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Gene regulatory networks that control the specification of neural-crest cells in the lamprey
Biochimica et Biophysica Acta 1789 (2009) 274-278
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g r m
Review
Gene regulatory networks that control the specification of neural-crest cells in the lamprey Natalya V. Nikitina, Marianne Bronner-Fraser ⁎ Division of Biology, 139-74 Beckman Institute, California Institute of Technology, Pasadena, CA, 91125, USA
a r t i c l e
i n f o
Article history: Received 14 January 2008 Received in revised form 4 March 2008 Accepted 18 March 2008 Available online 30 March 2008 Keywords: Gene regulatory network Neural crest Lamprey
a b s t r a c t The lamprey is the only basal vertebrate in which large-scale gene perturbation analyses are feasible at present. Studies on this unique animal model promise to contribute both to the understanding of the basic neural-crest gene regulatory network architecture, and evolution of the neural crest. In this review, we summarize the currently known regulatory relationships underlying formation of the vertebrate neural crest, and discuss new ways of addressing the many remaining questions using lamprey as an experimental model. © 2008 Published by Elsevier B.V.
The neural crest (NC) is a transient embryonic cell population that is present only in vertebrates, and gives rise to many of the structures that are vertebrate-specific, including parts of the cranium and jaws. As such, the neural crest is an important innovation that is considered to be responsible for the evolutionary success of vertebrates. These cells are ectoderm-derived, yet form a wide variety of both neural and non-neural derivatives, including melanocytes, cranial bone, cartilage and connective tissue of the head, dentine of the teeth, meninges of the brain, peripheral neurons and glia, smooth muscle cells of some blood vessels, and cells of the adrenal medulla. While the extent to which pre-migratory and migrating NC cells are fate-restricted is still subject to debate [1], and may vary between species, it is clear that these cells represent a highly plastic and regulative cell population. The presumptive neural-crest forms from two bands of ectodermal cells located on either side of the neural plate (called the neural plate border), between the prospective neural and ectodermal cells. In most vertebrates, these cells delaminate and migrate away from the neural tube after neurulation is completed. In the mouse, however, the emigration of the NC cells commences from the open neural folds at mid to late neurula which suggests that neural crest and neural plate specification might be occurring simultaneously in this animal. The neural plate border cells are not committed exclusively to forming NC, but also form neurogenic placodes, sensory neurons of the dorsal neural tube, Rohon–Beard neurons in zebrafish and Xenopus, and hatching gland in Xenopus [2–5]. Intriguing and open questions include how the neural crest forms, what gives this multipotent cell population its unique properties, and ⁎ Corresponding author. Fax: +1 626 395 7717. E-mail address: [email protected] (M. Bronner-Fraser). 1874-9399/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.bbagrm.2008.03.006
how does an ectoderm cell decide to become a NC cell, and then a melanocyte or a cartilage cell? Insights into the answers to these questions may come from defining the gene regulatory network underlying neural crest formation. Below, we discuss our current knowledge of the NC gene regulatory network in several vertebrate model organisms. 1. The NC gene regulatory network: how much do we really know? Gene regulatory network (GRN) models are highly useful tools that depict the temporal sequence of the sum of all molecular interactions underlying events in embryonic development. Even when only partially complete, these logic diagrams can be of great help in directing the design of further experiments. Well-characterized GRNs can help understand genetic mechanisms responsible for evolutionary changes and design approaches for cell/tissue engineering [6,7]. Experimental data obtained from a number of different laboratories using a range of vertebrate experimental models have recently been synthesized into a hypothetical GRN model for NC development [8]. In brief, the future neural crest is induced at the border between the neural and non-neural ectoderm by a number of signaling molecules, including Wnts, intermediate levels of Bmp, FGFs and Notch (reviewed in [9–12]). The combination of these signaling molecules turns on transcription factors that are expressed early in the neural plate border and have been shown to be important for the formation of both the NC and other neural plate border derivatives. These genes, termed the neural plate border specifiers, include Zic/Opl genes, Pax3 and possibly Pax7 (in chick), Msx1 and 2 and Blimp1/ prdm1 (in zebrafish). The term neural plate border specifiers refers to genes that encode transcription factors that: 1) are important for the
N.V. Nikitina, M. Bronner-Fraser / Biochimica et Biophysica Acta 1789 (2009) 274-278
formation of a range of neural plate border derivatives, 2) lie downstream of signaling molecules such as Bmps, Wnts and FGFs, 3) act upstream of the neural crest specifiers [8]. In this review, we extend this definition to encompass only genes expressed in the neural plate border cells. For example, Dlx5, though important for the positioning of the neural plate [13,14], is expressed only in the ectoderm at the time when specification of the neural plate border occurs, and therefore will not be considered here to be a neural plate border specifier, since its action would require an intermediate signaling event to affect neural crest specification. The transcription factors listed above are by no means expressed exclusively in the prospective neural plate border, but it is their co-expression in this region that is important for their ability to initiate neural crest specific differentiation by upregulating the expression of NC specifier genes [8]. After this putative NC-GRN model was proposed, new work in Xenopus demonstrated that hierarchical regulatory interactions are established at least between some of these early neural plate genes. Msx was shown to be upstream of Pax3 and Zic1 [15], which cooperate with each other to turn on NC-specific genes Snail2 and FoxD3 and induce NC formation [15,16]. However, it is not possible to conclude whether these regulatory interactions are direct or mediated by NC specifier genes, because all morpholino-injected embryos were analysed at stage 13, by which time all of the NC specifiers are expressed (Fig. 1). The next step of NC formation is driven by the expression of a set of transcription factors which include AP-2, c-Myc, Id, Snail1 and 2, FoxD3, Sox 8, 9 and 10 and Twist. Most of these “neural crest specifier” genes (with the exception of Id and AP-2) are expressed in the premigratory NC precursors (not in other derivatives of the neural plate border, or in neural plate or ectoderm) by mid-neurula and later in migrating NC cells, and are regulated by the neural plate border specifiers. Moreover, these genes appear to form a highly interconnected network of cross-regulatory interactions, with each of these genes regulating every other gene (reviewed in [8,17]). These crest
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specifier genes initiate NC migration and differentiation into various derivatives, by activating regulatory sub-networks responsible for these processes. Unlike the sea urchin endomesoderm gene regulatory network, and many other developmental networks discussed in this volume, the NC-GRN is still in the early stages of assembly. This is largely due to the fact that most of the perturbation data currently available was obtained by the “classical” single-gene approach. Because the neural-crest cell population is not morphologically distinguishable until it starts to migrate, and few NC-specific markers are turned on shortly before the migration is initiated, the induction of neural crest was until recently thought to occur around the time of the neurulation. By demonstrating that a portion of the chick epiblast at stage 3–4 is capable of forming crest cells in culture under noninducing conditions, Basch et al. provided the first evidence that NC induction is under way as early as mid-gastrula[18]. Interestingly, expression of all neural plate border specifiers and also of some of the NC specifier genes (Id, c-Myc, AP-2) is initiated at mid-gastrula (Fig. 1). According to the current putative GRN, however, these last three genes are downstream of the neural plate border specifiers. Though the effects of AP-2 loss of function were investigated in mouse, frog and fish, the formation of the NC in these animals was assessed at and after neurula stage by evaluating the expression of pre-migratory crest markers FoxD3, Snail2 and Sox9 [19–22]. Light et al. used a combination of loss-of-function, rescue and cis-regulatory approaches to demonstrate that Id3 in Xenopus is a direct transcriptional target of c-Myc [23]. However, transcription of Id3 is initiated before c-Myc is turned on (Fig. 1), suggesting that factors other than c-Myc are responsible for the initial onset of Id expression. Thus, placing the timing and function of these three genes accurately into the network is not yet possible, since there is currently insufficient information regarding their regulatory relationships with other members of the network. Regulatory relationships between the NC specifiers Snail 1 and 2, Sox 8, 9 and 10, FoxD3 have been extensively studied at the time of
Fig. 1. Onset of expression of neural plate border and NC specifier genes in Xenopus and lamprey. Question mark indicates that the expression of MsxA, Zic1, Pax3/7, n-Myc, AP-2, Id and snail in the lamprey before day 4 of embryonic development has not yet been determined. This figure is based on the expression pattern reported in [15,16,20,23– 25,28,30,32,44,45].
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pre-migratory crest formation and in the migrating crest, mostly using Xenopus as the model organism [24–29]. However, the lack of cisregulatory data, and the fact that every one of these genes seems to regulate every other gene at that stage makes it difficult to assemble them into a definite network. One approach to figuring out the hierarchical relationships between these genes would be to study the loss-of function effects of a subset of the genes at an earlier time point, when only some of the NC specifier genes are expressed. Because NC development from induction to migration occurs over a course of just a few hours, and specifier genes are turned on in quick succession, this approach may not be feasible in standard vertebrate embryological models that develop quickly. For instance in Xenopus the difference in time between the onset of cMyc/ Zic and Sox8 is about 45 min, and the embryos at these stages are not easily morphologically distinguishable [16,28,30].
2. The NC-GRN in the lamprey Lampreys and hagfishes are the most basal living vertebrates. These animals have most of the characteristic features of true vertebrates, with the exceptions of the jaws and much of the cranium. Classical embryological studies demonstrated that a migratory NC population was present in the lamprey. This population was shown to delaminate from the dorsal neural tube and to migrate along routes similar to those seen in other vertebrates [31]. However, it was not clear whether this cell population was truly homologous to the neural crest of higher vertebrates at the gene regulatory level. In order to address this question, Sauka-Spengler et al. constructed a lamprey cDNA library and identified the lamprey homologues of all currently known genes involved in the crest induction, delamination, migration and differentiation [32]. In the most comprehensive analysis of the
Fig. 2. Comparison between the putative NC-GRN of the lamprey (A) to the generalized vertebrate NC-GRN model. (A) is based on the data reported in [32], (B) is based on [8]. Question marks indicate that exact hierarchical relationships and feedback regulatory loops, though suggested by the data, cannot as yet be precisely assigned.
N.V. Nikitina, M. Bronner-Fraser / Biochimica et Biophysica Acta 1789 (2009) 274-278
NC-GRN done to date in any species, it was demonstrated that the deployment of the NC inducing signals and the transcription factors involved in the crest formation are essentially conserved between the lamprey and higher vertebrates (Fig. 2). Several important differences between the organization of the lamprey NC-GRN and that of other vertebrates have been uncovered by the above analysis. In the lamprey, the NC specifier genes are turned on in two discrete phases that are separated from each other by more than 24 h. AP-2, c-Myc, Id and Snail transcripts are found in the neural plate border of the early neurula at day 4.0 of development, while FoxD3 and SoxE are deployed a day and a half later specifically in pre-migratory neural crest (Fig. 1). While this sequence of transcription factor activation is consistent with what is seen in other vertebrates such as Xenopus, the large time gap between the expression of the early and late NC specifier genes was not previously discerned in any other vertebrate species (Fig. 1). This makes the lamprey an unexpectedly useful experimental model for the study of the regulatory relationships between early neural crest genes. The lamprey NC-GRN displays more conservation at the early steps of the network, while the late steps are more divergent. For instance, the lamprey Snail homologue is not expressed in an exclusively neural-crest-specific manner [32]. In craniates, Snail genes are expressed in paraxial mesoderm during gastrulation and later in the pre-migratory NC [26,33,34]. Interestingly, in invertebrate chordates (Ciona, amphioxus) lacking NC, the expression of Snail is similarly restricted to the mesoderm and later ectoderm bordering the neural plate [35,36]. The expression pattern of Snail in the lamprey is quite unlike that of both of these groups. Snail first appears throughout the neural plate at E4, but soon becomes ubiquitously expressed throughout the ectoderm and mesoderm of the embryo. A similar ubiquitous expression pattern is seen in hagfish, another jawless vertebrate [37]. It remains to be investigated whether this expansion of expression is connected to the appearance of the neural crest. Twist and Ets are two other transcription factors whose expression in the lamprey differs markedly from that of other vertebrates. In Xenopus, zebrafish and chick, Ets expression is initiated in premigratory crest, while Twist comes on in early migrating crest [33,38– 41]. In lamprey, four homologues of Twist and three of Ets have been identified; all of these genes are expressed in postmigratory crest cells [32]. This is rather surprising, as functional work in mouse and chick embryos demonstrated that Twist plays a role in neural-crest migration, while Ets is important for promoting NC delamination [42,43]. Perhaps the lamprey Twist and Ets homologues play roles in differentiation of NC derivatives but this has yet to be explored. We are at an exciting juncture in beginning to dissect the vertebrate NC gene regulatory network. We now know the extent of conservation of this network between the lamprey and higher vertebrates. The data suggest deep conservation of the core of this network for over 500 million years. We currently have all the necessary genomic tools and experimental approaches established to further explore the NC-GRN in this animal. Future work will use this basal vertebrate to advance both our understanding of the basic NC-GRN architecture and the evolution of the neural crest and its derivatives. References [1] J.F. Crane, P.A. Trainor, Neural crest stem and progenitor cells, Annu. Rev. Cell Dev. Biol. 22 (2006) 267–286. [2] C.V. Baker, M. Bronner-Fraser, Vertebrate cranial placodes I. Embryonic induction, Dev. Biol. 232 (2001) 1–61. [3] L. Hernandez-Lagunas, I.F. Choi, T. Kaji, P. Simpson, C. Hershey, Y. Zhou, L. Zon, M. Mercola, K.B. Artinger, Zebrafish narrowminded disrupts the transcription factor prdm1 and is required for neural crest and sensory neuron specification, Dev. Biol. 278 (2005) 347–357. [4] C.S. Hong, J.P. Saint-Jeannet, The activity of Pax3 and Zic1 regulates three distinct cell fates at the neural plate border, Mol. Biol. Cell 18 (2007) 2192–2202. [5] T. Kaji, K.B. Artinger, dlx3b and dlx4b function in the development of Rohon-Beard sensory neurons and trigeminal placode in the zebrafish neurula, Dev. Biol. 276 (2004) 523–540.
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Streit, DLX5 positions the neural crest and preplacode region at the border of the neural plate, Dev. Biol 259 (2003) 34–47. [14] J.M. Woda, J. Pastagia, M. Mercola, K.B. Artinger, Dlx proteins position the neural plate border and determine adjacent cell fates, Development 130 (2003) 331–342. [15] A.H. Monsoro-Burq, E. Wang, R. Harland, Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction, Dev. Cell 8 (2005) 167–178. [16] T. Sato, N. Sasai, Y. Sasai, Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm, Development 132 (2005) 2355–2363. [17] B. Steventon, C. Carmona-Fontaine, R. Mayor, Genetic network during neural crest induction: from cell specification to cell survival, Semin. Cell Dev. Biol. 16 (2005) 647–654. [18] M.L. Basch, M. Bronner-Fraser, M.I. Garcia-Castro, Specification of the neural crest occurs during gastrulation and requires Pax7, Nature 441 (2006) 218–222. [19] R.D. Knight, S. Nair, S.S. Nelson, A. Afshar, Y. Javidan, R. Geisler, G.J. Rauch, T.F. Schilling, Lockjaw encodes a zebrafish tfap2a required for early neural crest development, Development 130 (2003) 5755–5768. [20] T. Luo, Y.H. Lee, J.P. Saint-Jeannet, T.D. Sargent, Induction of neural crest in Xenopus by transcription factor AP2alpha, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 532–537. [21] H. Schorle, P. Meier, M. Buchert, R. Jaenisch, P.J. Mitchell, Transcription factor AP-2 essential for cranial closure and craniofacial development, Nature 381 (1996) 235–238. [22] J. Zhang, S. Hagopian-Donaldson, G. Serbedzija, J. Elsemore, D. Plehn-Dujowich, A.P. McMahon, R.A. Flavell, T. Williams, Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2, Nature 381 (1996) 238–241. [23] W. Light, A.E. Vernon, A. Lasorella, A. Iavarone, C. LaBonne, Xenopus Id3 is required downstream of Myc for the formation of multipotent neural crest progenitor cells, Development 132 (2005) 1831–1841. [24] Y. Aoki, N. Saint-Germain, M. Gyda, E. Magner-Fink, Y.H. Lee, C. Credidio, J.P. SaintJeannet, Sox10 regulates the development of neural crest-derived melanocytes in Xenopus, Dev. Biol. 259 (2003) 19–33. [25] S.M. Honore, M.J. Aybar, R. Mayor, Sox10 is required for the early development of the prospective neural crest in Xenopus embryos, Dev. Biol. 260 (2003) 79–96. [26] M.J. Aybar, M.A. Nieto, R. Mayor, Snail precedes slug in the genetic cascade required for the specification and migration of the Xenopus neural crest, Development 130 (2003) 483–494. [27] N. Sasai, K. Mizuseki, Y. Sasai, Requirement of FoxD3-class signaling for neural crest determination in Xenopus, Development 128 (2001) 2525–2536. [28] M. O'Donnell, C.S. Hong, X. Huang, R.J. Delnicki, J.P. Saint-Jeannet, Functional analysis of Sox8 during neural crest development in Xenopus, Development 133 (2006) 3817–3826. [29] Y.H. Lee, Y. Aoki, C.S. Hong, N. Saint-Germain, C. Credidio, J.P. Saint-Jeannet, Early requirement of the transcriptional activator Sox9 for neural crest specification in Xenopus, Dev. Biol 275 (2004) 93–103. [30] A. Bellmeyer, J. Krase, J. Lindgren, C. LaBonne, The protooncogene c-myc is an essential regulator of neural crest formation in Xenopus, Dev. Cell 4 (2003) 827–839. [31] D.W. McCauley, M. Bronner-Fraser, Neural crest contributions to the lamprey head, Development 130 (2003) 2317–2327. [32] T. Sauka-Spengler, D. Meulemans, M. Jones, M. Bronner-Fraser, Ancient evolutionary origin of the neural crest gene regulatory network, Dev. Cell 13 (2007) 405–420. [33] C. Linker, M. Bronner-Fraser, R. Mayor, Relationship between gene expression domains of Xsnail, Xslug, and Xtwist and cell movement in the prospective neural crest of Xenopus, Dev. Biol. 224 (2000) 215–225. [34] M.A. Nieto, M.G. Sargent, D.G. Wilkinson, J. Cooke, Control of cell behavior during vertebrate development by Slug, a zinc finger gene, Science 264 (1994) 835–839. [35] J.A. 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during Xenopus laevis oogenesis and embryogenesis, Int. J. Dev. Biol. 41 (1997) 607–620. [40] N.D. Hopwood, A. Pluck, J.B. Gurdon, A Xenopus mRNA related to Drosophila twist is expressed in response to induction in the mesoderm and the neural crest, Cell 59 (1989) 893–903. [41] I. Germanguz, D. Lev, T. Waisman, C.H. Kim, I. Gitelman, Four twist genes in zebrafish, four expression patterns, Dev. Dyn. 236 (2007) 2615–2626. [42] E. Theveneau, J.L. Duband, M. Altabef, Ets-1 confers cranial features on neural crest delamination, PLoS ONE 2 (2007) e1142.
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Contents lists available at ScienceDirect
Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g r m
Review
Gene regulatory networks that control the specification of neural-crest cells in the lamprey Natalya V. Nikitina, Marianne Bronner-Fraser ⁎ Division of Biology, 139-74 Beckman Institute, California Institute of Technology, Pasadena, CA, 91125, USA
a r t i c l e
i n f o
Article history: Received 14 January 2008 Received in revised form 4 March 2008 Accepted 18 March 2008 Available online 30 March 2008 Keywords: Gene regulatory network Neural crest Lamprey
a b s t r a c t The lamprey is the only basal vertebrate in which large-scale gene perturbation analyses are feasible at present. Studies on this unique animal model promise to contribute both to the understanding of the basic neural-crest gene regulatory network architecture, and evolution of the neural crest. In this review, we summarize the currently known regulatory relationships underlying formation of the vertebrate neural crest, and discuss new ways of addressing the many remaining questions using lamprey as an experimental model. © 2008 Published by Elsevier B.V.
The neural crest (NC) is a transient embryonic cell population that is present only in vertebrates, and gives rise to many of the structures that are vertebrate-specific, including parts of the cranium and jaws. As such, the neural crest is an important innovation that is considered to be responsible for the evolutionary success of vertebrates. These cells are ectoderm-derived, yet form a wide variety of both neural and non-neural derivatives, including melanocytes, cranial bone, cartilage and connective tissue of the head, dentine of the teeth, meninges of the brain, peripheral neurons and glia, smooth muscle cells of some blood vessels, and cells of the adrenal medulla. While the extent to which pre-migratory and migrating NC cells are fate-restricted is still subject to debate [1], and may vary between species, it is clear that these cells represent a highly plastic and regulative cell population. The presumptive neural-crest forms from two bands of ectodermal cells located on either side of the neural plate (called the neural plate border), between the prospective neural and ectodermal cells. In most vertebrates, these cells delaminate and migrate away from the neural tube after neurulation is completed. In the mouse, however, the emigration of the NC cells commences from the open neural folds at mid to late neurula which suggests that neural crest and neural plate specification might be occurring simultaneously in this animal. The neural plate border cells are not committed exclusively to forming NC, but also form neurogenic placodes, sensory neurons of the dorsal neural tube, Rohon–Beard neurons in zebrafish and Xenopus, and hatching gland in Xenopus [2–5]. Intriguing and open questions include how the neural crest forms, what gives this multipotent cell population its unique properties, and ⁎ Corresponding author. Fax: +1 626 395 7717. E-mail address: [email protected] (M. Bronner-Fraser). 1874-9399/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.bbagrm.2008.03.006
how does an ectoderm cell decide to become a NC cell, and then a melanocyte or a cartilage cell? Insights into the answers to these questions may come from defining the gene regulatory network underlying neural crest formation. Below, we discuss our current knowledge of the NC gene regulatory network in several vertebrate model organisms. 1. The NC gene regulatory network: how much do we really know? Gene regulatory network (GRN) models are highly useful tools that depict the temporal sequence of the sum of all molecular interactions underlying events in embryonic development. Even when only partially complete, these logic diagrams can be of great help in directing the design of further experiments. Well-characterized GRNs can help understand genetic mechanisms responsible for evolutionary changes and design approaches for cell/tissue engineering [6,7]. Experimental data obtained from a number of different laboratories using a range of vertebrate experimental models have recently been synthesized into a hypothetical GRN model for NC development [8]. In brief, the future neural crest is induced at the border between the neural and non-neural ectoderm by a number of signaling molecules, including Wnts, intermediate levels of Bmp, FGFs and Notch (reviewed in [9–12]). The combination of these signaling molecules turns on transcription factors that are expressed early in the neural plate border and have been shown to be important for the formation of both the NC and other neural plate border derivatives. These genes, termed the neural plate border specifiers, include Zic/Opl genes, Pax3 and possibly Pax7 (in chick), Msx1 and 2 and Blimp1/ prdm1 (in zebrafish). The term neural plate border specifiers refers to genes that encode transcription factors that: 1) are important for the
N.V. Nikitina, M. Bronner-Fraser / Biochimica et Biophysica Acta 1789 (2009) 274-278
formation of a range of neural plate border derivatives, 2) lie downstream of signaling molecules such as Bmps, Wnts and FGFs, 3) act upstream of the neural crest specifiers [8]. In this review, we extend this definition to encompass only genes expressed in the neural plate border cells. For example, Dlx5, though important for the positioning of the neural plate [13,14], is expressed only in the ectoderm at the time when specification of the neural plate border occurs, and therefore will not be considered here to be a neural plate border specifier, since its action would require an intermediate signaling event to affect neural crest specification. The transcription factors listed above are by no means expressed exclusively in the prospective neural plate border, but it is their co-expression in this region that is important for their ability to initiate neural crest specific differentiation by upregulating the expression of NC specifier genes [8]. After this putative NC-GRN model was proposed, new work in Xenopus demonstrated that hierarchical regulatory interactions are established at least between some of these early neural plate genes. Msx was shown to be upstream of Pax3 and Zic1 [15], which cooperate with each other to turn on NC-specific genes Snail2 and FoxD3 and induce NC formation [15,16]. However, it is not possible to conclude whether these regulatory interactions are direct or mediated by NC specifier genes, because all morpholino-injected embryos were analysed at stage 13, by which time all of the NC specifiers are expressed (Fig. 1). The next step of NC formation is driven by the expression of a set of transcription factors which include AP-2, c-Myc, Id, Snail1 and 2, FoxD3, Sox 8, 9 and 10 and Twist. Most of these “neural crest specifier” genes (with the exception of Id and AP-2) are expressed in the premigratory NC precursors (not in other derivatives of the neural plate border, or in neural plate or ectoderm) by mid-neurula and later in migrating NC cells, and are regulated by the neural plate border specifiers. Moreover, these genes appear to form a highly interconnected network of cross-regulatory interactions, with each of these genes regulating every other gene (reviewed in [8,17]). These crest
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specifier genes initiate NC migration and differentiation into various derivatives, by activating regulatory sub-networks responsible for these processes. Unlike the sea urchin endomesoderm gene regulatory network, and many other developmental networks discussed in this volume, the NC-GRN is still in the early stages of assembly. This is largely due to the fact that most of the perturbation data currently available was obtained by the “classical” single-gene approach. Because the neural-crest cell population is not morphologically distinguishable until it starts to migrate, and few NC-specific markers are turned on shortly before the migration is initiated, the induction of neural crest was until recently thought to occur around the time of the neurulation. By demonstrating that a portion of the chick epiblast at stage 3–4 is capable of forming crest cells in culture under noninducing conditions, Basch et al. provided the first evidence that NC induction is under way as early as mid-gastrula[18]. Interestingly, expression of all neural plate border specifiers and also of some of the NC specifier genes (Id, c-Myc, AP-2) is initiated at mid-gastrula (Fig. 1). According to the current putative GRN, however, these last three genes are downstream of the neural plate border specifiers. Though the effects of AP-2 loss of function were investigated in mouse, frog and fish, the formation of the NC in these animals was assessed at and after neurula stage by evaluating the expression of pre-migratory crest markers FoxD3, Snail2 and Sox9 [19–22]. Light et al. used a combination of loss-of-function, rescue and cis-regulatory approaches to demonstrate that Id3 in Xenopus is a direct transcriptional target of c-Myc [23]. However, transcription of Id3 is initiated before c-Myc is turned on (Fig. 1), suggesting that factors other than c-Myc are responsible for the initial onset of Id expression. Thus, placing the timing and function of these three genes accurately into the network is not yet possible, since there is currently insufficient information regarding their regulatory relationships with other members of the network. Regulatory relationships between the NC specifiers Snail 1 and 2, Sox 8, 9 and 10, FoxD3 have been extensively studied at the time of
Fig. 1. Onset of expression of neural plate border and NC specifier genes in Xenopus and lamprey. Question mark indicates that the expression of MsxA, Zic1, Pax3/7, n-Myc, AP-2, Id and snail in the lamprey before day 4 of embryonic development has not yet been determined. This figure is based on the expression pattern reported in [15,16,20,23– 25,28,30,32,44,45].
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pre-migratory crest formation and in the migrating crest, mostly using Xenopus as the model organism [24–29]. However, the lack of cisregulatory data, and the fact that every one of these genes seems to regulate every other gene at that stage makes it difficult to assemble them into a definite network. One approach to figuring out the hierarchical relationships between these genes would be to study the loss-of function effects of a subset of the genes at an earlier time point, when only some of the NC specifier genes are expressed. Because NC development from induction to migration occurs over a course of just a few hours, and specifier genes are turned on in quick succession, this approach may not be feasible in standard vertebrate embryological models that develop quickly. For instance in Xenopus the difference in time between the onset of cMyc/ Zic and Sox8 is about 45 min, and the embryos at these stages are not easily morphologically distinguishable [16,28,30].
2. The NC-GRN in the lamprey Lampreys and hagfishes are the most basal living vertebrates. These animals have most of the characteristic features of true vertebrates, with the exceptions of the jaws and much of the cranium. Classical embryological studies demonstrated that a migratory NC population was present in the lamprey. This population was shown to delaminate from the dorsal neural tube and to migrate along routes similar to those seen in other vertebrates [31]. However, it was not clear whether this cell population was truly homologous to the neural crest of higher vertebrates at the gene regulatory level. In order to address this question, Sauka-Spengler et al. constructed a lamprey cDNA library and identified the lamprey homologues of all currently known genes involved in the crest induction, delamination, migration and differentiation [32]. In the most comprehensive analysis of the
Fig. 2. Comparison between the putative NC-GRN of the lamprey (A) to the generalized vertebrate NC-GRN model. (A) is based on the data reported in [32], (B) is based on [8]. Question marks indicate that exact hierarchical relationships and feedback regulatory loops, though suggested by the data, cannot as yet be precisely assigned.
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NC-GRN done to date in any species, it was demonstrated that the deployment of the NC inducing signals and the transcription factors involved in the crest formation are essentially conserved between the lamprey and higher vertebrates (Fig. 2). Several important differences between the organization of the lamprey NC-GRN and that of other vertebrates have been uncovered by the above analysis. In the lamprey, the NC specifier genes are turned on in two discrete phases that are separated from each other by more than 24 h. AP-2, c-Myc, Id and Snail transcripts are found in the neural plate border of the early neurula at day 4.0 of development, while FoxD3 and SoxE are deployed a day and a half later specifically in pre-migratory neural crest (Fig. 1). While this sequence of transcription factor activation is consistent with what is seen in other vertebrates such as Xenopus, the large time gap between the expression of the early and late NC specifier genes was not previously discerned in any other vertebrate species (Fig. 1). This makes the lamprey an unexpectedly useful experimental model for the study of the regulatory relationships between early neural crest genes. The lamprey NC-GRN displays more conservation at the early steps of the network, while the late steps are more divergent. For instance, the lamprey Snail homologue is not expressed in an exclusively neural-crest-specific manner [32]. In craniates, Snail genes are expressed in paraxial mesoderm during gastrulation and later in the pre-migratory NC [26,33,34]. Interestingly, in invertebrate chordates (Ciona, amphioxus) lacking NC, the expression of Snail is similarly restricted to the mesoderm and later ectoderm bordering the neural plate [35,36]. The expression pattern of Snail in the lamprey is quite unlike that of both of these groups. Snail first appears throughout the neural plate at E4, but soon becomes ubiquitously expressed throughout the ectoderm and mesoderm of the embryo. A similar ubiquitous expression pattern is seen in hagfish, another jawless vertebrate [37]. It remains to be investigated whether this expansion of expression is connected to the appearance of the neural crest. Twist and Ets are two other transcription factors whose expression in the lamprey differs markedly from that of other vertebrates. In Xenopus, zebrafish and chick, Ets expression is initiated in premigratory crest, while Twist comes on in early migrating crest [33,38– 41]. In lamprey, four homologues of Twist and three of Ets have been identified; all of these genes are expressed in postmigratory crest cells [32]. This is rather surprising, as functional work in mouse and chick embryos demonstrated that Twist plays a role in neural-crest migration, while Ets is important for promoting NC delamination [42,43]. Perhaps the lamprey Twist and Ets homologues play roles in differentiation of NC derivatives but this has yet to be explored. We are at an exciting juncture in beginning to dissect the vertebrate NC gene regulatory network. We now know the extent of conservation of this network between the lamprey and higher vertebrates. The data suggest deep conservation of the core of this network for over 500 million years. We currently have all the necessary genomic tools and experimental approaches established to further explore the NC-GRN in this animal. Future work will use this basal vertebrate to advance both our understanding of the basic NC-GRN architecture and the evolution of the neural crest and its derivatives. References [1] J.F. Crane, P.A. Trainor, Neural crest stem and progenitor cells, Annu. Rev. Cell Dev. Biol. 22 (2006) 267–286. [2] C.V. Baker, M. Bronner-Fraser, Vertebrate cranial placodes I. Embryonic induction, Dev. Biol. 232 (2001) 1–61. [3] L. Hernandez-Lagunas, I.F. Choi, T. Kaji, P. Simpson, C. Hershey, Y. Zhou, L. Zon, M. Mercola, K.B. Artinger, Zebrafish narrowminded disrupts the transcription factor prdm1 and is required for neural crest and sensory neuron specification, Dev. Biol. 278 (2005) 347–357. [4] C.S. Hong, J.P. Saint-Jeannet, The activity of Pax3 and Zic1 regulates three distinct cell fates at the neural plate border, Mol. Biol. Cell 18 (2007) 2192–2202. [5] T. Kaji, K.B. Artinger, dlx3b and dlx4b function in the development of Rohon-Beard sensory neurons and trigeminal placode in the zebrafish neurula, Dev. Biol. 276 (2004) 523–540.
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