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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.
Neural crest cells are the great explorers of the verte- sequencing projects in chick, frog and zebrafish, this is brate embryo. Although they have the potential to rapidly changing. remain in the central nervous system at the site of their In this article, we will provide an overview of early birth, they strike off on their own and colonize the far neural crest development, paying particular attention to reaches of the embryo. These wandering cells form, our current understanding of the gene network that among other things, peripheral neurons, glia, connective regulates this process. We will then consider how tissue, bone, secretory cells and the outflow tract of the genomic techniques are changing this view, describing heart. We are only just beginning to understand how recent advances and highlighting future possibilities. migratory neural crest cells arise and maintain their ability to form such diverse cell types. Life at the margins Many of the properties that make neural crest cells In the earliest phase of neural development, neural so interesting to study also make their molecular tissue is induced in the ectodermal (outer) layer of the characterization a difficult task. As the neural crest is a embryo. As a consequence of neural induction, the vertebrate invention1, it cannot be studied in yeast, ectoderm becomes divided into three different regions worms or flies, which are the traditional organisms for — the neural ectoderm or neural plate, which will give rapid, large scale genetic screens. Furthermore, neural rise to the central nervous system; the non-neural crest cells form during the early stages of embryonic ectoderm, which will form epidermis; and the cells at development, when mouse embryos are small and diffi- the border between neural and non-neural ectoderm, cult to manipulate. Historically, the neural crest has which for the most part will become the neural crest been studied in accessible, externally developing avian (FIG. 1).This border region was first appreciated in 1868 and amphibian embryos. The resulting classical embry- by His, who described chick neural crest as a ‘zwischen- ological literature provides us with a detailed account of strang’ or ‘in-between strip’,lying between the neural where neural crest cells arise, which tissues send signals and non-neural ectoderm2.Neural tissue folds in on Division of Biology 139-74, to cause neural crest formation, and what cell types they itself to form the neural tube in a process called California Institute of will form1.Until recently, however, this rich descriptive neurulation. During neurulation, the neural plate border Technology, Pasadena, history of the neural crest has been counterbalanced by cells bend to form the neural folds and eventually California 91125, USA. a paucity of molecular information, owing largely to become the dorsal aspect of the neural tube. Depending e-mails: the scarcity of established molecular techniques in on the organism and the axial level, neural crest cells [email protected]; [email protected]. birds and genomic approaches in vertebrates other initiate migration from the closing neural folds or from doi:10.1038/nrn1219 than the mouse. However, with the advent of genome the dorsal neural tube.
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Non-neural Neural plate border shown that the neural plate border and neural crest cells ectoderm Neuroectoderm 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 inter- actions occur coincidentally or sequentially, or whether Neural fold 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 Paraxial Neural plate Dlx5 in the neural plate results in non-cell-autonomous mesoderm induction of neural plate border gene expression without the induction of neural crest markers18.Furthermore, when Dlx activity is inhibited in the non-neural ecto- derm, 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 (BMPs) and WNT PROTEINS FIBROBLAST GROWTH FACTORS (FGFs) have all been shown in A family of highly conserved Neural crest cells various assays to mimic the tissue interactions that secreted signalling molecules, which are related to the Somite induce neural crest. Another factor, Noelin,regulates the Drosophila wingless protein and Neural tube timing of neural crest production. This topic has regulate cell–cell interactions Notochord recently been reviewed in detail elsewhere20, and will be during embryogenesis. Wnt Figure 1 | Border induction and neurulation. The neural summarized only briefly here. proteins bind on the cell surface plate border (green) is induced by signalling between the In birds, BMPs are both necessary21 and sufficient22 to receptors of the Frizzled family. neuroectoderm (purple) and the non-neural ectoderm (blue) and from the underlying paraxial mesoderm (yellow). During to induce neural crest and other dorsal neural tube cell BONE MORPHOGENETIC neurulation, the neural plate borders (neural folds) elevate, types from the neural plate, and for several years they PROTEINS causing the neural plate to roll into a neural tube. Neural crest were believed to be the signal from the non-neural ecto- (BMPs). Multifunctional secreted cells (green) delaminate from the neural folds or the dorsal derm that mediates the neural/non-neural ectoderm proteins of the transforming neural tube (shown), depending on the species and axial level. growth factor-β superfamily. In interaction. However, additional work has shown that the early embryo, they participate neural crest formation requires BMP signalling only in dorsoventral patterning. after the initial induction step, indicating that BMPs Although the neural folds are viewed as ‘premigratory’ might serve a maintenance role in the induction FIBROBLAST GROWTH FACTORS 23 (FGFs). Multifunctional factors neural crest, only a subset of these cells will actually process ,or that they signal the emigration of neural 24 that are involved in embryonic migrate. Cell-marking experiments have shown that crest cells from the neural tube .It now seems that the development. More than 20 progeny of individual cells within the neural folds can inducing signal from the non-neural ectoderm is a Wnt FGFs and 4 FGF receptors have contribute to the neural tube3–6 and epidermis5, as well protein. Wnts are both necessary and sufficient for been described. Their as to the neural crest. Even if cells are marked shortly robust induction of neural crest in isolated neural tissue, coordinated activity controls cell proliferation, migration, survival before migration initiates, labelled progeny are found in and in birds, Wnt6 is expressed at the correct time and differentiation. FGFs both the neural tube and neural crest5.So, the neural and in the right place in the non-neural ectoderm25. regulate growth and crest is not a defined cell population until the cells begin In support of this idea, components of the Wnt morphogenesis by an early to migrate. Instead, the earliest events in neural crest signalling pathway have been shown to be important action on regional patterning, and a later effect on the growth development result in a population of cells in the neural for neural crest formation in several different assays 26 of progenitor cells of the folds with the potential to become migratory neural and organisms . forebrain. crest cells. However, the role of Wnts and BMPs seems to be The formation of neural crest precursors at the slightly different in Xenopus and zebrafish than in birds. ANAMNIOTES neural plate border involves several signalling events. In ANAMNIOTES,neural-inducing BMP antagonists, such as Vertebrates, such as fish and amphibians, that do not develop The neural plate forms first, followed by induction of its Noggin and Chordin,generate a BMP signalling gradient inside an amnion. border7.Several groups, using various organisms, have that specifies dorsoventral pattern in the ectoderm,
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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 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 inter- initiate neural crest development. As one might expect mediate levels of BMP signalling27.So,in Xenopus,partial from lineage analyses3–6, this list includes epidermal, inhibition of BMP signalling together with activation of neural and neural crest markers7.However,as we Wnt signalling seems to mediate neural crest induc- will discuss, the relationships between these genes are tion14,28,29.Work in zebrafish also supports a role for not clear. both a BMP gradient30,31 and Wnts32 during neural crest cell specification. Neural crest markers. The first category of neural crest Also in Xenopus,FGF signalling can induce neural genes is expressed almost exclusively in the neural folds. crest in neuralized ectoderm14,33,34,albeit through a Wnt These genes are typically used as markers of premigratory intermediary14, and seems to be a component of neural crest. the neural crest-inducing signal from the paraxial The best understood neural crest marker is the mesoderm15.Expression of FGF3, FGF4 and FGF8 has transcription factor Slug.Slug, and its close relative been observed in the paraxial mesoderm7,15,35–38, Snail,comprise a family of ZINC-FINGER transcriptional although only FGF8 can induce a subset of neural crest repressors40.Slug and Snail are functionally equiva- markers in isolated Xenopus ectoderm without lent41,42 and, depending on the vertebrate, one or the additional factors15. Finally, Noelin is a secreted glyco- other is highly specific to the neural crest — in the protein that seems to regulate the competence of the premigratory neural crest, chickens express only Slug, neural folds to give rise to neural crest in avians39. mice and fish express only Snail43,and Xenopus 44 ZINC FINGER expresses both . Slug expression seems to be a direct A protein module in which …and their molecular targets target of neural crest induction, as a functional Slug pro- cysteine or cysteine–histidine Less is known about the events downstream of the moter contains a lymphoid enhancer-binding factor/T- residues coordinate a zinc ion. signals that induce neural crest. A growing list of cell factor (LEF/TCF) binding site45,which has been Zinc fingers are often used in DNA recognition and in genes (TABLE 1) has been found to be expressed in neural shown to mediate the transcriptional response to Wnt protein–protein interactions. crest precursors and to be necessary and/or sufficient to signalling in various systems26.
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Slug/Snail activity is crucial at several stages during nuclear import and export might provide a mechanism early neural crest development. Overexpression expands to regulate the transcriptional activity of these proteins. the neural crest-forming region14,41,whereas blocking Slug and/or Snail function inhibits neural crest specifi- Markers for neural crest and neural plate. The next cation42,46 and migration46–48.The targets of Slug/Snail category of early neural crest genes is more broadly during premigratory neural crest development expressed than Slug, Foxd3 and the Sox genes, but is are not known. However, Slug and Snail have been sufficient and/or required for aspects of early neural shown to regulate EPITHELIAL–MESENCHYMAL TRANSITIONS crest development. In this section, we will consider the (EMT) in cultured cells through direct repression of genes that are expressed in the neural crest and neigh- E-cadherin49–51 and claudins/Occludin52, causing the bouring neural plate, and in the next section, we will break-up of ADHERENS and TIGHT JUNCTIONS,respectively. consider those that are expressed in the neural crest and The ability of Slug/Snail to regulate the junctional neighbouring non-neural ectoderm. proteins that are expressed in the neural crest has not The Zic genes (Zic1, Zicr1, Zic2, Zic3 and Zic5) are a been examined, but it is likely that they are also direct family of zinc-finger transcription factors that are regulators of EMT in the neural crest. Curiously, Slug expressed in the neural crest and neural plate/dorsal RNA is expressed in the neural folds long before migra- spinal cord75–83,although Zic5 is the most specific to tion actually initiates, and not all Slug-expressing cells neural crest precursors77.The onset of Zic gene expres- EPITHELIAL–MESENCHYMAL will become migratory neural crest cells44.Either a signal sion occurs early in the neural ectoderm, and this is TRANSITIONS 75,78 (EMT). The transformation of or a newly expressed cofactor activates Slug function in a likely to be an early response to neural inducers . an epithelial cell into a subset of premigratory neural crest cells when it is time Overexpression of any Zic gene induces both neural and mesenchymal cell with to migrate. Indeed, there are probably factors in addition neural crest-marker gene expression75–79,and inhibits migratory and invasive to Slug/Snail that regulate EMT at trunk levels41. differentiation while promoting proliferation79,84,85.A properties. Foxd3 is another gene whose expression is specific to role in regulating proliferation and/or differentiation is
ADHERENS JUNCTION neural crest precursors in the ectoderm of all verte- confirmed in Zic1 and Zic2 mutant mice, which exhibit A cell–cell junction also known brates53–56.In addition, it is weakly expressed in the parax- decreased proliferation84,86 and impaired differentiation of as zonula adherens, which is ial mesoderm. Like Slug,Foxd3 gain-of-function expands the dorsal neural plate87 and cerebellum86,although these characterized by the intracellular the neural crest field53–55 and loss-of-function ablates mice do not have obvious neural crest defects except for insertion of microfilaments. If 53,54 –/– intermediate filaments are neural crest precursors .Foxd3 is expressed in undiffer- a reduction in the size of the DORSAL ROOT GANGLIA in Zic2 57 87 inserted in lieu of entiated embryonic stem cells , and is required for mice .It is not clear how the neural crest-specification microfilaments, the resulting embryonic stem cell establishment and maintenance58. activity that is observed in Xenopus overexpression junction is referred to as a On the basis of their homology to linker histones, it has assays fits with the mouse mutant phenotypes, but gene desmosome. been postulated that winged-helix transcription factors redundancy could mask these effects in mice.
TIGHT JUNCTIONS like Foxd3 bind to nucleosomes and open compacted Pax3 (REFS 22,88–90) and Pax7 (REFS 88,91,92) are Belt-like regions of adhesion chromatin to potentiate transcription of target genes59. transcriptional activators93 that are expressed in both the between adjacent epithelial or Indeed, a protein related to Foxd3, FoxA, has been neural crest and neural plate, with Pax3 being expressed endothelial cells. Tight junctions shown to have such activity60.So, Foxd3 might regulate earlier than Pax7 in mice92.Mice that are mutant for regulate paracellular flux, and contribute to the maintenance of the transcriptional accessibility of a collection of genes Pax3 or Pax7 display defects in various neural crest 92,94 cell polarity by stopping that are responsible for the multipotency of neural crest derivatives , and Pax3 mutants exhibit a decrease or molecules from diffusing within and other stem cells. loss of migratory neural crest cells caudal to the otic the plane of the membrane. 6 Sox9 and Sox10 are HIGH MOBILITY GROUP (HMG)-DOMAIN vesicle .However, the requirement for Pax3 in the early transcriptional activators61,62 that are specific to the neural crest is non-cell-autonomous: neural crest cells HIGH MOBILITY GROUP (HMG) –/– DOMAIN premigratory neural crest and otic placode in frog and migrate from Pax3 neural tubes that have been trans- A conserved domain that is fish63–67.Murine Sox9 is also expressed in premigratory planted to chick hosts6 or in vitro substrates95, and present in HMG proteins, which and migratory neural crest68,whereas Sox10 is initially Pax3–/– migratory neural crest cells are observed in wild- are non-histone proteins expressed just as neural crest migration initiates in type/Pax3–/– chimeric mice89. Foxd3 is not expressed in involved in chromatin structure 69 69,70 55 and gene regulation. chick and mouse .In Xenopus, Sox9 (REF.65) or Sox10 caudal regions of Pax3 mutant mice ,correlating with (REF. 67) MORPHOLINO knockdown inhibits neural crest the failure to form neural crest in these regions6. MORPHOLINO specification. However, mouse or fish Sox9 mutants However, Foxd3 expression has not been examined An antisense oligonucleotide have no defects in neural crest formation or migra- in wild-type/Pax3–/– chimaeras. As migratory neural that acts specifically to block the tion64,68, and in mouse or fish Sox10 mutants, neural crest is observed in chimaeras, it would be interesting initiation of translation. crest cells are specified properly71,but undergo apoptosis to see if a wild-type environment rescues Foxd3 63,72 72,73 –/– DORSAL ROOT GANGLIA at the start of or during migration. It is not clear if expression in Pax3 premigratory and migratory The cell bodies of neural crest- these differences are due to experimental conditions, or neural crest, or if neural crest cells are migrating without derived sensory neurons are whether they represent true mechanistic variation expressing Foxd3. collected together in paired ganglia that lie alongside the among vertebrates. Sox10 inhibits differentiation and Notch1 is broadly expressed in the neural plate, 74 96–99 spinal cord. These cell bodies are maintains stem cell potential , and can synergize with although its expression is elevated in neural crest . surrounded by satellite glial cells, Pax3 in some neural crest-derived lineages71, although When the Notch receptor binds one of its ligands such which share much in common premigratory neural crest has not been analysed in this as Delta, the intracellular domain is cleaved and translo- with the Schwann cells that regard. Curiously, Sox10 undergoes nucleocytoplasmic cates into the nucleus to activate transcription100. ensheath peripheral axons.Very few synapses have been observed shuttling, and Sox9 contains identical regulatory Overexpressing an activated Notch ablates neural in these ganglia. sequences for shuttling61.So, regulating the balance of crest markers and results in a loss of neural crest
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derivatives97,98,yet Delta/Notch signalling is required for although the severity of the phenotype is probably neural crest formation97,101.So, finely tuned levels diminished by redundancy in Msx1 and Msx2 expression of Delta/Notch signalling and/or exquisite temporal and function123. Msx1 and Msx2 inhibit differentiation regulation of Notch activity are needed in the neural without promoting proliferation124. Like Ap2, Msx1 has crest97.In zebrafish at least, Notch promotes neural also been implicated in epidermal induction, and is crest formation by inhibiting neurogenesis through induced by BMP121 and Wnt104 signalling. repression of Neurogenin-1 (Ngn1)101. The proto-oncogene c-Myc,which stimulates prolif- Miscellaneous neural crest genes. Id2,a helix–loop–helix eration and prevents differentiation102, was recently (HLH) protein that negatively regulates basic HLH shown to be expressed in neural crest precursors and (BHLH) proteins125, is expressed in rostral but not caudal anterior neural plate in Xenopus,with expression in the neural folds126,although we know little about the mecha- neural crest temporally preceding that of Slug103.Loss nisms that determine rostral/caudal differences in of c-Myc activity inhibits neural crest-marker gene the neural crest. Id2 has been implicated in neural expression and results in the loss of various neural crest crest formation from the non-neural ectoderm126, and derivatives. In tissue culture, c-Myc is a target of Wnt in cultured cells it is a target of BMP127 and Wnt signalling104,105 and its expression in the neural crest signalling104, and of Pax3 (REF.128) and Myc activity129. depends on Wnt signals103.c-Myc activates signal-depen- Like many other neural crest factors, Id genes stimulate dent target gene expression through E-BOX binding and proliferation and inhibit differentiation125. histone H4 acetylation106, so like Foxd3, it might regulate Tw ist is a bHLH protein that is expressed in the the transcriptional accessibility of a cohort of genes that neural crest, SOMITES and lateral plate mesoderm130–132. are necessary for early neural crest development. Tw ist is an early neural crest marker in Xenopus130, Another recently identified neural crest marker with a whereas in the mouse, Tw ist is expressed in migrating neural plate component is the NK-1 HOMEOBOX gene Nbx. neural crest cells in the head133.Mouse mutants show When overexpressed in Xenopus, this transcriptional that Tw ist is required for neural crest migration and repressor expands neural crest at the expense of neural differentiation, but not for neural crest specification132. plate, whereas a DOMINANT-NEGATIVE construct inhibits Tw ist is a direct target of Wnt signalling in murine E-BOX neural crest formation107. Meis1b,a cofactor that mammary epithelial cells134. The conserved nucleotide sequence CANNTG that is regulates the transcriptional activity of Hox proteins, also In zebrafish, a molecule called Crestin,which is a recognized and bound by basic seems to be sufficient to promote the formation of member of a multi-copy family of RETROELEMENTS, is a helix–loop–helix and other neural crest — overexpression induces anterior neural very specific marker of neural crest135.In addition, large proteins. and neural crest markers in Xenopus108.Finally, the RNA- scale genetic screens have generated numerous mutant helicase translation initiation factor eif4a2 is expressed fish lines with neural crest defects, although the genes HOMEOBOX 136 A sequence of about 180 base in the neural plate and its border in Xenopus, and is responsible have not been identified . pairs that encodes a DNA- sufficient to induce neural and neural crest markers109. Finally, Rhob is a target of BMP signalling that is binding protein sequence known expressed in the dorsal neural tube137 and migrating as the homeodomain. The 60- Markers for neural crest and non-neural ectoderm. The neural crest137,138.Rho activity is not required for neural amino-acid homeodomain comprises three α-helices. transcription factor Ap2 is an example of a neural crest crest specification, but is necessary for the delamination gene that is initially expressed throughout the neural of neural crest cells137.Although Rho GTPases are DOMINANT-NEGATIVE plate border and non-neural ectoderm, and that is later typically viewed as regulators of the actin cytoskeleton, A mutant molecule that enhanced in the neural folds in all vertebrates110–113. they have been implicated in many other processes, interferes with and inhibits the Mice that are mutant for Ap2 show defects in neural including transcription and cell cycle progression139. activation of normal molecules. crest derivatives114,115, and in Xenopus, Ap2 is necessary BASIC HELIX–LOOP–HELIX and sufficient to promote Slug and Sox9 gene A minimal ‘network’? (bHLH). A structural motif expression111.Ap2 is also required for epidermal develop- As the list of genes expressed by premigratory neural present in many transcription ment116.Expression of Ap2 in neural crest depends on crest cells grows, there is an increasing need to define factors that is characterized by 111 two α-helices separated by a Wnt signalling , indicating that it is a target of inducing the interrelationships between these genes (FIG. 2). loop. The helices mediate signals from the non-neural ectoderm. Ap2 fosters Overexpression analyses have not been altogether helpful dimerization, and the adjacent proliferation by repressing genes that promote terminal in this regard. For example, in Xenopus animal cap basic region is required for DNA differentiation117.Ap2 and c-Myc interact to regulate assays, Foxd3 can activate Slug and Zicr1 expression, and binding. gene expression; for example, they activate or repress Zicr1 can induce Foxd3 and Slug expression53.Which
SOMITES transcription of E-cadherin depending on the relative comes first, Foxd3 or Zicr1? To add to the confusion, Paired blocks of mesoderm cells expression levels of the two different c-Myc isoforms118. investigators tend to look at the same markers — Slug, along the vertebrate body axis Msx1 and Msx2 are homeobox transcriptional Sox9 and Foxd3.In many cases, including Foxd3 overex- that form during early vertebrate repressors119 that are also expressed in the neural folds pression53,neural tissue is induced along with neural development and differentiate into dermal skin, bone and and, at lower levels, in neighbouring non-neural crest. How do we know that neural crest is not induced muscle. ectoderm, with Msx2 being more restricted in its expres- as a secondary consequence of the formation of ectopic sion pattern119,120 than Msx1 (REFS 7,119,121).Zebrafish neural tissue, which itself induces neural crest? RETROELEMENTS Msx genes are not orthologous to Msx genes in other Furthermore, overexpression assays have mostly been Segments of genetic material vertebrates, although Msxb and Msxc also show expres- performed in Xenopus, in which all neural crest genes that transpose around the 122 genome using an RNA sion at the borders of the neural plate . Msx1 mutant seem to turn on other neural crest genes. How can we intermediate. mice exhibit a loss of neural crest derivatives in the face, find order in this chaos?
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premigratory neural crest expresses N-cadherin and 153 Neural crest Premigratory Neural crest cadherin-6b in lieu of E-cadherin .During EMT, induction neural crest emigration including the transition from a tumour to a metastatic Wnt Slug/Snail Rhob cancer cell, E-cadherin is downregulated154.Likewise, FGF Foxd3 N-cadherin N-cadherin and cadherin-6b expression are down- BMP Sox9, Sox10 Zic genes regulated during EMT at the onset of neural crest Pax3, Pax7 153 c-Myc, Notch1 migration .E-cadherin and N-cadherin are functionally Ap2, Nbx equivalent155,although the mechanisms that regulate their Msx2 expression have not been compared. As Slug/Snail49–51, Dlx5 Msx1 Ap2 and c-Myc118 directly regulate E-cadherin expression, Border Neural plate it is possible that they also modulate N-cadherin and/ induction border or cadherin-6b expression to initiate EMT at the start of neural crest migration. Figure 2 | The current status of a neural crest gene Although they provide a framework for formulating regulatory network. The neural crest (yellow) and neural plate future experiments, these correlations do not describe border (orange) are induced as separate events regulated by overlapping secreted factors. A complex set of genes are the molecular mechanism by which migratory neural expressed in the neural folds (green and blue) as a crest cells are generated from the neural folds. How consequence of these inductions. This developmental can we distill this complex array of information into a programme leads to changes in gene expression (purple) that meaningful regulatory network? result in the emigration of neural crest cells from the neural tube. However, the interrelationships between neural crest Filling in the gaps genes are only just beginning to be elucidated. One way to create a clearer picture of early neural crest development is to take a comprehensive approach. As a starting point, it is possible to identify trends in Genomic-level screens could potentially identify the full our gene list (TABLE 1).In terms of function, many neural collection of genes that are involved in early neural crest crest genes have been shown to stimulate proliferation development. These genes can then be assembled into and prevent differentiation (Zic genes79,84,85, Pax3 (REF. functional networks. Researchers have been using 140), c-Myc102, Ap2 (REF.117), Msx1 and Msx2 (REF.124), Id2 microarrays to achieve these goals for several years (REF.125), Notch1 (REF.101) and Tw ist 134) or maintain stem now156.The advantage of using this approach for neural cell potential (Foxd3 (REF.58) and Sox10 (REF.74)), both of crest development is that increasingly sophisticated which are key characteristics of the neural crest lineage. bioinformatic tools are constantly being generated to The list includes several transcriptional repressors identify relationships and infer pathway models from (Slug/Snail40,Zic1 (REF. 85),Nbx107, Msx1 and Msx2 array data156.What is truly exciting about the chick and (REF. 119) and Id2 (REF. 125)), as well as transcriptional Xenopus genomic era, however, is the possibility of activators (Sox9 and Sox10 (REFS 61,62),Pax3 (REF. 93), combining powerful array technologies with the ability c-Myc102,Ap2 (REF.141) and Notch1 (REF.100)), indicating to do experimental embryology. Equally enticing is the that the formation of neural crest cells requires repres- intersection of vertebrate genetics, transgenics and sion as well as activation of new gene expression. genomics in zebrafish. Interestingly, with the exception of Pax3, all of the As we alluded to earlier, documenting the complete transcriptional activators have been shown to bind to gene expression profile of a premigratory neural crest CREB-binding protein (CBP)/p300 (REFS 142–146),as cell is not a straightforward endeavour. The availability does β-catenin,a downstream effector of Wnt of genomic tools for studying neural crest is not the only signalling147,148.Furthermore, CBP/p300 is a target obstacle. The neural folds contain a heterogeneous of Wnt signalling104. CBP and p300 are closely related population of cells, and neural crest precursors within transcriptional co-activators that connect sequence- this population are multipotent, with the ability to give specific transcription factors to the general transcrip- rise to neural crest, neural tube and epidermis3–6.So,it is tional machinery149.They are histone acetyltransferases not possible to simply purify cells from the neural folds (HATs) and, along with Foxd3 and c-Myc, might have and characterize gene expression in those cells. roles in regulating the chromatin structure of neural One study recently circumvented this problem to crest target genes. In another twist, the HAT activity of identify a collection of genes that are expressed in neural p300 is inhibited by Twist150. CBP/p300 also regulate the crest precursors at a single time-point following neural cell cycle in a complex containing Mdm2 (REF. 149), crest induction157. First, by co-culturing pieces of avian which is preferentially expressed in the neural folds non-neural ectoderm and neural plate, neural crest pre- and migrating neural crest151. Finally, Cited2 is a protein cursors were induced in vitro5,10.Then, genes that were that interacts with Ap2 and CBP/p300 to activate expressed as a consequence of neural crest induction transcription, and Cited2 mutant mice have various were enriched in the cDNA population by subtracting neural crest defects152. cDNA from non-neural ectoderm and neural Another interesting correlation in the premigratory plate that had been cultured in isolation157.Because neural crest gene list is the direct regulation of E-cadherin chick microarrays were not available, the subtracted expression. Expression of the cell adhesion molecule cDNA was used to screen ‘macroarrays’ containing a E-cadherin characterizes most epithelial cells, although cDNA library synthesized from 4 to 12 somite embryos,
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which was arrayed and spotted on nylon membranes. been defined in the past few years were initially identi- Only clones of interest were sequenced on the macro- fied and characterized in Xenopus,owing to the ease of array, circumventing the need for the databases of ectopic molecular manipulation in this organism. existing sequence information that are required for Furthermore, regulatory pathways can be tested in microarray production. zebrafish and Xenopus using transgenics and/or genetic The results of this screen have provided new markers approaches. and regulators of neural crest development. For example, Second, neural crest can be induced in various ways, the cDNA with the most specific expression pattern, and the subsequent changes in gene expression can be provisionally called precrest-1,has no homology to any documented using microarrays. Although we do not sequences in the database157.This gene is expressed at the know which events most closely mimic neural crest borders of the neural plate as soon as they are apparent, induction in vivo, the use of different neural crest-induc- earlier than Slug or Foxd3, and is probably a direct target ing strategies should nevertheless allow the identification of early neural crest-inducing signals. Neuropilin-2a1,a of the most complete neural crest gene repertoire that is receptor for semaphorins and vascular endothelial possible. For example, neural crest can be induced by growth factor158, is also expressed in head and trunk co-culturing neural and non-neural tissue, by treating neural folds at very early stages in neural crest develop- competent neural tissue with Wnt or BMP,or by forcing ment, implying that this signalling pathway is involved the expression of a key transcription factor (FIG. 3).In in neural crest cell specification and migration157. addition, one could compare gene expression profiles The macroarray screen also emphasized the importance between wild type and mutant zebrafish or mouse of proliferation, chromatin remodelling, nucleocyto- embryos with neural crest defects (FIG. 3). Data from plasmic export, post-translational regulation and the each successive screen can be assembled in the form of a Rho pathway for the generation of migratory neural gene expression profile database for every gene in the crest cells. catalogue, giving an indication of how each neural crest gene responds to different conditions, and allowing Defining a neural crest gene regulatory network coordinately regulated genes to be recognized. These The macroarray screen did not, however, identify the experiments will not only identify target genes, but will full complement of genes that are expressed at different also determine whether there are qualitative differences in steps in early neural crest development, nor did it orga- the neural crest induced by different treatments and tran- nize the neural crest gene list into a cascade or network. scription factors. Compiling these expression data and To achieve these goals, a combination of approaches will comparing them with in vivo expression patterns might be required. help us to clarify what factors are required and when. First, we can adopt a comparative strategy for the Third, the response to tissue interactions and treat- molecular analysis of neural crest formation, taking ments that induce neural crest could be documented advantage of the microarrays and other genomic over time. If the activity of an inducing factor is tempo- resources that are beginning to be developed for all rally regulated through the use of inducible forms of vertebrates. Just as Slug and Snail gene expression pat- proteins167,or if tissue is harvested at regular time inter- terns differ among vertebrates43, there are likely to be vals, immediate early responses (direct targets) can be variations in the molecular cascade of genes that specify characterized along with downstream changes in gene the neural crest. By comparing and contrasting verte- expression. Furthermore, when array data is collected as brate neural crest development, we can define the con- a function of time, co-regulated genes can be identified served events. The potential for mechanistic variation is using cluster analysis, in which genes are organized into best exemplified by comparing neural induction in frog groups with similar expression profiles156.These and chick, where differences in the importance and relationships can then be used to recognize regulatory EXPRESSED SEQUENCE TAGS timing of the various factors that are involved help us to interactions. (ESTs). Short (200–500 base understand the entire process more fully159.The chicken The combination of tissue-specific and temporal pairs) DNA sequences that 160 represent the sequences genome project is well underway , and chick EXPRESSED microarray data is already being applied to develop- expressed in an organism under SEQUENCE TAG (EST) databases are rapidly being gener- mental problems. This approach was elegantly used in a given condition. They are ated161, so commercially produced microarrays of chick Drosophila to define the networks of signalling path- generated from the 3′- and 5′- ESTs are on the horizon. For future avian genomic ways that regulate the response to ecdysone during ends of randomly selected cDNA screens, it will probably be faster to screen avian metamorphosis168.With regard to the neural crest, the clones. The purpose of EST 104 sequencing is to scan for all the microarrays by differential hybridization, rather than transcriptional response to Wnt and Pax3 (REF.128) protein-coding genes, and to screening macroarrays with subtracted probe157. has been documented in tissue culture cells using provide a tag for each gene on Furthermore, there have been important advances human cDNA and oligonucleotide arrays, respectively. the genome. recently in the toolbox that is available for chick experi- By performing a time course of Wnt3a treatment in ELECTROPORATION ELECTROPORATION mentation. For example, allows ectopic embryonic carcinoma cells, it was possible to identify The transient generation of expression of DNA, including morpholino antisense genes that are likely to be direct targets, based on the pores in a cell membrane by oligonucleotides and RNA-interference constructs for presence of TCF binding sites in almost all of the target exposing the cell to a high field loss-of-function analyses162.Microarrays that have gene promoters104.Several neural crest genes were iden- strength electrical pulse. This recently been developed in Xenopus163,164 and tified, including c-Myc, Id2, Msx1 and Msx2,confirming allows the entry of large 165,166 molecules, such as DNA zebrafish could be screened in a similar manner. the utility of this approach. The transcriptional constructs, into the cell. Many of the neural crest regulatory molecules that have response to Pax3 was slightly more complex, as the cells
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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)
Cy3/Cy5- labelled RNA Xenopus animal cap Microinjected (For example, Slug, Foxd3 and Sox9) +/– inducing factor (For example, Wnt and BMP inhibition) +/– inducing tissue (For example, paraxial mesoderm 1. Create a gene expression profile database of all and neural plate) genes upregulated by neural crest-inducing treatments 2. Identify groups of genes with similar expression profiles to predict direct targets and co-regulated genes Zebrafish Neural crest mutant 3. Test interrelationships with additional microarray versus wild type 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). Caenorhabditis elegans mutant embryos have identified However, many of the genes that were upregulated in target genes in an analogous manner170,171.The most this screen contained putative Pax3 binding sites. These comprehensive example of a gene regulatory network to screens emphasize the need to perform time courses to be synthesized from this type of information is the net- characterize both direct targets and the subsequent work that underlies endomesoderm specification in the downstream effects. sea urchin172.This complex network of transcription Once a cohort of neural crest genes has been identi- factors, signalling pathways and their targets has been fied, an early neural crest-specific microarray can be compiled by experimentally testing interconnections by generated. This reagent would facilitate future experi- loss-of-function approaches. ments to define the neural crest gene regulatory network, Finally, to definitively characterize regulatory interac- as only the genes that are involved in the process under tions, it will be necessary to dissect elements in the investigation would be included. This type of reagent has regulatory regions of key neural crest gene targets. been created to study neural crest-derived melanocyte Promoter constructs can be assayed quite effectively by differentiation by selecting genes with particular expres- electroporation into a tissue of interest in chicken sion profiles from mouse EST databases169.A neural embryos85,173.Meanwhile, cis regulatory elements can be crest-specific microarray is not an essential requirement, assessed using transient and stable transgenic approaches however, as it is possible to analyse only those clones that in frogs174 and fish175.In addition, the chick160,Xenopus176, are of interest on a global microarray. and zebrafish177 genome projects will place the genomic To determine how different factors are interrelated, sequence of all genes and their flanking regions into the the effect of one gene or condition on the expression of public domain. This will allow defined enhancer all neural crest genes could be tested. Gain- and loss-of- elements to be annotated and putative regulatory regions function techniques should allow the identification to be computationally identified as shared sequence of downstream targets, as well as potential regulatory motifs in coordinately regulated genes168,178.Complete interactions. Microarray screens of Drosophila and genomic sequences will also permit the construction of
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intergenic sequence microarrays, which can be used understand human neural crest development for the to identify protein binding sites and reveal direct purpose of clinical intervention. To this end, the avian regulatory interactions on a genome scale179,180. system might be the best available, as early chick neural development more closely resembles that of humans Conclusions than does early rodent neural development. Regardless, To date, neural crest development has been best studied it is clearly important to examine several different in chick and frog due to their accessibility and ease of model organisms to establish the conserved mecha- manipulation. Although differences exist between nisms. As neural crest genes and networks are defined in species, the assumption is that the most crucial mecha- zebrafish, frog and chick, it will be important to generate nisms must be conserved across vertebrates, including mouse mutants to determine whether gene regulatory mammals. In general, mouse null mutations have models apply to the murine example as well. shown that neural crest genes have largely homologous The genome era is an exciting time for biologists, roles in the chick, fish and frog (for example, transcrip- especially those who are interested in previously impen- tion factor Ap2 (REFS 111,114,115), Sox10 (REFS 63,67,71–73) etrable problems like the specification of premigratory and the Zic gene family75–79,84–87), although interesting neural crest. Although it is likely to occupy us for many switches in gene usage occur between paralogues years to come, the possibility of defining an early neural (for example, Slug/Snail43). The ultimate goal is to crest gene regulatory network is on the horizon.
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