NEURAL CREST SPECIFICATION: MIGRATING INTO GENOMICS

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1 NEURAL CREST SPECIFICATION: MIGRATING INTO GENOMICS Laura S. Gammill and Marianne Bronner-Fraser The bones in your face, the pigment in your skin and the neural circuitry that controls your digestive tract have one thing in common: they are all derived from neural crest cells. The formation of these migratory multipotent cells poses an interesting developmental problem, as neural crest cells are not a distinct cell type until they migrate away from the central nervous system. What defines the pool of cells with neural crest potential, and why do only some of these cells become migratory? New genomic approaches in chick, zebrafish and Xenopus might hold the key. Division of Biology , California Institute of Technology, Pasadena, California 91125, USA. s: lgammill@caltech.edu; mbronner@caltech.edu. doi: /nrn1219 Neural crest cells are the great explorers of the vertebrate embryo. Although they have the potential to remain in the central nervous system at the site of their birth, they strike off on their own and colonize the far reaches of the embryo. These wandering cells form, among other things, peripheral neurons, glia, connective tissue, bone, secretory cells and the outflow tract of the heart. We are only just beginning to understand how migratory neural crest cells arise and maintain their ability to form such diverse cell types. Many of the properties that make neural crest cells so interesting to study also make their molecular characterization a difficult task. As the neural crest is a vertebrate invention 1, it cannot be studied in yeast, worms or flies, which are the traditional organisms for rapid, large scale genetic screens. Furthermore, neural crest cells form during the early stages of embryonic development, when mouse embryos are small and difficult to manipulate. Historically, the neural crest has been studied in accessible, externally developing avian and amphibian embryos. The resulting classical embryological literature provides us with a detailed account of where neural crest cells arise, which tissues send signals to cause neural crest formation, and what cell types they will form 1.Until recently, however, this rich descriptive history of the neural crest has been counterbalanced by a paucity of molecular information, owing largely to the scarcity of established molecular techniques in birds and genomic approaches in vertebrates other than the mouse. However, with the advent of genome sequencing projects in chick, frog and zebrafish, this is rapidly changing. In this article, we will provide an overview of early neural crest development, paying particular attention to our current understanding of the gene network that regulates this process. We will then consider how genomic techniques are changing this view, describing recent advances and highlighting future possibilities. Life at the margins In the earliest phase of neural development, neural tissue is induced in the ectodermal (outer) layer of the embryo. As a consequence of neural induction, the ectoderm becomes divided into three different regions the neural ectoderm or neural plate, which will give rise to the central nervous system; the non-neural ectoderm, which will form epidermis; and the cells at the border between neural and non-neural ectoderm, which for the most part will become the neural crest (FIG. 1). This border region was first appreciated in 1868 by His, who described chick neural crest as a zwischenstrang or in-between strip, lying between the neural and non-neural ectoderm 2.Neural tissue folds in on itself to form the neural tube in a process called neurulation. During neurulation, the neural plate border cells bend to form the neural folds and eventually become the dorsal aspect of the neural tube. Depending on the organism and the axial level, neural crest cells initiate migration from the closing neural folds or from the dorsal neural tube. NATURE REVIEWS NEUROSCIENCE VOLUME 4 OCTOBER

2 Non-neural ectoderm Paraxial mesoderm Neural plate border Neural plate Neuroectoderm Neural fold shown that the neural plate border and neural crest cells form in response to signalling between newly induced neural tissue and the neighbouring non-neural ectoderm 5,7 10.Signals from the underlying paraxial mesoderm are also involved in inducing the border region 5,11 15.It is not clear, however, whether these interactions occur coincidentally or sequentially, or whether they normally serve inductive or maintenance roles in the embryo. To complicate matters further, recent evidence indicates that border induction and neural crest induction are not necessarily the same process. The transcription factor Dlx5 is one of the earliest markers of the neural plate border 16 18, and ectopic expression of Dlx5 in the neural plate results in non-cell-autonomous induction of neural plate border gene expression without the induction of neural crest markers 18.Furthermore, when Dlx activity is inhibited in the non-neural ectoderm, neural plate border genes are expressed in their normal patterns, but are shifted laterally 19.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 crest 19.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. WNT PROTEINS A family of highly conserved secreted signalling molecules, which are related to the Drosophila wingless protein and regulate cell cell interactions during embryogenesis. Wnt proteins bind on the cell surface to receptors of the Frizzled family. BONE MORPHOGENETIC PROTEINS (BMPs). Multifunctional secreted proteins of the transforming growth factor-β superfamily. In the early embryo, they participate in dorsoventral patterning. FIBROBLAST GROWTH FACTORS (FGFs). Multifunctional factors that are involved in embryonic development. More than 20 FGFs and 4 FGF receptors have been described. Their coordinated activity controls cell proliferation, migration, survival and differentiation. FGFs regulate growth and morphogenesis by an early action on regional patterning, and a later effect on the growth of progenitor cells of the forebrain. ANAMNIOTES Vertebrates, such as fish and amphibians, that do not develop inside an amnion. Neural crest cells Somite Neural tube Notochord Figure 1 Border induction and neurulation. The neural plate border (green) is induced by signalling between the neuroectoderm (purple) and the non-neural ectoderm (blue) and from the underlying paraxial mesoderm (yellow). During neurulation, the neural plate borders (neural folds) elevate, causing the neural plate to roll into a neural tube. Neural crest cells (green) delaminate from the neural folds or the dorsal neural tube (shown), depending on the species and axial level. Although the neural folds are viewed as premigratory neural crest, only a subset of these cells will actually migrate. Cell-marking experiments have shown that progeny of individual cells within the neural folds can contribute to the neural tube 3 6 and epidermis 5, as well as to the neural crest. Even if cells are marked shortly before migration initiates, labelled progeny are found in both the neural tube and neural crest 5.So, the neural crest is not a defined cell population until the cells begin to migrate. Instead, the earliest events in neural crest development result in a population of cells in the neural folds with the potential to become migratory neural crest cells. The formation of neural crest precursors at the neural plate border involves several signalling events. The neural plate forms first, followed by induction of its border 7.Several groups, using various organisms, have Secreted factors that induce neural crest WNT PROTEINS, BONE MORPHOGENETIC PROTEINS (BMPs) and FIBROBLAST GROWTH FACTORS (FGFs) have all been shown in various assays to mimic the tissue interactions that induce neural crest. Another factor, Noelin, regulates the timing of neural crest production. This topic has recently been reviewed in detail elsewhere 20, and will be summarized only briefly here. In birds, BMPs are both necessary 21 and sufficient 22 to induce neural crest and other dorsal neural tube cell types from the neural plate, and for several years they were believed to be the signal from the non-neural ectoderm that mediates the neural/non-neural ectoderm interaction. However, additional work has shown that neural crest formation requires BMP signalling only after the initial induction step, indicating that BMPs might serve a maintenance role in the induction process 23,or that they signal the emigration of neural crest cells from the neural tube 24.It now seems that the inducing signal from the non-neural ectoderm is a Wnt protein. Wnts are both necessary and sufficient for robust induction of neural crest in isolated neural tissue, and in birds, Wnt6 is expressed at the correct time and in the right place in the non-neural ectoderm 25. 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 organisms 26. However, the role of Wnts and BMPs seems to be slightly different in Xenopus and zebrafish than in birds. In ANAMNIOTES, neural-inducing BMP antagonists, such as Noggin and Chordin,generate a BMP signalling gradient that specifies dorsoventral pattern in the ectoderm, 796 OCTOBER 2003 VOLUME 4

3 Table 1 Genes expressed in premigratory neural crest Gene Mouse Chick Fish Frog NP NF EPI NC K/O function function Ap X X 114, Crestin 135 X 135 eif4a2 109 X X 109 Foxd X ND 53, Id2 126 X NP 126 Meis1b 108 X X 108 Msx 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 Notch X X ,98 Pax , X X 6,94 Pax X X 92 Rhob X NP 137 Slug X ,41 Snail X ND 42 41,42 Sox X 64,68 65 Sox ,67 X 63,72, Twist X 132 Zic , ,80 X X NP 76 Zic ,79 X X 87 76,79 Zic 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. ZINC FINGER A protein module in which cysteine or cysteine histidine residues coordinate a zinc ion. Zinc fingers are often used in DNA recognition and in protein protein interactions. with neural plate border cell types forming at intermediate levels of BMP signalling 27.So,in Xenopus, partial inhibition of BMP signalling together with activation of Wnt signalling seems to mediate neural crest induction 14,28,29.Work in zebrafish also supports a role for both a BMP gradient 30,31 and Wnts 32 during neural crest cell specification. Also in Xenopus,FGF signalling can induce neural crest in neuralized ectoderm 14,33,34,albeit through a Wnt intermediary 14, and seems to be a component of the neural crest-inducing signal from the paraxial mesoderm 15.Expression of FGF3, FGF4 and FGF8 has been observed in the paraxial mesoderm 7,15,35 38, although only FGF8 can induce a subset of neural crest markers in isolated Xenopus ectoderm without additional factors 15. Finally, Noelin is a secreted glycoprotein that seems to regulate the competence of the neural folds to give rise to neural crest in avians 39. and their molecular targets Less is known about the events downstream of the signals that induce neural crest. A growing list of genes (TABLE 1) has been found to be expressed in neural crest precursors and to be necessary and/or sufficient to initiate neural crest development. As one might expect from lineage analyses 3 6, this list includes epidermal, neural and neural crest markers 7.However,as we will discuss, the relationships between these genes are not clear. Neural crest markers. The first category of neural crest genes is expressed almost exclusively in the neural folds. These genes are typically used as markers of premigratory neural crest. The best understood neural crest marker is the transcription factor Slug.Slug, and its close relative Snail,comprise a family of ZINC-FINGER transcriptional repressors 40.Slug and Snail are functionally equivalent 41,42 and, depending on the vertebrate, one or the other is highly specific to the neural crest in the premigratory neural crest, chickens express only Slug, mice and fish express only Snail 43, and Xenopus expresses both 44. Slug expression seems to be a direct target of neural crest induction, as a functional Slug promoter contains a lymphoid enhancer-binding factor/tcell factor (LEF/TCF) binding site 45,which has been shown to mediate the transcriptional response to Wnt signalling in various systems 26. NATURE REVIEWS NEUROSCIENCE VOLUME 4 OCTOBER

4 EPITHELIAL MESENCHYMAL TRANSITIONS (EMT). The transformation of an epithelial cell into a mesenchymal cell with migratory and invasive properties. ADHERENS JUNCTION A cell cell junction also known as zonula adherens, which is characterized by the intracellular insertion of microfilaments. If intermediate filaments are inserted in lieu of microfilaments, the resulting junction is referred to as a desmosome. TIGHT JUNCTIONS Belt-like regions of adhesion between adjacent epithelial or endothelial cells. Tight junctions regulate paracellular flux, and contribute to the maintenance of cell polarity by stopping molecules from diffusing within the plane of the membrane. HIGH MOBILITY GROUP (HMG) DOMAIN A conserved domain that is present in HMG proteins, which are non-histone proteins involved in chromatin structure and gene regulation. MORPHOLINO An antisense oligonucleotide that acts specifically to block the initiation of translation. DORSAL ROOT GANGLIA The cell bodies of neural crestderived sensory neurons are collected together in paired ganglia that lie alongside the spinal cord. These cell bodies are surrounded by satellite glial cells, which share much in common with the Schwann cells that ensheath peripheral axons. Very few synapses have been observed in these ganglia. Slug/Snail activity is crucial at several stages during early neural crest development. Overexpression expands the neural crest-forming region 14,41,whereas blocking Slug and/or Snail function inhibits neural crest specification 42,46 and migration The targets of Slug/Snail during premigratory neural crest development are not known. However, Slug and Snail have been shown to regulate EPITHELIAL MESENCHYMAL TRANSITIONS (EMT) in cultured cells through direct repression of E-cadherin and claudins/occludin 52, causing the break-up of ADHERENS and TIGHT JUNCTIONS,respectively. The ability of Slug/Snail to regulate the junctional proteins that are expressed in the neural crest has not been examined, but it is likely that they are also direct regulators of EMT in the neural crest. Curiously, Slug RNA is expressed in the neural folds long before migration actually initiates, and not all Slug-expressing cells will become migratory neural crest cells 44.Either a signal or a newly expressed cofactor activates Slug function in a subset of premigratory neural crest cells when it is time to migrate. Indeed, there are probably factors in addition to Slug/Snail that regulate EMT at trunk levels 41. Foxd3 is another gene whose expression is specific to neural crest precursors in the ectoderm of all vertebrates In addition, it is weakly expressed in the paraxial mesoderm. Like Slug, Foxd3 gain-of-function expands the neural crest field and loss-of-function ablates neural crest precursors 53,54. Foxd3 is expressed in undifferentiated embryonic stem cells 57, and is required for embryonic stem cell establishment and maintenance 58. On the basis of their homology to linker histones, it has been postulated that winged-helix transcription factors like Foxd3 bind to nucleosomes and open compacted chromatin to potentiate transcription of target genes 59. Indeed, a protein related to Foxd3, FoxA, has been shown to have such activity 60.So, Foxd3 might regulate the transcriptional accessibility of a collection of genes that are responsible for the multipotency of neural crest and other stem cells. Sox9 and Sox10 are HIGH MOBILITY GROUP (HMG)-DOMAIN transcriptional activators 61,62 that are specific to the premigratory neural crest and otic placode in frog and fish Murine Sox9 is also expressed in premigratory and migratory neural crest 68,whereas Sox10 is initially expressed just as neural crest migration initiates in chick 69 and mouse 69,70.In Xenopus, Sox9 (REF. 65) or Sox10 (REF. 67) MORPHOLINO knockdown inhibits neural crest specification. However, mouse or fish Sox9 mutants have no defects in neural crest formation or migration 64,68, and in mouse or fish Sox10 mutants, neural crest cells are specified properly 71,but undergo apoptosis at the start of 63,72 or during 72,73 migration. It is not clear if these differences are due to experimental conditions, or whether they represent true mechanistic variation among vertebrates. Sox10 inhibits differentiation and maintains stem cell potential 74, and can synergize with Pax3 in some neural crest-derived lineages 71, although premigratory neural crest has not been analysed in this regard. Curiously, Sox10 undergoes nucleocytoplasmic shuttling, and Sox9 contains identical regulatory sequences for shuttling 61.So, regulating the balance of nuclear import and export might provide a mechanism to regulate the transcriptional activity of these proteins. Markers for neural crest and neural plate. The next category of early neural crest genes is more broadly expressed than Slug, Foxd3 and the Sox genes, but is sufficient and/or required for aspects of early neural crest development. In this section, we will consider the genes that are expressed in the neural crest and neighbouring neural plate, and in the next section, we will consider those that are expressed in the neural crest and neighbouring non-neural ectoderm. The Zic genes (Zic1, Zicr1, Zic2, Zic3 and Zic5) are a family of zinc-finger transcription factors that are expressed in the neural crest and neural plate/dorsal spinal cord 75 83,although Zic5 is the most specific to neural crest precursors 77.The onset of Zic gene expression occurs early in the neural ectoderm, and this is likely to be an early response to neural inducers 75,78. Overexpression of any Zic gene induces both neural and neural crest-marker gene expression 75 79,and inhibits differentiation while promoting proliferation 79,84,85.A role in regulating proliferation and/or differentiation is confirmed in Zic1 and Zic2 mutant mice, which exhibit decreased proliferation 84,86 and impaired differentiation of the dorsal neural plate 87 and cerebellum 86, although these mice do not have obvious neural crest defects except for a reduction in the size of the DORSAL ROOT GANGLIA in Zic2 / mice 87.It is not clear how the neural crest-specification activity that is observed in Xenopus overexpression assays fits with the mouse mutant phenotypes, but gene redundancy could mask these effects in mice. Pax3 (REFS 22,88 90) and Pax7 (REFS 88,91,92) are transcriptional activators 93 that are expressed in both the neural crest and neural plate, with Pax3 being expressed earlier than Pax7 in mice 92.Mice that are mutant for Pax3 or Pax7 display defects in various neural crest derivatives 92,94, and Pax3 mutants exhibit a decrease or loss of migratory neural crest cells caudal to the otic vesicle 6.However, the requirement for Pax3 in the early neural crest is non-cell-autonomous: neural crest cells migrate from Pax3 / neural tubes that have been transplanted to chick hosts 6 or in vitro substrates 95, and Pax3 / migratory neural crest cells are observed in wildtype/pax3 / chimeric mice 89. Foxd3 is not expressed in caudal regions of Pax3 mutant mice 55,correlating with the failure to form neural crest in these regions 6. However, Foxd3 expression has not been examined in wild-type/pax3 / chimaeras. As migratory neural crest is observed in chimaeras, it would be interesting to see if a wild-type environment rescues Foxd3 expression in Pax3 / premigratory and migratory neural crest, or if neural crest cells are migrating without expressing Foxd3. Notch1 is broadly expressed in the neural plate, although its expression is elevated in neural crest When the Notch receptor binds one of its ligands such as Delta, the intracellular domain is cleaved and translocates into the nucleus to activate transcription 100. Overexpressing an activated Notch ablates neural crest markers and results in a loss of neural crest 798 OCTOBER 2003 VOLUME 4

5 E-BOX The conserved nucleotide sequence CANNTG that is recognized and bound by basic helix loop helix and other proteins. HOMEOBOX A sequence of about 180 base pairs that encodes a DNAbinding protein sequence known as the homeodomain. The 60- amino-acid homeodomain comprises three α-helices. DOMINANT-NEGATIVE A mutant molecule that interferes with and inhibits the activation of normal molecules. BASIC HELIX LOOP HELIX (bhlh). A structural motif present in many transcription factors that is characterized by two α-helices separated by a loop. The helices mediate dimerization, and the adjacent basic region is required for DNA binding. SOMITES Paired blocks of mesoderm cells along the vertebrate body axis that form during early vertebrate development and differentiate into dermal skin, bone and muscle. RETROELEMENTS Segments of genetic material that transpose around the genome using an RNA intermediate. derivatives 97,98,yet Delta/Notch signalling is required for neural crest formation 97,101.So, finely tuned levels of Delta/Notch signalling and/or exquisite temporal regulation of Notch activity are needed in the neural crest 97.In zebrafish at least, Notch promotes neural crest formation by inhibiting neurogenesis through repression of Neurogenin-1 (Ngn1) 101. The proto-oncogene c-myc,which stimulates proliferation and prevents differentiation 102, was recently shown to be expressed in neural crest precursors and anterior neural plate in Xenopus,with expression in the neural crest temporally preceding that of Slug 103.Loss of c-myc activity inhibits neural crest-marker gene expression and results in the loss of various neural crest derivatives. In tissue culture, c-myc is a target of Wnt signalling 104,105 and its expression in the neural crest depends on Wnt signals 103. c-myc activates signal-dependent target gene expression through E-BOX binding and histone H4 acetylation 106, so like Foxd3, it might regulate the transcriptional accessibility of a cohort of genes that are necessary for early neural crest development. Another recently identified neural crest marker with a neural plate component is the NK-1 HOMEOBOX gene Nbx. When overexpressed in Xenopus, this transcriptional repressor expands neural crest at the expense of neural plate, whereas a DOMINANT-NEGATIVE construct inhibits neural crest formation 107. Meis1b, a cofactor that regulates the transcriptional activity of Hox proteins, also seems to be sufficient to promote the formation of neural crest overexpression induces anterior neural and neural crest markers in Xenopus 108.Finally, the RNAhelicase translation initiation factor eif4a2 is expressed in the neural plate and its border in Xenopus, and is sufficient to induce neural and neural crest markers 109. Markers for neural crest and non-neural ectoderm. The transcription factor Ap2 is an example of a neural crest gene that is initially expressed throughout the neural plate border and non-neural ectoderm, and that is later enhanced in the neural folds in all vertebrates Mice that are mutant for Ap2 show defects in neural crest derivatives 114,115, and in Xenopus, Ap2 is necessary and sufficient to promote Slug and Sox9 gene expression 111. Ap2 is also required for epidermal development 116.Expression of Ap2 in neural crest depends on Wnt signalling 111, indicating that it is a target of inducing signals from the non-neural ectoderm. Ap2 fosters proliferation by repressing genes that promote terminal differentiation 117.Ap2 and c-myc interact to regulate gene expression; for example, they activate or repress transcription of E-cadherin depending on the relative expression levels of the two different c-myc isoforms 118. Msx1 and Msx2 are homeobox transcriptional repressors 119 that are also expressed in the neural folds and, at lower levels, in neighbouring non-neural ectoderm, with Msx2 being more restricted in its expression pattern 119,120 than Msx1 (REFS 7,119,121).Zebrafish Msx genes are not orthologous to Msx genes in other vertebrates, although Msxb and Msxc also show expression at the borders of the neural plate 122. Msx1 mutant mice exhibit a loss of neural crest derivatives in the face, although the severity of the phenotype is probably diminished by redundancy in Msx1 and Msx2 expression and function 123. Msx1 and Msx2 inhibit differentiation without promoting proliferation 124. Like Ap2, Msx1 has also been implicated in epidermal induction, and is induced by BMP 121 and Wnt 104 signalling. Miscellaneous neural crest genes. Id2,a helix loop helix (HLH) protein that negatively regulates basic HLH (BHLH) proteins 125, is expressed in rostral but not caudal neural folds 126, although we know little about the mechanisms that determine rostral/caudal differences in the neural crest. Id2 has been implicated in neural crest formation from the non-neural ectoderm 126, and in cultured cells it is a target of BMP 127 and Wnt signalling 104, and of Pax3 (REF. 128) and Myc activity 129. Like many other neural crest factors, Id genes stimulate proliferation and inhibit differentiation 125. Twist is a bhlh protein that is expressed in the neural crest, SOMITES and lateral plate mesoderm Twist is an early neural crest marker in Xenopus 130, whereas in the mouse, Twist is expressed in migrating neural crest cells in the head 133.Mouse mutants show that Twist is required for neural crest migration and differentiation, but not for neural crest specification 132. Twist is a direct target of Wnt signalling in murine mammary epithelial cells 134. In zebrafish, a molecule called Crestin,which is a member of a multi-copy family of RETROELEMENTS, is a very specific marker of neural crest 135.In addition, large scale genetic screens have generated numerous mutant fish lines with neural crest defects, although the genes responsible have not been identified 136. Finally, Rhob is a target of BMP signalling that is expressed in the dorsal neural tube 137 and migrating neural crest 137,138.Rho activity is not required for neural crest specification, but is necessary for the delamination of neural crest cells 137.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 progression 139. A minimal network? As the list of genes expressed by premigratory neural crest cells grows, there is an increasing need to define the interrelationships between these genes (FIG. 2). Overexpression analyses have not been altogether helpful in this regard. For example, in Xenopus animal cap assays, Foxd3 can activate Slug and Zicr1 expression, and Zicr1 can induce Foxd3 and Slug expression 53.Which comes first, Foxd3 or Zicr1? To add to the confusion, investigators tend to look at the same markers Slug, Sox9 and Foxd3. In many cases, including Foxd3 overexpression 53,neural tissue is induced along with neural crest. How do we know that neural crest is not induced as a secondary consequence of the formation of ectopic neural tissue, which itself induces neural crest? Furthermore, overexpression assays have mostly been performed in Xenopus, in which all neural crest genes seem to turn on other neural crest genes. How can we find order in this chaos? NATURE REVIEWS NEUROSCIENCE VOLUME 4 OCTOBER

6 Neural crest induction Wnt FGF BMP Dlx5 Border induction Premigratory neural crest Slug/Snail Foxd3 Sox9, Sox10 Zic genes Pax3, Pax7 c-myc, Notch1 Ap2, Nbx Msx2 Msx1 Neural plate border Neural crest emigration Rhob N-cadherin Figure 2 The current status of a neural crest gene regulatory network. The neural crest (yellow) and neural plate border (orange) are induced as separate events regulated by overlapping secreted factors. A complex set of genes are expressed in the neural folds (green and blue) as a consequence of these inductions. This developmental programme leads to changes in gene expression (purple) that result in the emigration of neural crest cells from the neural tube. However, the interrelationships between neural crest genes are only just beginning to be elucidated. As a starting point, it is possible to identify trends in our gene list (TABLE 1).In terms of function, many neural crest genes have been shown to stimulate proliferation and prevent differentiation (Zic genes 79,84,85, Pax3 (REF. 140), c-myc 102, Ap2 (REF. 117), Msx1 and Msx2 (REF. 124), Id2 (REF. 125), Notch1 (REF. 101) and Twist 134 ) or maintain stem cell potential (Foxd3 (REF. 58) and Sox10 (REF. 74)), both of which are key characteristics of the neural crest lineage. The list includes several transcriptional repressors (Slug/Snail 40,Zic1 (REF. 85), Nbx 107, Msx1 and Msx2 (REF. 119) and Id2 (REF. 125)), as well as transcriptional activators (Sox9 and Sox10 (REFS 61,62),Pax3 (REF. 93), c-myc 102,Ap2 (REF. 141) and Notch1 (REF. 100)), indicating that the formation of neural crest cells requires repression as well as activation of new gene expression. Interestingly, with the exception of Pax3, all of the transcriptional activators have been shown to bind to CREB-binding protein (CBP)/p300 (REFS ),as does β-catenin, a downstream effector of Wnt signalling 147,148.Furthermore, CBP/p300 is a target of Wnt signalling 104. CBP and p300 are closely related transcriptional co-activators that connect sequencespecific transcription factors to the general transcriptional machinery 149.They are histone acetyltransferases (HATs) and, along with Foxd3 and c-myc, might have roles in regulating the chromatin structure of neural crest target genes. In another twist, the HAT activity of p300 is inhibited by Twist 150. CBP/p300 also regulate the cell cycle in a complex containing Mdm2 (REF. 149), which is preferentially expressed in the neural folds and migrating neural crest 151. Finally, Cited2 is a protein that interacts with Ap2 and CBP/p300 to activate transcription, and Cited2 mutant mice have various neural crest defects 152. Another interesting correlation in the premigratory neural crest gene list is the direct regulation of E-cadherin expression. Expression of the cell adhesion molecule E-cadherin characterizes most epithelial cells, although premigratory neural crest expresses N-cadherin and cadherin-6b in lieu of E-cadherin 153.During EMT, including the transition from a tumour to a metastatic cancer cell, E-cadherin is downregulated 154.Likewise, N-cadherin and cadherin-6b expression are downregulated during EMT at the onset of neural crest migration 153. E-cadherin and N-cadherin are functionally equivalent 155, although the mechanisms that regulate their expression have not been compared. As Slug/Snail 49 51, Ap2 and c-myc 118 directly regulate E-cadherin expression, it is possible that they also modulate N-cadherin and/ or cadherin-6b expression to initiate EMT at the start of neural crest migration. Although they provide a framework for formulating future experiments, these correlations do not describe the molecular mechanism by which migratory neural crest cells are generated from the neural folds. How can we distill this complex array of information into a meaningful regulatory network? Filling in the gaps One way to create a clearer picture of early neural crest development is to take a comprehensive approach. Genomic-level screens could potentially identify the full collection of genes that are involved in early neural crest development. These genes can then be assembled into functional networks. Researchers have been using microarrays to achieve these goals for several years now 156.The advantage of using this approach for neural crest development is that increasingly sophisticated bioinformatic tools are constantly being generated to identify relationships and infer pathway models from array data 156.What is truly exciting about the chick and Xenopus genomic era, however, is the possibility of combining powerful array technologies with the ability to do experimental embryology. Equally enticing is the intersection of vertebrate genetics, transgenics and genomics in zebrafish. As we alluded to earlier, documenting the complete gene expression profile of a premigratory neural crest cell is not a straightforward endeavour. The availability of genomic tools for studying neural crest is not the only obstacle. The neural folds contain a heterogeneous population of cells, and neural crest precursors within this population are multipotent, with the ability to give rise to neural crest, neural tube and epidermis 3 6.So,it is not possible to simply purify cells from the neural folds and characterize gene expression in those cells. One study recently circumvented this problem to identify a collection of genes that are expressed in neural crest precursors at a single time-point following neural crest induction 157. First, by co-culturing pieces of avian non-neural ectoderm and neural plate, neural crest precursors were induced in vitro 5,10.Then, genes that were expressed as a consequence of neural crest induction were enriched in the cdna population by subtracting cdna from non-neural ectoderm and neural plate that had been cultured in isolation 157.Because chick microarrays were not available, the subtracted cdna was used to screen macroarrays containing a cdna library synthesized from 4 to 12 somite embryos, 800 OCTOBER 2003 VOLUME 4

7 EXPRESSED SEQUENCE TAGS (ESTs). Short ( base pairs) DNA sequences that represent the sequences expressed in an organism under a given condition. They are generated from the 3 - and 5 - ends of randomly selected cdna clones. The purpose of EST sequencing is to scan for all the protein-coding genes, and to provide a tag for each gene on the genome. ELECTROPORATION The transient generation of pores in a cell membrane by exposing the cell to a high field strength electrical pulse. This allows the entry of large molecules, such as DNA constructs, into the cell. which was arrayed and spotted on nylon membranes. Only clones of interest were sequenced on the macroarray, circumventing the need for the databases of existing sequence information that are required for microarray production. The results of this screen have provided new markers and regulators of neural crest development. For example, the cdna with the most specific expression pattern, provisionally called precrest-1,has no homology to any sequences in the database 157.This gene is expressed at the borders of the neural plate as soon as they are apparent, earlier than Slug or Foxd3, and is probably a direct target of early neural crest-inducing signals. Neuropilin-2a1,a receptor for semaphorins and vascular endothelial growth factor 158, is also expressed in head and trunk neural folds at very early stages in neural crest development, implying that this signalling pathway is involved in neural crest cell specification and migration 157. The macroarray screen also emphasized the importance of proliferation, chromatin remodelling, nucleocytoplasmic export, post-translational regulation and the Rho pathway for the generation of migratory neural crest cells. Defining a neural crest gene regulatory network The macroarray screen did not, however, identify the full complement of genes that are expressed at different steps in early neural crest development, nor did it organize the neural crest gene list into a cascade or network. To achieve these goals, a combination of approaches will be required. First, we can adopt a comparative strategy for the molecular analysis of neural crest formation, taking advantage of the microarrays and other genomic resources that are beginning to be developed for all vertebrates. Just as Slug and Snail gene expression patterns differ among vertebrates 43, there are likely to be variations in the molecular cascade of genes that specify the neural crest. By comparing and contrasting vertebrate neural crest development, we can define the conserved events. The potential for mechanistic variation is best exemplified by comparing neural induction in frog and chick, where differences in the importance and timing of the various factors that are involved help us to understand the entire process more fully 159.The chicken genome project is well underway 160, and chick EXPRESSED SEQUENCE TAG (EST) databases are rapidly being generated 161, so commercially produced microarrays of chick ESTs are on the horizon. For future avian genomic screens, it will probably be faster to screen avian microarrays by differential hybridization, rather than screening macroarrays with subtracted probe 157. Furthermore, there have been important advances recently in the toolbox that is available for chick experimentation. For example, ELECTROPORATION allows ectopic expression of DNA, including morpholino antisense oligonucleotides and RNA-interference constructs for loss-of-function analyses 162.Microarrays that have recently been developed in Xenopus 163,164 and zebrafish 165,166 could be screened in a similar manner. Many of the neural crest regulatory molecules that have been defined in the past few years were initially identified and characterized in Xenopus,owing to the ease of ectopic molecular manipulation in this organism. Furthermore, regulatory pathways can be tested in zebrafish and Xenopus using transgenics and/or genetic approaches. Second, neural crest can be induced in various ways, and the subsequent changes in gene expression can be documented using microarrays. Although we do not know which events most closely mimic neural crest induction in vivo, the use of different neural crest-inducing strategies should nevertheless allow the identification of the most complete neural crest gene repertoire that is possible. For example, neural crest can be induced by co-culturing neural and non-neural tissue, by treating competent neural tissue with Wnt or BMP, or by forcing the expression of a key transcription factor (FIG. 3).In addition, one could compare gene expression profiles between wild type and mutant zebrafish or mouse embryos with neural crest defects (FIG. 3). Data from each successive screen can be assembled in the form of a gene expression profile database for every gene in the catalogue, giving an indication of how each neural crest gene responds to different conditions, and allowing coordinately regulated genes to be recognized. These experiments will not only identify target genes, but will also determine whether there are qualitative differences in the neural crest induced by different treatments and transcription factors. Compiling these expression data and comparing them with in vivo expression patterns might help us to clarify what factors are required and when. Third, the response to tissue interactions and treatments that induce neural crest could be documented over time. If the activity of an inducing factor is temporally regulated through the use of inducible forms of proteins 167,or if tissue is harvested at regular time intervals, immediate early responses (direct targets) can be characterized along with downstream changes in gene expression. Furthermore, when array data is collected as a function of time, co-regulated genes can be identified using cluster analysis, in which genes are organized into groups with similar expression profiles 156. These relationships can then be used to recognize regulatory interactions. The combination of tissue-specific and temporal microarray data is already being applied to developmental problems. This approach was elegantly used in Drosophila to define the networks of signalling pathways that regulate the response to ecdysone during metamorphosis 168.With regard to the neural crest, the transcriptional response to Wnt 104 and Pax3 (REF. 128) has been documented in tissue culture cells using human cdna and oligonucleotide arrays, respectively. By performing a time course of Wnt3a treatment in embryonic carcinoma cells, it was possible to identify genes that are likely to be direct targets, based on the presence of TCF binding sites in almost all of the target gene promoters 104.Several neural crest genes were identified, including c-myc, Id2, Msx1 and Msx2, confirming the utility of this approach. The transcriptional response to Pax3 was slightly more complex, as the cells NATURE REVIEWS NEUROSCIENCE VOLUME 4 OCTOBER

8 Chick intermediate neural plate Electroporated (For example, Slug, Foxd3 and Sox9) +/ inducing factor (For example, Wnt and BMP) +/ inducing tissue (For example, non-neural ectoderm) Array hybridization and analysis Xenopus animal cap Microinjected (For example, Slug, Foxd3 and Sox9) +/ inducing factor (For example, Wnt and BMP inhibition) +/ inducing tissue (For example, paraxial mesoderm and neural plate) Zebrafish Neural crest mutant versus wild type Cy3/Cy5- labelled RNA 1. Create a gene expression profile database of all genes upregulated by neural crest-inducing treatments 2. Identify groups of genes with similar expression profiles to predict direct targets and co-regulated genes 3. Test interrelationships with additional microarray experiments Morpholino injected (For example, Snail, Foxd3 and Sox9) Figure 3 Integrating embryology and genomics to define a neural crest gene regulatory network. Examples of various protocols for inducing neural crest in chick and Xenopus. Microarray hybridization can be used to compare gene expression in control tissue and induced neural crest tissue; for example, in chick intermediate neural plate that has been electroporated with a control construct or with a transcription factor that initiates the neural crest programme. Gene expression in zebrafish neural crest mutants can also be compared. The resulting gene expression profiles can then be compiled, analysed and tested further. that were used were stably transfected with Pax3 (REF. 128). However, many of the genes that were upregulated in this screen contained putative Pax3 binding sites. These screens emphasize the need to perform time courses to characterize both direct targets and the subsequent downstream effects. Once a cohort of neural crest genes has been identified, an early neural crest-specific microarray can be generated. This reagent would facilitate future experiments to define the neural crest gene regulatory network, as only the genes that are involved in the process under investigation would be included. This type of reagent has been created to study neural crest-derived melanocyte differentiation by selecting genes with particular expression profiles from mouse EST databases 169.A neural crest-specific microarray is not an essential requirement, however, as it is possible to analyse only those clones that are of interest on a global microarray. To determine how different factors are interrelated, the effect of one gene or condition on the expression of all neural crest genes could be tested. Gain- and loss-offunction techniques should allow the identification of downstream targets, as well as potential regulatory interactions. Microarray screens of Drosophila and Caenorhabditis elegans mutant embryos have identified target genes in an analogous manner 170,171.The most comprehensive example of a gene regulatory network to be synthesized from this type of information is the network that underlies endomesoderm specification in the sea urchin 172.This complex network of transcription factors, signalling pathways and their targets has been compiled by experimentally testing interconnections by loss-of-function approaches. Finally, to definitively characterize regulatory interactions, it will be necessary to dissect elements in the regulatory regions of key neural crest gene targets. Promoter constructs can be assayed quite effectively by electroporation into a tissue of interest in chicken embryos 85,173.Meanwhile, cis regulatory elements can be assessed using transient and stable transgenic approaches in frogs 174 and fish 175.In addition, the chick 160, Xenopus 176, and zebrafish 177 genome projects will place the genomic sequence of all genes and their flanking regions into the public domain. This will allow defined enhancer elements to be annotated and putative regulatory regions to be computationally identified as shared sequence motifs in coordinately regulated genes 168,178.Complete genomic sequences will also permit the construction of 802 OCTOBER 2003 VOLUME 4

9 intergenic sequence microarrays, which can be used to identify protein binding sites and reveal direct regulatory interactions on a genome scale 179,180. Conclusions To date, neural crest development has been best studied in chick and frog due to their accessibility and ease of manipulation. Although differences exist between species, the assumption is that the most crucial mechanisms must be conserved across vertebrates, including mammals. In general, mouse null mutations have shown that neural crest genes have largely homologous roles in the chick, fish and frog (for example, transcription factor Ap2 (REFS 111,114,115), Sox10 (REFS 63,67,71 73) and the Zic gene family 75 79,84 87 ), although interesting switches in gene usage occur between paralogues (for example, Slug/Snail 43 ). The ultimate goal is to understand human neural crest development for the purpose of clinical intervention. To this end, the avian system might be the best available, as early chick neural development more closely resembles that of humans than does early rodent neural development. Regardless, it is clearly important to examine several different model organisms to establish the conserved mechanisms. As neural crest genes and networks are defined in zebrafish, frog and chick, it will be important to generate mouse mutants to determine whether gene regulatory models apply to the murine example as well. The genome era is an exciting time for biologists, especially those who are interested in previously impenetrable problems like the specification of premigratory neural crest. Although it is likely to occupy us for many years to come, the possibility of defining an early neural crest gene regulatory network is on the horizon. 1. LeDouarin, N. & Kalcheim, C. The Neural Crest (eds. Bard, J., Barlow, P. & Kirk, D.) (Cambridge Univ. Press, 1999). 2. His, W. 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