiation andmaintains stem cell potential74, and can synergize withPax3 in some neural crest-derived lineages71, althoughpremigratory neural crest has not been analysed in thisregard. Curiously, Sox10 undergoes nucleocytoplasmicshuttling, and Sox9 contains identical regulatorysequences for shuttling61. So, regulating the balance of

EPITHELIAL–MESENCHYMAL

TRANSITIONS

(EMT). The transformation ofan epithelial cell into amesenchymal cell withmigratory and invasiveproperties.

ADHERENS JUNCTION

A cell–cell junction also knownas zonula adherens, which ischaracterized by the intracellularinsertion of microfilaments. Ifintermediate filaments areinserted in lieu ofmicrofilaments, the resultingjunction is referred to as adesmosome.

TIGHT JUNCTIONS

Belt-like regions of adhesionbetween adjacent epithelial orendothelial cells. Tight junctionsregulate paracellular flux, andcontribute to the maintenance ofcell polarity by stoppingmolecules from diffusing withinthe plane of the membrane.

HIGH MOBILITY GROUP (HMG)

DOMAIN

A conserved domain that ispresent in HMG proteins, whichare non-histone proteinsinvolved in chromatin structureand gene regulation.

MORPHOLINO

An antisense oligonucleotidethat acts specifically to block theinitiation of translation.

DORSAL ROOT GANGLIA

The cell bodies of neural crest-derived sensory neurons arecollected together in pairedganglia that lie alongside thespinal cord. These cell bodies aresurrounded by satellite glial cells,which share much in commonwith the Schwann cells thatensheath peripheral axons.Veryfew synapses have been observedin these ganglia.

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although the severity of the phenotype is probablydiminished by redundancy in Msx1 and Msx2 expressionand function123. Msx1 and Msx2 inhibit differentiationwithout promoting proliferation124. Like Ap2, Msx1 hasalso been implicated in epidermal induction, and isinduced 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 caudalneural folds126, although we know little about the mecha-nisms 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 stimulateproliferation and inhibit differentiation125.

Twist is a bHLH protein that is expressed in theneural crest, SOMITES and lateral plate mesoderm130–132.Twist is an early neural crest marker in Xenopus130,whereas in the mouse, Twist is expressed in migratingneural crest cells in the head133. Mouse mutants showthat Twist is required for neural crest migration and differentiation, but not for neural crest specification132.Twist is a direct target of Wnt signalling in murinemammary epithelial cells134.

In zebrafish, a molecule called Crestin, which is amember of a multi-copy family of RETROELEMENTS, is avery specific marker of neural crest135. In addition, largescale genetic screens have generated numerous mutantfish lines with neural crest defects, although the genesresponsible have not been identified136.

Finally, Rhob is a target of BMP signalling that isexpressed in the dorsal neural tube137 and migratingneural crest137,138. Rho activity is not required for neuralcrest specification, but is necessary for the delaminationof 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 neuralcrest cells grows, there is an increasing need to define the interrelationships between these genes (FIG. 2).Overexpression analyses have not been altogether helpfulin this regard. For example, in Xenopus animal capassays, Foxd3 can activate Slug and Zicr1 expression, andZicr1 can induce Foxd3 and Slug expression53. Whichcomes 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 overex-pression53, neural tissue is induced along with neuralcrest. How do we know that neural crest is not inducedas a secondary consequence of the formation of ectopicneural tissue, which itself induces neural crest?Furthermore, overexpression assays have mostly beenperformed in Xenopus, in which all neural crest genesseem to turn on other neural crest genes. How can wefind order in this chaos?

derivatives97,98, yet Delta/Notch signalling is required forneural crest formation97,101. So, finely tuned levels of Delta/Notch signalling and/or exquisite temporal regulation of Notch activity are needed in the neuralcrest97. In zebrafish at least, Notch promotes neural crest formation by inhibiting neurogenesis throughrepression of Neurogenin-1 (Ngn1)101.

The proto-oncogene c-Myc, which stimulates prolif-eration and prevents differentiation102, was recentlyshown to be expressed in neural crest precursors andanterior neural plate in Xenopus, with expression in theneural crest temporally preceding that of Slug103. Loss of c-Myc activity inhibits neural crest-marker geneexpression and results in the loss of various neural crestderivatives. In tissue culture, c-Myc is a target of Wnt signalling104,105 and its expression in the neural crestdepends on Wnt signals103. c-Myc activates signal-depen-dent target gene expression through E-BOX binding andhistone H4 acetylation106, so like Foxd3, it might regulatethe transcriptional accessibility of a cohort of genes thatare necessary for early neural crest development.

Another recently identified neural crest marker with aneural plate component is the NK-1 HOMEOBOX gene Nbx.When overexpressed in Xenopus, this transcriptionalrepressor expands neural crest at the expense of neuralplate, whereas a DOMINANT-NEGATIVE construct inhibitsneural crest formation107. Meis1b, a cofactor that regulates the transcriptional activity of Hox proteins, alsoseems to be sufficient to promote the formation ofneural crest — overexpression induces anterior neuraland neural crest markers in Xenopus108. Finally, the RNA-helicase 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. Thetranscription factor Ap2 is an example of a neural crestgene that is initially expressed throughout the neuralplate border and non-neural ectoderm, and that is laterenhanced in the neural folds in all vertebrates110–113.Mice that are mutant for Ap2 show defects in neuralcrest derivatives114,115, and in Xenopus, Ap2 is necessaryand sufficient to promote Slug and Sox9 geneexpression111. Ap2 is also required for epidermal develop-ment116. Expression of Ap2 in neural crest depends onWnt signalling111, indicating that it is a target of inducingsignals from the non-neural ectoderm. Ap2 fosters proliferation by repressing genes that promote terminaldifferentiation117. Ap2 and c-Myc interact to regulategene expression; for example, they activate or repress transcription of E-cadherin depending on the relativeexpression levels of the two different c-Myc isoforms118.

Msx1 and Msx2 are homeobox transcriptionalrepressors119 that are also expressed in the neural foldsand, at lower levels, in neighbouring non-neural ectoderm, with Msx2 being more restricted in its expres-sion pattern119,120 than Msx1 (REFS 7,119,121). ZebrafishMsx genes are not orthologous to Msx genes in othervertebrates, although Msxb and Msxc also show expres-sion at the borders of the neural plate122. Msx1 mutantmice exhibit a loss of neural crest derivatives in the face,

E-BOX

The conserved nucleotidesequence CANNTG that isrecognized and bound by basichelix–loop–helix and otherproteins.

HOMEOBOX

A sequence of about 180 basepairs that encodes a DNA-binding protein sequence knownas the homeodomain. The 60-amino-acid homeodomaincomprises three α-helices.

DOMINANT-NEGATIVE

A mutant molecule thatinterferes with and inhibits theactivation of normal molecules.

BASIC HELIX–LOOP–HELIX

(bHLH). A structural motifpresent in many transcriptionfactors that is characterized bytwo α-helices separated by aloop. The helices mediatedimerization, and the adjacentbasic region is required for DNAbinding.

SOMITES

Paired blocks of mesoderm cellsalong the vertebrate body axisthat form during early vertebratedevelopment and differentiateinto dermal skin, bone andmuscle.

RETROELEMENTS

Segments of genetic materialthat transpose around thegenome using an RNAintermediate.

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premigratory neural crest expresses N-cadherin and cadherin-6b in lieu of E-cadherin153. During EMT,including the transition from a tumour to a metastaticcancer cell, E-cadherin is downregulated154. Likewise,N-cadherin and cadherin-6b expression are down-regulated during EMT at the onset of neural crest migration153. E-cadherin and N-cadherin are functionallyequivalent155, although the mechanisms that regulate theirexpression 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 formulatingfuture experiments, these correlations do not describethe molecular mechanism by which migratory neuralcrest cells are generated from the neural folds. How can we distill this complex array of information into ameaningful regulatory network?

Filling in the gapsOne way to create a clearer picture of early neural crestdevelopment is to take a comprehensive approach.Genomic-level screens could potentially identify the fullcollection of genes that are involved in early neural crestdevelopment. These genes can then be assembled intofunctional networks. Researchers have been usingmicroarrays to achieve these goals for several yearsnow156. The advantage of using this approach for neuralcrest development is that increasingly sophisticatedbioinformatic tools are constantly being generated toidentify relationships and infer pathway models fromarray data156. What is truly exciting about the chick andXenopus genomic era, however, is the possibility ofcombining powerful array technologies with the abilityto do experimental embryology. Equally enticing is theintersection of vertebrate genetics, transgenics andgenomics in zebrafish.

As we alluded to earlier, documenting the completegene expression profile of a premigratory neural crestcell is not a straightforward endeavour. The availabilityof genomic tools for studying neural crest is not the onlyobstacle. The neural folds contain a heterogeneous population of cells, and neural crest precursors withinthis population are multipotent, with the ability to giverise to neural crest, neural tube and epidermis3–6. So, it isnot possible to simply purify cells from the neural foldsand characterize gene expression in those cells.

One study recently circumvented this problem toidentify a collection of genes that are expressed in neuralcrest precursors at a single time-point following neuralcrest induction157. First, by co-culturing pieces of aviannon-neural ectoderm and neural plate, neural crest pre-cursors were induced in vitro5,10. Then, genes that wereexpressed as a consequence of neural crest inductionwere enriched in the cDNA population by subtractingcDNA from non-neural ectoderm and neural plate that had been cultured in isolation157. Because chick microarrays were not available, the subtractedcDNA was used to screen ‘macroarrays’ containing acDNA library synthesized from 4 to 12 somite embryos,

As a starting point, it is possible to identify trends inour gene list (TABLE 1). In terms of function, many neuralcrest genes have been shown to stimulate proliferationand 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 stemcell potential (Foxd3 (REF. 58) and Sox10 (REF. 74)), both ofwhich 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)), indicatingthat the formation of neural crest cells requires repres-sion as well as activation of new gene expression.

Interestingly, with the exception of Pax3, all of thetranscriptional activators have been shown to bind toCREB-binding protein (CBP)/p300 (REFS 142–146), asdoes β-catenin, a downstream effector of Wntsignalling147,148. Furthermore, CBP/p300 is a target of Wnt signalling104. CBP and p300 are closely relatedtranscriptional co-activators that connect sequence-specific transcription factors to the general transcrip-tional machinery149. They are histone acetyltransferases(HATs) and, along with Foxd3 and c-Myc, might haveroles in regulating the chromatin structure of neuralcrest target genes. In another twist, the HAT activity ofp300 is inhibited by Twist150. CBP/p300 also regulate thecell cycle in a complex containing Mdm2 (REF. 149),which is preferentially expressed in the neural folds and migrating neural crest151. Finally, Cited2 is a proteinthat interacts with Ap2 and CBP/p300 to activate transcription, and Cited2 mutant mice have variousneural crest defects152.

Another interesting correlation in the premigratoryneural crest gene list is the direct regulation of E-cadherinexpression. Expression of the cell adhesion molecule E-cadherin characterizes most epithelial cells, although

Neural crestinduction

WntFGFBMP

Dlx5

Borderinduction

Premigratoryneural crest

Slug/SnailFoxd3

Sox9, Sox10Zic genes

Pax3, Pax7c-Myc, Notch1

Ap2, NbxMsx2

Msx1

Neural plateborder

Neural crestemigration

RhobN-cadherin

Figure 2 | The current status of a neural crest generegulatory network. The neural crest (yellow) and neural plateborder (orange) are induced as separate events regulated byoverlapping secreted factors. A complex set of genes areexpressed in the neural folds (green and blue) as aconsequence of these inductions. This developmentalprogramme leads to changes in gene expression (purple) thatresult in the emigration of neural crest cells from the neuraltube. However, the interrelationships between neural crestgenes are only just beginning to be elucidated.

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been defined in the past few years were initially identi-fied and characterized in Xenopus, owing to the ease ofectopic molecular manipulation in this organism.Furthermore, regulatory pathways can be tested inzebrafish and Xenopus using transgenics and/or geneticapproaches.

Second, neural crest can be induced in various ways,and the subsequent changes in gene expression can bedocumented using microarrays. Although we do notknow which events most closely mimic neural crestinduction in vivo, the use of different neural crest-induc-ing strategies should nevertheless allow the identificationof the most complete neural crest gene repertoire that ispossible. For example, neural crest can be induced by co-culturing neural and non-neural tissue, by treatingcompetent neural tissue with Wnt or BMP, or by forcingthe expression of a key transcription factor (FIG. 3). Inaddition, one could compare gene expression profilesbetween wild type and mutant zebrafish or mouseembryos with neural crest defects (FIG. 3). Data fromeach successive screen can be assembled in the form of agene expression profile database for every gene in thecatalogue, giving an indication of how each neural crestgene responds to different conditions, and allowingcoordinately regulated genes to be recognized. Theseexperiments will not only identify target genes, but willalso determine whether there are qualitative differences inthe neural crest induced by different treatments and tran-scription factors. Compiling these expression data andcomparing them with in vivo expression patterns mighthelp us to clarify what factors are required and when.

Third, the response to tissue interactions and treat-ments that induce neural crest could be documentedover time. If the activity of an inducing factor is tempo-rally regulated through the use of inducible forms ofproteins167, or if tissue is harvested at regular time inter-vals, immediate early responses (direct targets) can becharacterized along with downstream changes in geneexpression. Furthermore, when array data is collected asa function of time, co-regulated genes can be identifiedusing cluster analysis, in which genes are organized intogroups with similar expression profiles156. These relationships can then be used to recognize regulatoryinteractions.

The combination of tissue-specific and temporalmicroarray data is already being applied to develop-mental problems. This approach was elegantly used inDrosophila to define the networks of signalling path-ways that regulate the response to ecdysone duringmetamorphosis168. With regard to the neural crest, thetranscriptional response to Wnt104 and Pax3 (REF. 128)

has been documented in tissue culture cells usinghuman cDNA and oligonucleotide arrays, respectively.By performing a time course of Wnt3a treatment inembryonic carcinoma cells, it was possible to identifygenes that are likely to be direct targets, based on thepresence of TCF binding sites in almost all of the targetgene promoters104. Several neural crest genes were iden-tified, including c-Myc, Id2, Msx1 and Msx2, confirmingthe utility of this approach. The transcriptionalresponse to Pax3 was slightly more complex, as the cells

which was arrayed and spotted on nylon membranes.Only clones of interest were sequenced on the macro-array, circumventing the need for the databases ofexisting sequence information that are required formicroarray production.

The results of this screen have provided new markersand regulators of neural crest development. For example,the cDNA with the most specific expression pattern,provisionally called precrest-1, has no homology to anysequences in the database157. This gene is expressed at theborders of the neural plate as soon as they are apparent,earlier than Slug or Foxd3, and is probably a direct targetof early neural crest-inducing signals. Neuropilin-2a1, areceptor for semaphorins and vascular endothelialgrowth factor158, is also expressed in head and trunkneural folds at very early stages in neural crest develop-ment, implying that this signalling pathway is involvedin neural crest cell specification and migration157.The macroarray screen also emphasized the importanceof proliferation, chromatin remodelling, nucleocyto-plasmic export, post-translational regulation and theRho pathway for the generation of migratory neuralcrest cells.

Defining a neural crest gene regulatory networkThe macroarray screen did not, however, identify thefull complement of genes that are expressed at differentsteps in early neural crest development, nor did it orga-nize the neural crest gene list into a cascade or network.To achieve these goals, a combination of approaches willbe required.

First, we can adopt a comparative strategy for themolecular analysis of neural crest formation, takingadvantage of the microarrays and other genomicresources that are beginning to be developed for all vertebrates. Just as Slug and Snail gene expression pat-terns differ among vertebrates43, there are likely to bevariations in the molecular cascade of genes that specifythe neural crest. By comparing and contrasting verte-brate neural crest development, we can define the con-served events. The potential for mechanistic variation isbest exemplified by comparing neural induction in frogand chick, where differences in the importance and timing of the various factors that are involved help us tounderstand the entire process more fully159. The chickengenome project is well underway160, and chick EXPRESSED

SEQUENCE TAG (EST) databases are rapidly being gener-ated161, so commercially produced microarrays of chickESTs are on the horizon. For future avian genomicscreens, it will probably be faster to screen avianmicroarrays by differential hybridization, rather thanscreening macroarrays with subtracted probe157.Furthermore, there have been important advancesrecently in the toolbox that is available for chick experi-mentation. For example, ELECTROPORATION allows ectopicexpression of DNA, including morpholino antisenseoligonucleotides and RNA-interference constructs forloss-of-function analyses162. Microarrays that haverecently been developed in Xenopus163,164 andzebrafish165,166 could be screened in a similar manner.Many of the neural crest regulatory molecules that have

EXPRESSED SEQUENCE TAGS

(ESTs). Short (200–500 basepairs) DNA sequences thatrepresent the sequencesexpressed in an organism undera given condition. They aregenerated from the 3′- and 5′-ends of randomly selected cDNAclones. The purpose of ESTsequencing is to scan for all theprotein-coding genes, and toprovide a tag for each gene onthe genome.

ELECTROPORATION

The transient generation ofpores in a cell membrane byexposing the cell to a high fieldstrength electrical pulse. Thisallows the entry of largemolecules, such as DNAconstructs, into the cell.

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Caenorhabditis elegans mutant embryos have identifiedtarget genes in an analogous manner170,171. The mostcomprehensive example of a gene regulatory network tobe synthesized from this type of information is the net-work that underlies endomesoderm specification in thesea urchin172. This complex network of transcriptionfactors, signalling pathways and their targets has beencompiled by experimentally testing interconnections byloss-of-function approaches.

Finally, to definitively characterize regulatory interac-tions, it will be necessary to dissect elements in the regulatory regions of key neural crest gene targets.Promoter constructs can be assayed quite effectively byelectroporation into a tissue of interest in chickenembryos85,173. Meanwhile, cis regulatory elements can beassessed using transient and stable transgenic approachesin frogs174 and fish175. In addition, the chick160, Xenopus176,and zebrafish177 genome projects will place the genomicsequence of all genes and their flanking regions into thepublic domain. This will allow defined enhancer elements to be annotated and putative regulatory regionsto be computationally identified as shared sequencemotifs in coordinately regulated genes168,178. Completegenomic sequences will also permit the construction of

that were used were stably transfected with Pax3 (REF. 128).However, many of the genes that were upregulated inthis screen contained putative Pax3 binding sites. Thesescreens emphasize the need to perform time courses tocharacterize both direct targets and the subsequentdownstream effects.

Once a cohort of neural crest genes has been identi-fied, an early neural crest-specific microarray can be generated. This reagent would facilitate future experi-ments to define the neural crest gene regulatory network,as only the genes that are involved in the process underinvestigation would be included. This type of reagent hasbeen created to study neural crest-derived melanocytedifferentiation by selecting genes with particular expres-sion profiles from mouse EST databases169. A neuralcrest-specific microarray is not an essential requirement,however, as it is possible to analyse only those clones thatare of interest on a global microarray.

To determine how different factors are interrelated,the effect of one gene or condition on the expression ofall neural crest genes could be tested. Gain- and loss-of-function techniques should allow the identification of downstream targets, as well as potential regulatoryinteractions. Microarray screens of Drosophila and

Chick intermediate neural plate

Array hybridization and analysis

Electroporated(For example, Slug, Foxd3 and Sox9)

+/– inducing factor(For example, Wntand BMP)

+/– inducing tissue(For example, non-neural ectoderm)

Xenopus animal cap

Zebrafish

Microinjected(For example, Slug,Foxd3 and Sox9)

+/– inducing factor(For example, Wntand BMP inhibition)

+/– inducing tissue(For example,paraxial mesodermand neural plate)

Cy3/Cy5-labelled RNA

1. Create a gene expression profile database of allgenes upregulated by neural crest-inducing treatments

2. Identify groups of genes with similar expressionprofiles to predict direct targets and co-regulated genes

3. Test interrelationships with additional microarrayexperiments

Neural crest mutantversus wild type

Morpholino injected(For example, Snail,Foxd3 and Sox9)

Figure 3 | Integrating embryology and genomics to define a neural crest gene regulatory network. Examples of variousprotocols for inducing neural crest in chick and Xenopus. Microarray hybridization can be used to compare gene expression incontrol tissue and induced neural crest tissue; for example, in chick intermediate neural plate that has been electroporated with acontrol construct or with a transcription factor that initiates the neural crest programme. Gene expression in zebrafish neural crestmutants can also be compared. The resulting gene expression profiles can then be compiled, analysed and tested further.

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understand human neural crest development for thepurpose of clinical intervention. To this end, the aviansystem might be the best available, as early chick neuraldevelopment more closely resembles that of humansthan does early rodent neural development. Regardless,it is clearly important to examine several different model organisms to establish the conserved mecha-nisms.As neural crest genes and networks are defined inzebrafish, frog and chick, it will be important to generatemouse mutants to determine whether gene regulatorymodels apply to the murine example as well.

The genome era is an exciting time for biologists,especially those who are interested in previously impen-etrable problems like the specification of premigratoryneural crest. Although it is likely to occupy us for manyyears to come, the possibility of defining an early neuralcrest gene regulatory network is on the horizon.

intergenic sequence microarrays, which can be used to identify protein binding sites and reveal direct regulatory interactions on a genome scale179,180.

ConclusionsTo date, neural crest development has been best studiedin chick and frog due to their accessibility and ease ofmanipulation. Although differences exist betweenspecies, the assumption is that the most crucial mecha-nisms must be conserved across vertebrates, includingmammals. In general, mouse null mutations haveshown that neural crest genes have largely homologousroles in the chick, fish and frog (for example, transcrip-tion factor Ap2 (REFS 111,114,115), Sox10 (REFS 63,67,71–73)

and the Zic gene family75–79,84–87), although interestingswitches in gene usage occur between paralogues (for example, Slug/Snail43). The ultimate goal is to

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AcknowledgementsThe 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

DATABASESThe 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 | Zic5Xenbase: http://www.xenbase.org/nbxTIGR Gallus gallus Gene Index:http://www.tigr.org/tdb/tgi/gggi/Chordin | NogginTIGR Xenopus laevis Gene Index:http://www.tigr.org/tdb/tgi/xgi/eif4a2 | Meis1b | Zicr1 |Access to this interactive links box is free online.