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Gene-Regulatory Interactions in Neural Crest Evolution and Development
Developmental Cell, Vol. 7, 291–299, September, 2004, Copyright 2004 by Cell Press
Gene-Regulatory Interactions in Neural Crest Evolution and Development Daniel Meulemans and Marianne Bronner-Fraser* California Institute of Technology 1200 East California Boulevard Pasadena, California 91125
In this review, we outline the gene-regulatory interactions driving neural crest development and compare these to a hypothetical network operating in the embryonic ectoderm of the cephalochordate amphioxus. While the early stages of ectodermal patterning appear conserved between amphioxus and vertebrates, later activation of neural crest-specific factors at the neural plate border appears to be a vertebrate novelty. This difference may reflect co-option of genetic pathways which conferred novel properties upon the evolving vertebrate neural plate border, potentiating the evolution of definitive neural crest.
Introduction The neural crest is an embryonic cell population defined by its origins at the neural plate border, migratory capacity, multipotency, and characteristic gene expression profile. The appearance of this tissue was likely a turning point in vertebrate evolution since many of the structures which define the vertebrate clade are derived from neural crest cells (Gans and Northcutt, 1983; Northcutt and Gans, 1983). Consistent with a key role in vertebrate origins, a bona fide neural crest is present in every extant vertebrate species including the most basal jawless fish, but not in the most vertebrate-like invertebrates. Gene expression patterns and functional comparisons suggest that the gene-regulatory interactions driving neural crest formation are conserved among vertebrates. However, the mechanistic data at the core of the emerging neural crest gene network are derived from embryological models not particularly amenable to genetic manipulation. As a result, experimental analyses of neural crest gene-regulatory relationships have largely been restricted to gain- or loss-of-function studies using injected mRNA, cDNA, or antisense oligonucleotides. Further limiting a comprehensive dissection of the neural crest regulatory network is the fact that most vertebrates are poorly suited for cis-regulatory analyses due to the dysregulated and mosaic expression of injected reporter constructs and the difficulty of making transgenics. Thus, for a given “neural crest gene” it is only known that over- or underexpression in turn influences the expression of other neural crest genes. It is usually unclear whether this interdependency involves direct binding of a transcription factor to an enhancer or works through intermediate genes. In the first part of this review, we assemble current data from such gain- and loss-of-function analyses and outline the genetic interactions which initiate and maintain the neural crest cell gene-regulatory state—driving *Correspondence: [email protected]
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neural crest delamination from the neural tube, migration through the periphery, and differentiation into varied cell types (Figure 1A). In the second part, we compare these gene-regulatory circuits to putatively homologous networks operating in amphioxus—a vertebrate-like chordate that lacks neural crest (Figure 1B). From these comparisons, we identify some of the novel gene-regulatory relationships that may have potentiated the evolution of definitive neural crest. Wnts, Fgfs, and Various Levels of BMP Signaling Induce Epidermal, Neural Plate, and Neural Crest Gene Expression The neural crest regulatory state is initiated by inductive signals in the form of BMP, Wnt, and Fgf signaling molecules secreted from underlying mesoderm and adjacent nonneural ectoderm (Bonstein et al., 1998; Garcia-Castro et al., 2002; LaBonne and Bronner-Fraser, 1998; Liem et al., 1995; Marchant et al., 1998; Monsoro-Burq et al., 2003). These intercellular signals also function simultaneously to segregate neural from nonneural ectoderm during neural induction (Sasai and De Robertis, 1997; Weinstein and Hemmati-Brivanlou, 1999). In nonneural ectoderm, high levels of BMP signal activate the epidermal program via Msx1, Dlx3/5, and AP-2 (Feledy et al., 1999; Luo et al., 2001a, 2002; Pera et al., 1999). Msx1, an immediate early target of BMP2/4 signaling, is sufficient to activate keratin expression and repress the neural markers Zic3 and N-CAM (Suzuki et al., 1997). Dlx3 and 5 similarly repress neural factors such as Sox2 (Feledy et al., 1999; Luo et al., 2001a; McLarren et al., 2003) while ectodermal AP-2 activates epidermal keratin expression by direct binding of the keratin promoter (Luo et al., 2002; Snape et al., 1991). Inhibition of ectodermal BMP signaling induces expression of the transcription factors Sox2, Zicr-1, and Zic3 in the neural plate (Mizuseki et al., 1998; Nakata et al., 1997). Sox2 and Zic genes, in turn, activate neural differentiation genes such as N-CAM and N-tubulin (Mizuseki et al., 1998), probably through the neurogenic bHLH transcription factors Neurogenin, Neuro-D, and achaete-scute. Sox2 also indirectly represses expression of the neural crest specifier Slug in the avian neural plate (Wakamatsu et al., 2004). At the neural plate border of frogs and zebrafish, intermediate levels of BMP activate high-level expression of “neural crest specifiers” such as Snail, Slug, AP-2, Sox9, and FoxD3 (LaBonne and Bronner-Fraser, 1998; Luo et al., 2003; Marchant et al., 1998; Morgan and Sargent, 1997; Nguyen et al., 1998; Sasai et al., 2001). Establishment and maintenance of this attenuated signal likely depends on local Notch receptor-mediated signaling (Endo et al., 2002; Glavic et al., 2004). Interestingly, Notch pathway activation can dampen or amplify BMP signaling at the neural plate border, depending on the organism. This bifunctionality may have evolved to compensate for the different levels of ectodermal BMP observed in different vertebrate embryos (discussed in Glavic et al., 2004). In addition to an attenuated BMP signal, input from the Wnt pathway is also required for
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Figure 1. Comparison of Putative Gene-Regulatory and Signaling Interactions Operating at the Neural Plate Border of Vertebrates and Amphioxus Red arrows indicate proven direct regulatory interactions. Black arrows are genetic interactions suggested by gain- and loss-of-function analyses largely in Xenopus. Gray lines indicate repression.
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expression of Snail, Slug, AP-2, and FoxD3, as well as virtually every other described neural crest and neural plate border marker (Garcia-Castro et al., 2002; LaBonne and Bronner-Fraser, 1998; Luo et al., 2003; SaintJeannet et al., 1997; Sasai et al., 2001). Finally, Fgf signaling is both necessary and sufficient for neural crest specifier expression in cultured ectodermal explants and in vivo (LaBonne and Bronner-Fraser, 1998; Mayor et al., 1997; Monsoro-Burq et al., 2003). It is unknown if the influence of BMP, Wnt, and Fgf pathways on neural crest specifier expression is direct or controlled by other factors. Evidence presented below suggests that Zic factors, Msx1, and Pax3/7 may mediate the effects of BMPs and Wnts on neural crest specifier expression. However, data from Xenopus suggest that Slug expression in neural crest is dependent on binding of Wnt effector genes (Tcf/Lef transcription factors) to the Slug promoter, implying direct regulation of Slug by the Wnt pathway (Vallin et al., 2001). Neural Plate Border Specifiers Mediate the Influence of Wnts, BMPs, and Fgfs on Neural Crest Specifiers Initial signaling events that establish the neural plate border lead to expression of a small set of transcription factors designated here as “neural plate border specifiers.” This group of genes includes Zic factors, Pax3/7, Dlx5, and Msx1/2. Of these, Pax3 and Msx1/2 appear to be downstream of Wnt signaling (Bang et al., 1999), while Zic1, Zic3, Dlx5, and Msx1/2 become upregulated at the neural plate border in response to an attenuated BMP signal (Aruga et al., 2002; Luo et al., 2001b; Nakata et al., 1997; Tribulo et al., 2003). A third Zic gene, Zic5, is upregulated in response to ectopic Fgf8 in ectodermal explants (Monsoro-Burq et al., 2003). These genes display features that distinguish them from other neural plate border markers and suggest they may mediate the influence of Wnts, BMPs, and Fgfs on neural crest specifiers like Slug/Snail. First, these factors are expressed early and broadly at the neural plate border and precede activation of most definitive neural crest genes (Aruga et al., 2002; Luo et al., 2001b; Mizuseki et al., 1998; Nakata et al., 2000, 1997; Suzuki et al., 1997). Second, they are not general markers of migrating or post-migratory neural crest cells (though Msx2 and Dlx3/5 are expressed in specific subsets of cranial neural crest [Akimenko et al., 1994; Pera and Kessel, 1999; Robinson and Mahon, 1994; Takahashi et al., 2001; Yang et al., 1998] and Pax3 marks differentiating neural crestderived neurons and glia [Goulding et al., 1991]). Third, they function in neural plate border-derived cell types
aside from neural crest, including roof plate, dorsal interneurons, placodes, and Rohon-Beard cells (Aruga et al., 2002; Bang et al., 1999; Goulding et al., 1993; Liu et al., 2004; Mansouri and Gruss, 1998; McLarren et al., 2003; Tremblay et al., 1996; Woda et al., 2003). Finally, experimental evidence places these factors upstream of neural crest specifiers. Ectopic Zic is sufficient to activate expression of Snail, Slug, FoxD3, and Twist in Xenopus embryos and ectodermal explants (Brewster et al., 1998; Mizuseki et al., 1998; Nakata et al., 2000, 1997; Sasai et al., 2001). Pax3 is required for the expression of FoxD3 in mouse (Dottori et al., 2001). Msx1 is necessary and sufficient for the expression of Snail, Slug, and FoxD3 in frog (Tribulo et al., 2003). Furthermore, Msx1 cannot rescue disruption of Slug/Snail function, but dominant-negative disruption of Msx1 can be rescued by overexpression of Slug/Snail—implying Slug/Snail is downstream of Msx1 (Tribulo et al., 2003). Dlx5 has not been shown to directly regulate neural crest specifiers but is sufficient to drive ectopic expression of Msx1 (Woda et al., 2003). Thus, the timing and breadth of neural plate border specifier expression, as well as their demonstrated regulatory capacities, suggest they act upstream of neural crest markers—and downstream of Wnt, BMP, and Fgf signals. Neural Crest Specifiers Occupy a Distinct Position in the Neural Crest Gene Network and Crossregulate to Maintain Each Other’s Expression After the onset of neural plate border specifier expression, a suite of genes including Slug/Snail, AP-2, FoxD3, Sox10, Sox9, and c-Myc is activated in nascent neural crest cells (Aoki et al., 2003; Bellmeyer et al., 2003; Honore et al., 2003; LaBonne and Bronner-Fraser, 2000; Luo et al., 2003; Sasai et al., 2001; Spokony et al., 2002). These neural crest specifiers share several characteristics which suggest they operate downstream of Wnt/ BMP/Fgf inductive signals and the neural plate border specifiers Zic, Msx1/2, and Pax3/7. All neural crest specifiers are expressed in pre- and migratory neural crest cells, repress Sox2 expression, and require a Wnt signal for activation. In addition, Zic, Msx1/2, and Pax3/7 are necessary and/or sufficient for the expression of Slug/ Snail, FoxD3, and Sox10. Every neural crest specifier examined thus far also appears necessary and/or sufficient for the expression of the other specifiers in Xenopus. Thus, injection of a Sox10 antisense morpholino oligonucleotide causes loss of Sox9, Slug/Snail, and FoxD3, while Sox10 overexpression expands the expression domain of Slug and
(A) In vertebrates, dorsal ectoderm is segregated into presumptive epidermal, neural crest, and neural plate domains by distinct but interacting genetic cascades. The epidermal fate is specified early by high levels of BMP signaling which act through a battery of transcription factors to turn on epidermis-specific effector genes such as keratin. In the neural plate, BMP inhibition, as well as inductive signals from underlying mesoderm, leads to the expression of Zic and Sox1,2,3 (group B) genes, proneural bHLH transcription factors, and neural-specific effectors. At the neural plate border, Wnt and Fgf signals, as well as intermediate levels of BMPs, induce expression of neural plate border and neural crest specifiers. Gene-regulatory crosstalk between neural crest genes maintains their expression until migration and differentiation, when neural crest effector genes are expressed. (B) Ectodermal gene expression in amphioxus neurulae. While gene expression implies that the essential structure of the epidermal and neural regulatory cascades is conserved in amphioxus and vertebrates, only the earliest steps of neural plate border specification appear conserved between the two subphyla. Thus, amphioxus lacks a vertebrate-like neural plate border gene network below the level of Snail expression.
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Sox9 (Aoki et al., 2003; Honore et al., 2003). Similarly, ectopic Slug transcripts activate expression of AP-2, Sox9, Sox10, and FoxD3, while dominant-negative Slug suppresses expression of Sox10 and FoxD3 (Aoki et al., 2003; Aybar et al., 2003; del Barrio and Nieto, 2002; Sasai et al., 2001). It is unknown whether the neural crest specifiers influence each other via direct transcriptional activation or through secondary factors. However, Slug/ Snail and FoxD3 probably act through at least one unidentified secondary inhibitor as both are known transcriptional repressors (LaBonne and Bronner-Fraser, 2000; Sasai et al., 2001). In mouse, there appears to be less of a tight interdependence between the neural crest specifiers, as knockouts of Snail, Slug, Sox10, and AP-2␣ affect later differentiation rather than early inductive events (Britsch et al., 2001; Carver et al., 2001; Jiang et al., 1998; Schorle et al., 1996; Zhang et al., 1996). Similary, in zebrafish, AP-2␣ is not essential for early expression of neural crest specifiers like Snail and FoxD3 but is required to maintain Sox9a expression in migrating neural crest (Barrallo-Gimeno et al., 2004; Knight et al., 2003). The apparently disparate responses of mouse, fish, and frog embryos to loss-of-function perturbations most likely reflect species-specific differences in the deployment of paralogous genes rather than fundamental differences in the mechanisms of neural crest specification. Such expression domain shuffling has been described for Slug/Snail (Locascio et al., 2002), and would be expected to cause various levels of genetic redundancy depending on species and developmental time point. The extensive crossregulation of Slug/Snail, AP-2, FoxD3, Sox10, Sox9, and c-Myc expression makes assigning hierarchical relationships to these factors risky. Nevertheless, arguments have been made which alternately place AP-2, Slug/Snail, and FoxD3 upstream of all the other factors. Two pieces of evidence suggest AP-2 occupies a unique place among the other neural crest specifiers (Luo et al., 2003). First, AP-2 is earlier and more broadly deployed at the neural plate border than any of the other neural crest specifiers. Second, AP-2 upregulation in Wnt-sensitized ectoderm occurs with minimal BMP inhibition, approximately 30-fold less than is needed for Slug or Sox9. Evidence placing Snail and FoxD3 near the top of the hierarchy is that both genes, when misexpressed at high levels in naive ectodermal explants, are capable of inducing the expression of some of the other neural crest specifiers (Aybar et al., 2003; Sasai et al., 2001). Slug, AP-2, Sox9, and Sox10 do not seem to have this ability and can only induce neural crest in Wnt or BMP-sensitized ectoderm. The difficulty of imposing a simple linear hierarchy upon these factors is exacerbated by evidence that they also feed back on genes putatively “upstream” of themselves. For instance, FoxD3 is capable of inducing Zicr1 and neural markers (Sasai et al., 2001), while Sox9 is required for continued expression of the neural plate border specifier Pax3 (Spokony et al., 2002). Thus, rather than participating in a unidirectional regulatory cascade, the neural crest specifiers appear to act as more or less equal nodes in a gene-regulatory network initially established by inductive signals and neural plate border
specifiers. Maintenance of the network requires the continued function of every neural crest specifier or the neural crest cell regulatory state is lost. Twist and Id Two other transcriptional regulators are coexpressed with the neural crest specifiers, but likely lie outside their tightly interdependent network. Twist, a bHLH transcriptional regulator, and Id HLH transcriptional inhibitors are expressed in pre- and migratory neural crest cells (Ishii et al., 2003; Kee and Bronner-Fraser, 2001; Martinsen and Bronner-Fraser, 1998; Meulemans et al., 2003). Twist is activated by many of the neural crest specifier genes but has not been shown to crossregulate with them. Rather, Twist appears necessary for the differentiation of specific neural crest-derived pharyngeal arch structures and thus likely operates between neural crest specifiers and differentiation effector genes (Hopwood et al., 1989; Ishii et al., 2003; Soo et al., 2002). The regulatory relationship of Id genes to any other neural crest gene is unknown. It is suspected that the function of Id at the neural plate border and in neural crest cells may be suppression of pro-neural bHLHs and/or regulation of Twist (Martinsen and Bronner-Fraser, 1998). The Neural Crest Specifiers Regulate a Variety of Effector Genes that Control Different Aspects of the Neural Crest Phenotype Besides cross- and autoregulating to maintain their collective expression, neural crest specifiers control several downstream mediators of neural crest migration and differentiation. Two interacting targets of neural crest specifier regulation, Rho GTPases and cadherins, mediate neural crest cell delamination by altering cell shape and adhesion (Fukata and Kaibuchi, 2001). In chicken neural tube cells, RhoB function is necessary for neural crest cell delamination and is upregulated in response to ectopic Slug (del Barrio and Nieto, 2002; Liu and Jessell, 1998). In cultured mouse epidermal cells, E-cadherin prevents delamination and is directly repressed by binding of Snail to its promoter (Cano et al., 2000). Cadherin-7 is upregulated as neural crest cells begin migrating and can be induced by ectopic FoxD3 in the chicken neural tube (Dottori et al., 2001; Nakagawa and Takeichi, 1998). Interestingly, the cadherin/Rhomediated delamination program can also feed back to maintain specifier expression in migrating neural crest cells. Overexpression of cadherin-11 in Xenopus interferes with AP-2, Snail, and Twist expression as well as proper migration (Borchers et al., 2001). The neural crest specifiers Sox9 and Sox10 are also expressed in postmigratory neural crest where they regulate differentiation. Sox9 directly and positively regulates the expression of collagen in neural crest-derived cartilage (Ng et al., 1997), while Sox10 and Pax3 cooperate to activate cRet expression in enteric neurons by binding cRet enhancer elements (Lang and Epstein, 2003). Sox10 also activates several cell-type-specific differentiation genes in melanogenic and gliogenic neural crest cells including Trp2, Mitf, ckit, Cx32, P0, and Erbb3 (Aoki et al., 2003; Bondurand et al., 2001; Britsch et al., 2001; Elworthy et al., 2003; Honore et al., 2003; Peirano and Wegner, 2000; Watanabe et al., 2002). In
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the case of Mitf, P0, and Cx32, this regulation is via direct binding of Sox10 to transcriptional enhancers. Neural Crest Specifiers Interact with Components of the Neural Crest Patterning Network Operating in parallel with the gene-regulatory network driving neural crest cell induction, migration, and differentiation is a distinct set of transcription factors that pattern the neural crest along the anterior-posterior axis. These genes confer specific cellular fates and migratory pathways upon the nascent cells according to their anterior-posterior level of origin. Work in mouse and chick demonstrates that this neural crest patterning network is intimately linked to the network of neural crest specifiers. At the core of the patterning network are Hox genes, which are expressed in nested domains in migrating cranial and trunk neural crest cells (Hunt et al., 1991a, 1991b). Hoxa2 marks neural crest cells emanating from rhombomere 4 and migrating into the second branchial arch (Hunt et al., 1991a; Maconochie et al., 1999). This expression has been shown to require binding of the neural crest specifier AP-2 to a Hoxa2 enhancer element. The patterning gene Krox20 is expressed in rhombomeres 3 and 5 and a subset of neural crest cells migrating into the third branchial arch. Expression of Krox20 in these migrating neural crest cells is dependent on binding of Sox10 to the Krox20 neural crest enhancer (Ghislain et al., 2003). Interestingly, neural crest patterning genes also appear to feed back on neural crest specifiers. In mice deficient in ectodermal Hoxa1 and Hoxb1, second arch neural crest is lost and, with it, expression of the neural crest specifiers Sox10 and Twist (Gavalas et al., 2001). Thus, some level of crosstalk between the neural crest induction and patterning networks appears necessary for the proper maintenance of both generegulatory systems. Neural Crest Specifiers Are Downregulated in Postmigratory Neural Crest After neural crest cells have reached their destinations and begun to differentiate, expression of AP-2, Slug/ Snail, FoxD3, and Id is lost. The events that trigger this cessation of neural crest gene expression and lead to the end of migration and decision to differentiate remain unknown. It is possible that downregulation of Slug/ Snail allows repression of the other neural crest specifiers. Loss of the neural crest specifier expression then results in the loss of migratory ability and multipotentiality. Similarly, loss of Id expression may release bHLH transcription factors from inhibition, permitting differentiation into neural and mesodermal-type derivatives. Overview of the Neural Crest Gene Network The genetic interactions reviewed above are synthesized in Figure 1A. The neural crest regulatory state is initiated by Wnt and attenuated BMP signals and requires Fgf. These inductive signals activate neural plate border specifiers (Zic, Pax3/7, Dlx5, and Msx1/2) as well as neural crest specifiers (Slug/Snail, FoxD3, Sox9, Sox10, AP-2, cMyc, Twist, and Id). Zic factors are sufficient for the expression of Slug/Snail and FoxD3 while Pax3/7 and Msx are required for Slug/Snail and Sox10
expression, suggesting that neural plate border specifiers act upstream of neural crest specifiers. The neural crest specifiers cross- and autoregulate until neural crest cells begin differentiating. Little is known about the immediate downstream targets of neural crest specifiers, or the mechanisms that quash their expression and cue crest cells to stop migrating and lose their multipotency. The neural crest specifiers Sox9 and Sox10 have later roles in postmigratory neural crest where they are direct regulators of cartilage, melanocyte, and neural differentiation. Gene Expression in Amphioxus Implies Genetic Cascades in the Nonneural Ectoderm and Neural Plate Are Conserved with Vertebrates At the gene-regulatory level, neural crest evolution is the sum of the molecular alterations that drove the gradual assembly of the neural crest gene network outlined above. In the second portion of this review, we will attempt to define some of these novel regulatory interactions using amphioxus as a living approximation of the vertebrate ancestor. Amphioxus is particularly well suited for this task because—despite its own long evolutionary history—it likely retains many features shared with the ancestral prevertebrate chordate. Overall, the major genetic regulatory and signaling pathways of vertebrate neural induction appear to be utilized in amphioxus (Figure 1B). Consistent with conserved roles in patterning the early ectoderm, amphioxus BMPs, Wnts, Notch, and Hairy (a transcription factor downstream of Notch signaling) are expressed in a pattern similar to their vertebrate homologs (Holland et al., 2001; Minguillon et al., 2003; Panopoulou et al., 1998; Schubert et al., 2000, 2001). In the vertebrate epidermal lineage, high levels of BMP signaling induce expression of Dlx3/5, AP-2, and Msx1. These genes act directly upstream of epidermal effectors such as keratin. Consistent with conservation of this genetic cascade, BMP2/4, Dlx, and AP-2 are all expressed in presumptive epidermis of amphioxus (Holland et al., 1996; Meulemans and Bronner-Fraser, 2002; Panopoulou et al., 1998). In the developing vertebrate neural plate, inhibition of BMP signaling induces expression of SoxB and proneural bHLH genes. These, in turn, activate a battery of neural differentiation and patterning genes including neurotubulins, Islet, and Hu/Elav. Homologs of all these factors have been described in amphioxus and are temporally and spatially deployed in a manner identical to their vertebrate counterparts (Holland et al., 2000; Jackman et al., 1997; Satoh et al., 2001; Yasui et al., 1998a). Amphioxus and Vertebrates Utilize Some Genes Differently at the Neural Plate Border, Highlighting Potential Evolutionary Novelties In cells at the vertebrate neural plate border, intermediate levels of BMP signal, together with Wnts and Fgfs, induce high-level expression of early patterning genes such as Pax3/7, Msx1/2, Dlx5, and Zic. Homologs of Pax3/7, Msx1/2, Zic, and the neural crest specifier Slug/ Snail are present at the amphioxus neural plate border, suggesting conservation of these initial steps of border specification (Gostling and Shimeld, 2003; Holland et al., 1999; Langeland et al., 1998; Sharman et al., 1999).
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Figure 2. Putative Co-Option of Neural Crest Specifiers to the Neural Plate Border Early in Vertebrate Evolution as Inferred from Comparisons of Amphioxus and Vertebrate Gene Expression Patterns (A) Expression of neural crest marker homologs in amphioxus neurulae. In vertebrates, Id, Snail, and AP-2 genes are coexpressed in presumptive neural crest cells at the neural plate border. In amphioxus, these genes have almost completely nonoverlapping patterns of expression; Id is expressed in the endoderm and axial mesoderm, Snail is expressed in the paraxial mesoderm and neural plate, and AP-2 is expressed in the epidermal ectoderm. (B) In a hypothetical amphioxus-like vertebrate ancestor, FoxD, AP-2, Id, Twist, and SoxE are expressed in various nonneural tissues. (C) Early in the vertebrate lineage, all these factors became coexpressed in cells at the neural plate border. Figure 2A from Meulemans et al. (2003).
As neurulation proceeds in vertebrates, the neural plate border specifiers (Pax3/7, Zic, Msx1/2) and early inductive signals (BMP, Wnt, Fgf) activate a suite of transcriptional regulators that specify neural crest fate (AP-2, Slug/Snail, FoxD3, Sox9, Sox10, cMyc, Twist, Id). Amphioxus appears to lack this network of interacting transcription factors below the level of Slug/Snail. Thus, while early induction signals likely activate expression of the neural plate border specifiers Pax3/7, Msx, and Zic, expression of the neural crest specifiers AP-2 (Meulemans and Bronner-Fraser, 2002), SoxE (the amphioxus homolog of Sox9 and 10, our unpublished data), FoxD3 (Yu et al., 2002), Id (Meulemans et al., 2003), and Twist (Yasui et al., 1998b) is not induced at the amphioxus neural plate border (Figure 2). These differences suggest that regulation of neural crest specifiers by neural plate border specifiers and early inductive signals is a vertebrate innovation. These new interactions may have conferred novel properties upon the evolving vertebrate neural plate border by bringing together high-order transcription factors from other tissues and germ layers. Hints as to the functional consequences of this juxtaposition are suggested by the demonstrated roles and downstream targets of neural crest specifier genes in vertebrates. Virtually all neural crest specifiers have been shown to repress Sox2 expression in the neural plate border, preventing adoption of neural plate fates. In amphioxus, the Sox2 homolog Sox1/2/3 is not excluded from the neural plate border but rather extends to the edge of the nonneural ectoderm, implying that one consequence of neural crest specifier recruitment may have been suppression of neural fates. Besides suppressing Sox2, individual neural crest specifiers also confer different aspects of the neural crest phenotype. Slug/Snail and FoxD3 have been shown to mediate the delamination of neural crest cells from the neuroepithelium. AP-2, Sox9, and Twist each have essential roles in maintaining the chondrogenic
potential of cranial neural crest. Sox10 has been shown to initially hold neural crest cells in an undifferentiated state, but later to promote gliogenesis, melanogenesis, and enteric neuron formation. Id genes may also function as negative regulators of differentiation by repressing the activity of neurogenic bHLH transcription factors. Taken together, the functional capacities of the neural crest specifiers suggest that their co-option may have endowed the evolving vertebrate neural plate border with both multipotency and migratory ability—two defining properties of the neural crest. It is important to note, however, that gene co-option is not the only possible explanation of the observed differences between amphioxus and vertebrate neural crest specifier deployment. It is conceivable that absence of specifier gene expression at the amphioxus neural plate border reflects the loss of this expression domain by amphioxus neural crest specifier homologs. The loss of the same neural plate border expression domain by multiple genes could be explained by the loss of a primitive neural crest-like cell population in the cephalochordate lineage. The expression of neural crest specifier homologs in the third chordate subphylum, urochordata, would help establish the likelihood of such a scenario. To date, homologs of Msx, Pax3/7, Zic, and Slug/Snail have been described in ascidians and all are deployed in a manner virtually identical to their cephalochordate and vertebrate counterparts (Corbo et al., 1997; Gostling and Shimeld, 2003; Ma et al., 1996; Satou et al., 2002; Wada et al., 1996). Testing Conserved and Divergent Elements of the Amphioxus and Vertebrate Neural Plate Border Gene Networks Comparisons of amphioxus and vertebrate gene expression patterns provide the observational foundations of testable hypotheses regarding neural crest gene-regulatory evolution. Continued development of techniques for
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manipulating amphioxus embryos will eventually allow empirical verification of the regulatory differences inferred from gene expression data. Comparison of analogous gain- and loss-of-function perturbations in amphioxus and vertebrates could potentially elucidate the specific regulatory changes causal to neural crest evolution. An even deeper understanding of amphioxus and vertebrate neural plate border gene networks will become realistic as more is known about the cis-regulation of vertebrate neural crest genes. Critical questions yet to be answered are: (1) What are the specific cis-regulatory DNAs governing the relationships between early inductive signals and vertebrate neural plate border genes? (2) What are the elements controlling the response of neural crest specifiers to the inductive signals and neural plate border specifiers? (3) What cis-regulatory elements mediate the crossregulation of the neural crest specifiers? (4) What are the downstream targets of the neural crest specifiers, and the cis-regulatory elements mediating their influence? (5) Which of these relationships and regulatory elements are conserved, absent, or divergent in nonvertebrate chordates? Optimal strategies for addressing such questions will involve not only traditional cis-regulatory analyses, but interspecies testing of conserved and divergent cis-acting DNAs. Indeed, this approach has already begun to yield provocative insights into the evolution of vertebrate Hox gene regulation in the neural tube and neural crest (Manzanares et al., 2000).
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Acknowledgments
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Gene-Regulatory Interactions in Neural Crest Evolution and Development Daniel Meulemans and Marianne Bronner-Fraser* California Institute of Technology 1200 East California Boulevard Pasadena, California 91125
In this review, we outline the gene-regulatory interactions driving neural crest development and compare these to a hypothetical network operating in the embryonic ectoderm of the cephalochordate amphioxus. While the early stages of ectodermal patterning appear conserved between amphioxus and vertebrates, later activation of neural crest-specific factors at the neural plate border appears to be a vertebrate novelty. This difference may reflect co-option of genetic pathways which conferred novel properties upon the evolving vertebrate neural plate border, potentiating the evolution of definitive neural crest.
Introduction The neural crest is an embryonic cell population defined by its origins at the neural plate border, migratory capacity, multipotency, and characteristic gene expression profile. The appearance of this tissue was likely a turning point in vertebrate evolution since many of the structures which define the vertebrate clade are derived from neural crest cells (Gans and Northcutt, 1983; Northcutt and Gans, 1983). Consistent with a key role in vertebrate origins, a bona fide neural crest is present in every extant vertebrate species including the most basal jawless fish, but not in the most vertebrate-like invertebrates. Gene expression patterns and functional comparisons suggest that the gene-regulatory interactions driving neural crest formation are conserved among vertebrates. However, the mechanistic data at the core of the emerging neural crest gene network are derived from embryological models not particularly amenable to genetic manipulation. As a result, experimental analyses of neural crest gene-regulatory relationships have largely been restricted to gain- or loss-of-function studies using injected mRNA, cDNA, or antisense oligonucleotides. Further limiting a comprehensive dissection of the neural crest regulatory network is the fact that most vertebrates are poorly suited for cis-regulatory analyses due to the dysregulated and mosaic expression of injected reporter constructs and the difficulty of making transgenics. Thus, for a given “neural crest gene” it is only known that over- or underexpression in turn influences the expression of other neural crest genes. It is usually unclear whether this interdependency involves direct binding of a transcription factor to an enhancer or works through intermediate genes. In the first part of this review, we assemble current data from such gain- and loss-of-function analyses and outline the genetic interactions which initiate and maintain the neural crest cell gene-regulatory state—driving *Correspondence: [email protected]
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neural crest delamination from the neural tube, migration through the periphery, and differentiation into varied cell types (Figure 1A). In the second part, we compare these gene-regulatory circuits to putatively homologous networks operating in amphioxus—a vertebrate-like chordate that lacks neural crest (Figure 1B). From these comparisons, we identify some of the novel gene-regulatory relationships that may have potentiated the evolution of definitive neural crest. Wnts, Fgfs, and Various Levels of BMP Signaling Induce Epidermal, Neural Plate, and Neural Crest Gene Expression The neural crest regulatory state is initiated by inductive signals in the form of BMP, Wnt, and Fgf signaling molecules secreted from underlying mesoderm and adjacent nonneural ectoderm (Bonstein et al., 1998; Garcia-Castro et al., 2002; LaBonne and Bronner-Fraser, 1998; Liem et al., 1995; Marchant et al., 1998; Monsoro-Burq et al., 2003). These intercellular signals also function simultaneously to segregate neural from nonneural ectoderm during neural induction (Sasai and De Robertis, 1997; Weinstein and Hemmati-Brivanlou, 1999). In nonneural ectoderm, high levels of BMP signal activate the epidermal program via Msx1, Dlx3/5, and AP-2 (Feledy et al., 1999; Luo et al., 2001a, 2002; Pera et al., 1999). Msx1, an immediate early target of BMP2/4 signaling, is sufficient to activate keratin expression and repress the neural markers Zic3 and N-CAM (Suzuki et al., 1997). Dlx3 and 5 similarly repress neural factors such as Sox2 (Feledy et al., 1999; Luo et al., 2001a; McLarren et al., 2003) while ectodermal AP-2 activates epidermal keratin expression by direct binding of the keratin promoter (Luo et al., 2002; Snape et al., 1991). Inhibition of ectodermal BMP signaling induces expression of the transcription factors Sox2, Zicr-1, and Zic3 in the neural plate (Mizuseki et al., 1998; Nakata et al., 1997). Sox2 and Zic genes, in turn, activate neural differentiation genes such as N-CAM and N-tubulin (Mizuseki et al., 1998), probably through the neurogenic bHLH transcription factors Neurogenin, Neuro-D, and achaete-scute. Sox2 also indirectly represses expression of the neural crest specifier Slug in the avian neural plate (Wakamatsu et al., 2004). At the neural plate border of frogs and zebrafish, intermediate levels of BMP activate high-level expression of “neural crest specifiers” such as Snail, Slug, AP-2, Sox9, and FoxD3 (LaBonne and Bronner-Fraser, 1998; Luo et al., 2003; Marchant et al., 1998; Morgan and Sargent, 1997; Nguyen et al., 1998; Sasai et al., 2001). Establishment and maintenance of this attenuated signal likely depends on local Notch receptor-mediated signaling (Endo et al., 2002; Glavic et al., 2004). Interestingly, Notch pathway activation can dampen or amplify BMP signaling at the neural plate border, depending on the organism. This bifunctionality may have evolved to compensate for the different levels of ectodermal BMP observed in different vertebrate embryos (discussed in Glavic et al., 2004). In addition to an attenuated BMP signal, input from the Wnt pathway is also required for
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Figure 1. Comparison of Putative Gene-Regulatory and Signaling Interactions Operating at the Neural Plate Border of Vertebrates and Amphioxus Red arrows indicate proven direct regulatory interactions. Black arrows are genetic interactions suggested by gain- and loss-of-function analyses largely in Xenopus. Gray lines indicate repression.
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expression of Snail, Slug, AP-2, and FoxD3, as well as virtually every other described neural crest and neural plate border marker (Garcia-Castro et al., 2002; LaBonne and Bronner-Fraser, 1998; Luo et al., 2003; SaintJeannet et al., 1997; Sasai et al., 2001). Finally, Fgf signaling is both necessary and sufficient for neural crest specifier expression in cultured ectodermal explants and in vivo (LaBonne and Bronner-Fraser, 1998; Mayor et al., 1997; Monsoro-Burq et al., 2003). It is unknown if the influence of BMP, Wnt, and Fgf pathways on neural crest specifier expression is direct or controlled by other factors. Evidence presented below suggests that Zic factors, Msx1, and Pax3/7 may mediate the effects of BMPs and Wnts on neural crest specifier expression. However, data from Xenopus suggest that Slug expression in neural crest is dependent on binding of Wnt effector genes (Tcf/Lef transcription factors) to the Slug promoter, implying direct regulation of Slug by the Wnt pathway (Vallin et al., 2001). Neural Plate Border Specifiers Mediate the Influence of Wnts, BMPs, and Fgfs on Neural Crest Specifiers Initial signaling events that establish the neural plate border lead to expression of a small set of transcription factors designated here as “neural plate border specifiers.” This group of genes includes Zic factors, Pax3/7, Dlx5, and Msx1/2. Of these, Pax3 and Msx1/2 appear to be downstream of Wnt signaling (Bang et al., 1999), while Zic1, Zic3, Dlx5, and Msx1/2 become upregulated at the neural plate border in response to an attenuated BMP signal (Aruga et al., 2002; Luo et al., 2001b; Nakata et al., 1997; Tribulo et al., 2003). A third Zic gene, Zic5, is upregulated in response to ectopic Fgf8 in ectodermal explants (Monsoro-Burq et al., 2003). These genes display features that distinguish them from other neural plate border markers and suggest they may mediate the influence of Wnts, BMPs, and Fgfs on neural crest specifiers like Slug/Snail. First, these factors are expressed early and broadly at the neural plate border and precede activation of most definitive neural crest genes (Aruga et al., 2002; Luo et al., 2001b; Mizuseki et al., 1998; Nakata et al., 2000, 1997; Suzuki et al., 1997). Second, they are not general markers of migrating or post-migratory neural crest cells (though Msx2 and Dlx3/5 are expressed in specific subsets of cranial neural crest [Akimenko et al., 1994; Pera and Kessel, 1999; Robinson and Mahon, 1994; Takahashi et al., 2001; Yang et al., 1998] and Pax3 marks differentiating neural crestderived neurons and glia [Goulding et al., 1991]). Third, they function in neural plate border-derived cell types
aside from neural crest, including roof plate, dorsal interneurons, placodes, and Rohon-Beard cells (Aruga et al., 2002; Bang et al., 1999; Goulding et al., 1993; Liu et al., 2004; Mansouri and Gruss, 1998; McLarren et al., 2003; Tremblay et al., 1996; Woda et al., 2003). Finally, experimental evidence places these factors upstream of neural crest specifiers. Ectopic Zic is sufficient to activate expression of Snail, Slug, FoxD3, and Twist in Xenopus embryos and ectodermal explants (Brewster et al., 1998; Mizuseki et al., 1998; Nakata et al., 2000, 1997; Sasai et al., 2001). Pax3 is required for the expression of FoxD3 in mouse (Dottori et al., 2001). Msx1 is necessary and sufficient for the expression of Snail, Slug, and FoxD3 in frog (Tribulo et al., 2003). Furthermore, Msx1 cannot rescue disruption of Slug/Snail function, but dominant-negative disruption of Msx1 can be rescued by overexpression of Slug/Snail—implying Slug/Snail is downstream of Msx1 (Tribulo et al., 2003). Dlx5 has not been shown to directly regulate neural crest specifiers but is sufficient to drive ectopic expression of Msx1 (Woda et al., 2003). Thus, the timing and breadth of neural plate border specifier expression, as well as their demonstrated regulatory capacities, suggest they act upstream of neural crest markers—and downstream of Wnt, BMP, and Fgf signals. Neural Crest Specifiers Occupy a Distinct Position in the Neural Crest Gene Network and Crossregulate to Maintain Each Other’s Expression After the onset of neural plate border specifier expression, a suite of genes including Slug/Snail, AP-2, FoxD3, Sox10, Sox9, and c-Myc is activated in nascent neural crest cells (Aoki et al., 2003; Bellmeyer et al., 2003; Honore et al., 2003; LaBonne and Bronner-Fraser, 2000; Luo et al., 2003; Sasai et al., 2001; Spokony et al., 2002). These neural crest specifiers share several characteristics which suggest they operate downstream of Wnt/ BMP/Fgf inductive signals and the neural plate border specifiers Zic, Msx1/2, and Pax3/7. All neural crest specifiers are expressed in pre- and migratory neural crest cells, repress Sox2 expression, and require a Wnt signal for activation. In addition, Zic, Msx1/2, and Pax3/7 are necessary and/or sufficient for the expression of Slug/ Snail, FoxD3, and Sox10. Every neural crest specifier examined thus far also appears necessary and/or sufficient for the expression of the other specifiers in Xenopus. Thus, injection of a Sox10 antisense morpholino oligonucleotide causes loss of Sox9, Slug/Snail, and FoxD3, while Sox10 overexpression expands the expression domain of Slug and
(A) In vertebrates, dorsal ectoderm is segregated into presumptive epidermal, neural crest, and neural plate domains by distinct but interacting genetic cascades. The epidermal fate is specified early by high levels of BMP signaling which act through a battery of transcription factors to turn on epidermis-specific effector genes such as keratin. In the neural plate, BMP inhibition, as well as inductive signals from underlying mesoderm, leads to the expression of Zic and Sox1,2,3 (group B) genes, proneural bHLH transcription factors, and neural-specific effectors. At the neural plate border, Wnt and Fgf signals, as well as intermediate levels of BMPs, induce expression of neural plate border and neural crest specifiers. Gene-regulatory crosstalk between neural crest genes maintains their expression until migration and differentiation, when neural crest effector genes are expressed. (B) Ectodermal gene expression in amphioxus neurulae. While gene expression implies that the essential structure of the epidermal and neural regulatory cascades is conserved in amphioxus and vertebrates, only the earliest steps of neural plate border specification appear conserved between the two subphyla. Thus, amphioxus lacks a vertebrate-like neural plate border gene network below the level of Snail expression.
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Sox9 (Aoki et al., 2003; Honore et al., 2003). Similarly, ectopic Slug transcripts activate expression of AP-2, Sox9, Sox10, and FoxD3, while dominant-negative Slug suppresses expression of Sox10 and FoxD3 (Aoki et al., 2003; Aybar et al., 2003; del Barrio and Nieto, 2002; Sasai et al., 2001). It is unknown whether the neural crest specifiers influence each other via direct transcriptional activation or through secondary factors. However, Slug/ Snail and FoxD3 probably act through at least one unidentified secondary inhibitor as both are known transcriptional repressors (LaBonne and Bronner-Fraser, 2000; Sasai et al., 2001). In mouse, there appears to be less of a tight interdependence between the neural crest specifiers, as knockouts of Snail, Slug, Sox10, and AP-2␣ affect later differentiation rather than early inductive events (Britsch et al., 2001; Carver et al., 2001; Jiang et al., 1998; Schorle et al., 1996; Zhang et al., 1996). Similary, in zebrafish, AP-2␣ is not essential for early expression of neural crest specifiers like Snail and FoxD3 but is required to maintain Sox9a expression in migrating neural crest (Barrallo-Gimeno et al., 2004; Knight et al., 2003). The apparently disparate responses of mouse, fish, and frog embryos to loss-of-function perturbations most likely reflect species-specific differences in the deployment of paralogous genes rather than fundamental differences in the mechanisms of neural crest specification. Such expression domain shuffling has been described for Slug/Snail (Locascio et al., 2002), and would be expected to cause various levels of genetic redundancy depending on species and developmental time point. The extensive crossregulation of Slug/Snail, AP-2, FoxD3, Sox10, Sox9, and c-Myc expression makes assigning hierarchical relationships to these factors risky. Nevertheless, arguments have been made which alternately place AP-2, Slug/Snail, and FoxD3 upstream of all the other factors. Two pieces of evidence suggest AP-2 occupies a unique place among the other neural crest specifiers (Luo et al., 2003). First, AP-2 is earlier and more broadly deployed at the neural plate border than any of the other neural crest specifiers. Second, AP-2 upregulation in Wnt-sensitized ectoderm occurs with minimal BMP inhibition, approximately 30-fold less than is needed for Slug or Sox9. Evidence placing Snail and FoxD3 near the top of the hierarchy is that both genes, when misexpressed at high levels in naive ectodermal explants, are capable of inducing the expression of some of the other neural crest specifiers (Aybar et al., 2003; Sasai et al., 2001). Slug, AP-2, Sox9, and Sox10 do not seem to have this ability and can only induce neural crest in Wnt or BMP-sensitized ectoderm. The difficulty of imposing a simple linear hierarchy upon these factors is exacerbated by evidence that they also feed back on genes putatively “upstream” of themselves. For instance, FoxD3 is capable of inducing Zicr1 and neural markers (Sasai et al., 2001), while Sox9 is required for continued expression of the neural plate border specifier Pax3 (Spokony et al., 2002). Thus, rather than participating in a unidirectional regulatory cascade, the neural crest specifiers appear to act as more or less equal nodes in a gene-regulatory network initially established by inductive signals and neural plate border
specifiers. Maintenance of the network requires the continued function of every neural crest specifier or the neural crest cell regulatory state is lost. Twist and Id Two other transcriptional regulators are coexpressed with the neural crest specifiers, but likely lie outside their tightly interdependent network. Twist, a bHLH transcriptional regulator, and Id HLH transcriptional inhibitors are expressed in pre- and migratory neural crest cells (Ishii et al., 2003; Kee and Bronner-Fraser, 2001; Martinsen and Bronner-Fraser, 1998; Meulemans et al., 2003). Twist is activated by many of the neural crest specifier genes but has not been shown to crossregulate with them. Rather, Twist appears necessary for the differentiation of specific neural crest-derived pharyngeal arch structures and thus likely operates between neural crest specifiers and differentiation effector genes (Hopwood et al., 1989; Ishii et al., 2003; Soo et al., 2002). The regulatory relationship of Id genes to any other neural crest gene is unknown. It is suspected that the function of Id at the neural plate border and in neural crest cells may be suppression of pro-neural bHLHs and/or regulation of Twist (Martinsen and Bronner-Fraser, 1998). The Neural Crest Specifiers Regulate a Variety of Effector Genes that Control Different Aspects of the Neural Crest Phenotype Besides cross- and autoregulating to maintain their collective expression, neural crest specifiers control several downstream mediators of neural crest migration and differentiation. Two interacting targets of neural crest specifier regulation, Rho GTPases and cadherins, mediate neural crest cell delamination by altering cell shape and adhesion (Fukata and Kaibuchi, 2001). In chicken neural tube cells, RhoB function is necessary for neural crest cell delamination and is upregulated in response to ectopic Slug (del Barrio and Nieto, 2002; Liu and Jessell, 1998). In cultured mouse epidermal cells, E-cadherin prevents delamination and is directly repressed by binding of Snail to its promoter (Cano et al., 2000). Cadherin-7 is upregulated as neural crest cells begin migrating and can be induced by ectopic FoxD3 in the chicken neural tube (Dottori et al., 2001; Nakagawa and Takeichi, 1998). Interestingly, the cadherin/Rhomediated delamination program can also feed back to maintain specifier expression in migrating neural crest cells. Overexpression of cadherin-11 in Xenopus interferes with AP-2, Snail, and Twist expression as well as proper migration (Borchers et al., 2001). The neural crest specifiers Sox9 and Sox10 are also expressed in postmigratory neural crest where they regulate differentiation. Sox9 directly and positively regulates the expression of collagen in neural crest-derived cartilage (Ng et al., 1997), while Sox10 and Pax3 cooperate to activate cRet expression in enteric neurons by binding cRet enhancer elements (Lang and Epstein, 2003). Sox10 also activates several cell-type-specific differentiation genes in melanogenic and gliogenic neural crest cells including Trp2, Mitf, ckit, Cx32, P0, and Erbb3 (Aoki et al., 2003; Bondurand et al., 2001; Britsch et al., 2001; Elworthy et al., 2003; Honore et al., 2003; Peirano and Wegner, 2000; Watanabe et al., 2002). In
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the case of Mitf, P0, and Cx32, this regulation is via direct binding of Sox10 to transcriptional enhancers. Neural Crest Specifiers Interact with Components of the Neural Crest Patterning Network Operating in parallel with the gene-regulatory network driving neural crest cell induction, migration, and differentiation is a distinct set of transcription factors that pattern the neural crest along the anterior-posterior axis. These genes confer specific cellular fates and migratory pathways upon the nascent cells according to their anterior-posterior level of origin. Work in mouse and chick demonstrates that this neural crest patterning network is intimately linked to the network of neural crest specifiers. At the core of the patterning network are Hox genes, which are expressed in nested domains in migrating cranial and trunk neural crest cells (Hunt et al., 1991a, 1991b). Hoxa2 marks neural crest cells emanating from rhombomere 4 and migrating into the second branchial arch (Hunt et al., 1991a; Maconochie et al., 1999). This expression has been shown to require binding of the neural crest specifier AP-2 to a Hoxa2 enhancer element. The patterning gene Krox20 is expressed in rhombomeres 3 and 5 and a subset of neural crest cells migrating into the third branchial arch. Expression of Krox20 in these migrating neural crest cells is dependent on binding of Sox10 to the Krox20 neural crest enhancer (Ghislain et al., 2003). Interestingly, neural crest patterning genes also appear to feed back on neural crest specifiers. In mice deficient in ectodermal Hoxa1 and Hoxb1, second arch neural crest is lost and, with it, expression of the neural crest specifiers Sox10 and Twist (Gavalas et al., 2001). Thus, some level of crosstalk between the neural crest induction and patterning networks appears necessary for the proper maintenance of both generegulatory systems. Neural Crest Specifiers Are Downregulated in Postmigratory Neural Crest After neural crest cells have reached their destinations and begun to differentiate, expression of AP-2, Slug/ Snail, FoxD3, and Id is lost. The events that trigger this cessation of neural crest gene expression and lead to the end of migration and decision to differentiate remain unknown. It is possible that downregulation of Slug/ Snail allows repression of the other neural crest specifiers. Loss of the neural crest specifier expression then results in the loss of migratory ability and multipotentiality. Similarly, loss of Id expression may release bHLH transcription factors from inhibition, permitting differentiation into neural and mesodermal-type derivatives. Overview of the Neural Crest Gene Network The genetic interactions reviewed above are synthesized in Figure 1A. The neural crest regulatory state is initiated by Wnt and attenuated BMP signals and requires Fgf. These inductive signals activate neural plate border specifiers (Zic, Pax3/7, Dlx5, and Msx1/2) as well as neural crest specifiers (Slug/Snail, FoxD3, Sox9, Sox10, AP-2, cMyc, Twist, and Id). Zic factors are sufficient for the expression of Slug/Snail and FoxD3 while Pax3/7 and Msx are required for Slug/Snail and Sox10
expression, suggesting that neural plate border specifiers act upstream of neural crest specifiers. The neural crest specifiers cross- and autoregulate until neural crest cells begin differentiating. Little is known about the immediate downstream targets of neural crest specifiers, or the mechanisms that quash their expression and cue crest cells to stop migrating and lose their multipotency. The neural crest specifiers Sox9 and Sox10 have later roles in postmigratory neural crest where they are direct regulators of cartilage, melanocyte, and neural differentiation. Gene Expression in Amphioxus Implies Genetic Cascades in the Nonneural Ectoderm and Neural Plate Are Conserved with Vertebrates At the gene-regulatory level, neural crest evolution is the sum of the molecular alterations that drove the gradual assembly of the neural crest gene network outlined above. In the second portion of this review, we will attempt to define some of these novel regulatory interactions using amphioxus as a living approximation of the vertebrate ancestor. Amphioxus is particularly well suited for this task because—despite its own long evolutionary history—it likely retains many features shared with the ancestral prevertebrate chordate. Overall, the major genetic regulatory and signaling pathways of vertebrate neural induction appear to be utilized in amphioxus (Figure 1B). Consistent with conserved roles in patterning the early ectoderm, amphioxus BMPs, Wnts, Notch, and Hairy (a transcription factor downstream of Notch signaling) are expressed in a pattern similar to their vertebrate homologs (Holland et al., 2001; Minguillon et al., 2003; Panopoulou et al., 1998; Schubert et al., 2000, 2001). In the vertebrate epidermal lineage, high levels of BMP signaling induce expression of Dlx3/5, AP-2, and Msx1. These genes act directly upstream of epidermal effectors such as keratin. Consistent with conservation of this genetic cascade, BMP2/4, Dlx, and AP-2 are all expressed in presumptive epidermis of amphioxus (Holland et al., 1996; Meulemans and Bronner-Fraser, 2002; Panopoulou et al., 1998). In the developing vertebrate neural plate, inhibition of BMP signaling induces expression of SoxB and proneural bHLH genes. These, in turn, activate a battery of neural differentiation and patterning genes including neurotubulins, Islet, and Hu/Elav. Homologs of all these factors have been described in amphioxus and are temporally and spatially deployed in a manner identical to their vertebrate counterparts (Holland et al., 2000; Jackman et al., 1997; Satoh et al., 2001; Yasui et al., 1998a). Amphioxus and Vertebrates Utilize Some Genes Differently at the Neural Plate Border, Highlighting Potential Evolutionary Novelties In cells at the vertebrate neural plate border, intermediate levels of BMP signal, together with Wnts and Fgfs, induce high-level expression of early patterning genes such as Pax3/7, Msx1/2, Dlx5, and Zic. Homologs of Pax3/7, Msx1/2, Zic, and the neural crest specifier Slug/ Snail are present at the amphioxus neural plate border, suggesting conservation of these initial steps of border specification (Gostling and Shimeld, 2003; Holland et al., 1999; Langeland et al., 1998; Sharman et al., 1999).
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Figure 2. Putative Co-Option of Neural Crest Specifiers to the Neural Plate Border Early in Vertebrate Evolution as Inferred from Comparisons of Amphioxus and Vertebrate Gene Expression Patterns (A) Expression of neural crest marker homologs in amphioxus neurulae. In vertebrates, Id, Snail, and AP-2 genes are coexpressed in presumptive neural crest cells at the neural plate border. In amphioxus, these genes have almost completely nonoverlapping patterns of expression; Id is expressed in the endoderm and axial mesoderm, Snail is expressed in the paraxial mesoderm and neural plate, and AP-2 is expressed in the epidermal ectoderm. (B) In a hypothetical amphioxus-like vertebrate ancestor, FoxD, AP-2, Id, Twist, and SoxE are expressed in various nonneural tissues. (C) Early in the vertebrate lineage, all these factors became coexpressed in cells at the neural plate border. Figure 2A from Meulemans et al. (2003).
As neurulation proceeds in vertebrates, the neural plate border specifiers (Pax3/7, Zic, Msx1/2) and early inductive signals (BMP, Wnt, Fgf) activate a suite of transcriptional regulators that specify neural crest fate (AP-2, Slug/Snail, FoxD3, Sox9, Sox10, cMyc, Twist, Id). Amphioxus appears to lack this network of interacting transcription factors below the level of Slug/Snail. Thus, while early induction signals likely activate expression of the neural plate border specifiers Pax3/7, Msx, and Zic, expression of the neural crest specifiers AP-2 (Meulemans and Bronner-Fraser, 2002), SoxE (the amphioxus homolog of Sox9 and 10, our unpublished data), FoxD3 (Yu et al., 2002), Id (Meulemans et al., 2003), and Twist (Yasui et al., 1998b) is not induced at the amphioxus neural plate border (Figure 2). These differences suggest that regulation of neural crest specifiers by neural plate border specifiers and early inductive signals is a vertebrate innovation. These new interactions may have conferred novel properties upon the evolving vertebrate neural plate border by bringing together high-order transcription factors from other tissues and germ layers. Hints as to the functional consequences of this juxtaposition are suggested by the demonstrated roles and downstream targets of neural crest specifier genes in vertebrates. Virtually all neural crest specifiers have been shown to repress Sox2 expression in the neural plate border, preventing adoption of neural plate fates. In amphioxus, the Sox2 homolog Sox1/2/3 is not excluded from the neural plate border but rather extends to the edge of the nonneural ectoderm, implying that one consequence of neural crest specifier recruitment may have been suppression of neural fates. Besides suppressing Sox2, individual neural crest specifiers also confer different aspects of the neural crest phenotype. Slug/Snail and FoxD3 have been shown to mediate the delamination of neural crest cells from the neuroepithelium. AP-2, Sox9, and Twist each have essential roles in maintaining the chondrogenic
potential of cranial neural crest. Sox10 has been shown to initially hold neural crest cells in an undifferentiated state, but later to promote gliogenesis, melanogenesis, and enteric neuron formation. Id genes may also function as negative regulators of differentiation by repressing the activity of neurogenic bHLH transcription factors. Taken together, the functional capacities of the neural crest specifiers suggest that their co-option may have endowed the evolving vertebrate neural plate border with both multipotency and migratory ability—two defining properties of the neural crest. It is important to note, however, that gene co-option is not the only possible explanation of the observed differences between amphioxus and vertebrate neural crest specifier deployment. It is conceivable that absence of specifier gene expression at the amphioxus neural plate border reflects the loss of this expression domain by amphioxus neural crest specifier homologs. The loss of the same neural plate border expression domain by multiple genes could be explained by the loss of a primitive neural crest-like cell population in the cephalochordate lineage. The expression of neural crest specifier homologs in the third chordate subphylum, urochordata, would help establish the likelihood of such a scenario. To date, homologs of Msx, Pax3/7, Zic, and Slug/Snail have been described in ascidians and all are deployed in a manner virtually identical to their cephalochordate and vertebrate counterparts (Corbo et al., 1997; Gostling and Shimeld, 2003; Ma et al., 1996; Satou et al., 2002; Wada et al., 1996). Testing Conserved and Divergent Elements of the Amphioxus and Vertebrate Neural Plate Border Gene Networks Comparisons of amphioxus and vertebrate gene expression patterns provide the observational foundations of testable hypotheses regarding neural crest gene-regulatory evolution. Continued development of techniques for
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manipulating amphioxus embryos will eventually allow empirical verification of the regulatory differences inferred from gene expression data. Comparison of analogous gain- and loss-of-function perturbations in amphioxus and vertebrates could potentially elucidate the specific regulatory changes causal to neural crest evolution. An even deeper understanding of amphioxus and vertebrate neural plate border gene networks will become realistic as more is known about the cis-regulation of vertebrate neural crest genes. Critical questions yet to be answered are: (1) What are the specific cis-regulatory DNAs governing the relationships between early inductive signals and vertebrate neural plate border genes? (2) What are the elements controlling the response of neural crest specifiers to the inductive signals and neural plate border specifiers? (3) What cis-regulatory elements mediate the crossregulation of the neural crest specifiers? (4) What are the downstream targets of the neural crest specifiers, and the cis-regulatory elements mediating their influence? (5) Which of these relationships and regulatory elements are conserved, absent, or divergent in nonvertebrate chordates? Optimal strategies for addressing such questions will involve not only traditional cis-regulatory analyses, but interspecies testing of conserved and divergent cis-acting DNAs. Indeed, this approach has already begun to yield provocative insights into the evolution of vertebrate Hox gene regulation in the neural tube and neural crest (Manzanares et al., 2000).
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