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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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The Wnt Gene Family and the Evolutionary Conservation of Wnt Expression

and .

The Wnt gene family has undergone extensive gene duplications during metazoan evolution. To date, there is one Wnt gene known from the cnidarian Hydra, seven from Drosophila and nineteen from vertebrates. Phylogenetic and genome analyses group the vertebrate Wnt genes into thirteen subfamilies and suggest that the last common ancestor of protostomes and deuterostomes had at least six Wnt genes. The present review focuses on the roles of Wnt genes in early metazoan development, during the establishment of the major regions and chief axes of the body. Wnt genes signal through three pathways: Wnt/b-catenin (the canonical Wnt pathway), Wnt/JNK (involved in convergent extension), and Wnt/Ca2+ (involved in tissue separation). The earliest expression of Wnt and other genes of the canonical pathway is around the single gut opening of the cnidarian Hydra, at the posterior end of insect embryos, and around the forming blastopore in deuterostomes (sea urchins, amphioxus, and vertebrates). Experimental evidence shows that, in deuterostomes, this posterior Wnt signaling center establishes a gradient of Wnt activity that is high posterior and low anterior. Thus, the major conserved role of the canonical Wnt pathway in Metazoa appears to be patterning the longitudinal embryonic axis. It is likely that this role arose very early in metazoan evolution and that additional roles for Wnt signaling evolved later. Such later roles include specification of neural fate, boundary formation (between segments in insects and at the midbrain-hindbrain junction in vertebrates), somite formation, induction of neural crest, elongation of the body axis in amphioxus and vertebrates, and establishing the dorsoventral axis in amphibians.

Introduction

Wnt genes encode secreted glycoproteins, which mediate intercellular signaling either over short or long distances depending on the tissues where they are being expressed.1 In animals generally, Wnt signaling is involved in a wide variety of cell interactions from early development through the adult stage. The present review focuses on the early developmental roles of Wnt genes. The Wnt gene family, which has undergone extensive gene duplications during metazoan evolution, can be subdivided into thirteen subfamilies, which fall into three classes differing in the signal transduction cascades they trigger. Two of the signaling pathways are mediated by Dishevelled. In one, Dishevelled inhibits GSK3, which results in translocation of β-catenin to the nucleus. This pathway is involved in axial patterning and specification of cell fate. In the second, Dishevelled signals through Rho to JNK/SAPK (c-Jun N-terminal kinase/ stress-activated protein kinase). This pathway mediates planar cell polarity and convergent extension movements. The third Wnt signaling pathway involves G-proteins and results in changes in intracellular Ca2+ levels, which play roles in cell cycling and tissue separation. Wnt pathways will not be discussed here in detail. For comprehensive reviews see references 27

Wnt genes have been cloned from several animal phyla (Fig. 1). The most (nineteen) are known from vertebrates [see Roel Nusse's Wnt gene homepage at http://www.stanford.edu/˜rnusse/Wntwindow.html]. At least five of the vertebrate Wnt genes appear to signal predominantly through the canonical Wnt/β-catenin pathway (Wnt-1, Wnt-2b, Wnt-3a, Wnt-8a, and Wnt-8b), at least two signal through the Wnt/Ca2+ pathway (Wnt-4 and Wnt-5a),4,8 and one (Wnt-11) apparently signals preferentially through the Wnt/JNK pathway,7,9 although it can also signal through the Wnt/Ca2+ pathway.10 The signaling pathways of the remaining Wnt genes have not been well studied. Among invertebrate deuterostomes, (Wnt genes have been studied in sea urchins (four Wnt genes cloned),1114 ascidians (two cloned),1517 and amphioxus (nine cloned).1823 Among the protostomes, Wnt genes have been most studied in Drosophila (seven Wnt genes)24 and in Caenorhabditis elegans (five Wnt genes).2537 Most research on Drosophila Wnt genes has concerned wingless, the Wnt-1 subfamily ortholog, although developmental expression is known for five of the others.3850 Among other protostomes, one Wnt has been cloned from a brachiopod51 and one from leeches.52,53 In animals basal to the bilaterians, one Wnt gene has been isolated from the cnidarian Hydra.54

Figure 1. Metazoan phylogeny based on 18S rDNA.

Figure 1

Metazoan phylogeny based on 18S rDNA. Branch lengths are not indicative of phylogenetic distance. Asterisks indicate animal groups from which Wnt genes have been isolated. Modified from reference .

The availability of Wnt genes from several species provides a basis for molecular phylogenetic and comparative gene expression analyses. Molecular phylogenetics is useful for revealing the diversification of a gene family in the course of evolution,5558 while comparisons of how genetic pathways pattern the embryo can elucidate the evolution of developmental mechanisms. To understand the evolution of the roles of Wnt genes in embryonic patterning, it would be most useful if they had been studied in numerous phyla, including their most basal members. Regrettably, both Drosophila melanogaster and Caenorhabditis elegans are derived within their groups5961 and their genes tend to evolve more rapidly compared to those in more basal members of their respective phyla.62 In addition, it is not known if the one Hydra Wnt gene cloned, which belongs to the Wnt-3 subfamily,22 is the only Wnt gene in cnidarians. Consequently, phylogenetic analyses can only give a minimum value for the number of Wnt genes in the ancestral bilaterian. The major conserved role of Wnt gene function in early development of metazoan animals is apparently patterning along the longitudinal body axis. Later in development, Wnt genes play many roles: in elongation of the body axis, in induction of cell fates, in specification of cell identity, and in establishment of regional boundaries within a tissue.

Recent Phylogenetic Analyses Fail to Resolve the Evolutionary History of the Wnt Gene Family

Signaling of different Wnt genes through the three known pathways raises the question of which evolved first. Unfortunately, phylogenetic trees based on available sequences do not provide an answer. Most of the invertebrate Wnt genes (mainly from Drosophila and Caenorhabditis) group within the thirteen subfamilies of vertebrate Wnt genes30 (Table 1), and phylogenetic trees using either complete63 or partial sequences64 do not adequately resolve the evolutionary relations among these subfamilies (Fig. 2). Thus, the number of Wnt subfamilies in the last common ancestor of both protostomes and deuterostomes remains uncertain and the grouping in the tree of Wnt subfamilies signaling via different pathways, e.g., Wnt-5 with Wnt-8, may not reflect the true relationship. What phylogenetic analyses do show is that all the amphioxus sequences mark the base of probable vertebrate gene diversifications, suggesting that gene duplications occurred within the vertebrate lineage.22 Nevertheless, the overall number of Wnt genes in vertebrates is only about 2.7x higher than in Drosophila and approximately 2.1x higher than in amphioxus. These are not the expected ratios if two rounds of whole-genome duplications occurred during early vertebrate evolution,65,66 unless a massive secondary Wnt gene loss has taken place within the vertebrate lineage.

Table 1. Classification of Wnt genes from selected organisms.

Table 1

Classification of Wnt genes from selected organisms.

Figure 2. Phylogenetic tree of the Wnt gene family based on all full-length sequences available through the end of 1999.

Figure 2

Phylogenetic tree of the Wnt gene family based on all full-length sequences available through the end of 1999. The tree includes representatives of twelve of the thirteen Wnt subfamilies (Wnt-15 sequences were not included in the analysis). Bremer support (more...)

Wnt sequences from the protostomes Drosophila and Caenorhabditis tend to cluster together and form weakly supported clades with very long branches.63 This clustering results from the small sampling of protostome taxa in the tree and from long-branch attraction due to the high rate of evolution within these two species.62 Even so, since genome analyses show that the seven Drosophila Wnt genes are probably homologous to at least five of the vertebrate Wnt genes (Wnt-1, Wnt-5, Wnt-6, Wnt-7, and Wnt-10),67,68 it can be concluded that the genome of the last common ancestor of arthropods and deuterostomes probably contained at least these five Wnt genes. In addition, the presence of a Wnt-3 homolog in Hydra54 suggests that a Wnt-3 gene may have been lost in the Drosophila lineage (Table 1) and, therefore, that the last common ancestor of protostomes and deuterostomes probably had at least six Wnt genes. The Hydra Wnt-3 is the only Hydra Wnt gene cloned to date and appears to be closely related to the Wnt-1 subfamily.22 Thus, unless there are other Wnt genes in the Hydra genome, the root of the Wnt gene family probably falls close to or within the Wnt-1 or Wnt-3 subfamilies.

The Role of Wnt Genes in Establishing the Early Anteroposterior Axis in Deuterostomes

Deuterostomes include the echinoderm/hemichordate clade and the chordates, which comprise the vertebrates plus two invertebrate groups (ascidians and amphioxus). In deuterostomes except for ascidians, signaling by the Wnt/β-catenin pathway appears to be involved in early anteroposterior patterning during the late blastula and/or early gastrula stages.

Echinoderms and Hemichordates

Among echinoderms, sea urchins have been most studied in regard to the role of Wnt genes in anteroposterior (animal/vegetal) patterning during the late blastula and early gastrula. The first known indication of animal/vegetal polarity in sea urchins is the localization of maternal transcripts of SpSoxB1, which encodes a HMG box transcription factor, to animal blas- tomeres starting at the 4th cleavage.69 At the 16-cell stage maternal β-catenin becomes localized to vegetal nuclei. Vegetal cells begin to express Wnt-1 and Wnt-8 during late cleavage, reinforcing the nuclear localization of maternal β-catenin.12,13 Wnt-5, which preferentially signals through the Wnt/Ca2+ pathway, then turns on in the future blastoporal lip at the ectoderm-endoderm boundary.13 Wnt-4 has also been cloned from sea urchins, but its expression pattern has not been described.11 The role of the Wnt signaling pathway in specifying posterior identity in sea urchin embryos is well established.12,7077 Nuclear localization of β-catenin in vegetal cells at the late blastula is required for the specification of vegetal cell fates and endoderm formation.72,73 Moreover, manipulation of the pathway by up-regulation of Wnt signaling (e.g., by lithium treatment or by injection of dominant negative forms of GSK3 β) vegetalizes (posteriorizes) the embryo.71 Conversely, blocking Wnt signaling by injection of dominant negative forms of TCF/LEF inhibits formation of endoderm.74

In sea urchins, Wnt signaling is mediated by the Notch signaling pathway. Nuclear localization of β-catenin in macromeres is required for them to receive inductive signals, mediated by Notch, from the micromeres.75,78 Moreover, during the late blastula stage Notch becomes localized to the future blastoporal lip in a ring around the vegetal plate at the endoderm/ secondary mesoderm boundary,79,80 and both the position of this boundary and the localization of nuclear β-catenin in this region are changed by manipulating Notch signaling.81

In vertebrates, brachyury has been shown to be a target of signaling by Wnt-3a and an upstream activator of Wnt-11.9,82,83 Sea urchin brachyury is first expressed in the vegetal plate,12,84,85 and resolves into a ring around the blastopore as development proceeds.86 Since vegetal expression of brachyury depends on nuclear β-catenin in the macromeres, brachyury is probably downstream of Wnt signaling in sea urchins as well as in vertebrates.86 In starfish and hemichordates, brachyury is also expressed in a ring around the blastopore.87,88 Thus, although Wnt genes have not been cloned from starfish and hemichordates, a role in early anteroposterior patterning similar to that in sea urchins seems likely.

Ascidians

Early development of ascidian tunicates differs considerably from that in other deuterostomes. Ascidian embryos have relatively few cells and determinant cleavage. Depending on the cell lineage, cell fates are restricted between the 64-cell stage and the onset of gastrulation at the 110 cell-stage.89 Thus, in ascidians, maternal mRNAs play a dominant role in establishing the embryonic axes. The future posterior pole of the embryo is established during ooplasmic segregation, when, shortly after fertilization, the myoplasm migrates to one side of the vegetal pole. This posterior vegetal cytoplasm contains determinants for gastrulation and for posterior fate.90 Anterior fate evidently results from the absence of posterior cytoplasm.89 There is no blastocoel, and presumably because there are few cells, gastrulation appears to be highly modified. Cells at the dorsal (often called anterior) lip of the blastopore give rise to endoderm and notochord, while those at the ventral (often called posterior) lip of the blastopore give rise to endoderm and muscle. The anteroposterior axis in ascidian tunicates is not so closely allied to the animal/vegetal axis as it is in echinoderms. Instead, in early embryos it is roughly perpendicular to the animal/vegetal axis,91 but shifts during gastrulation because of morphogenetic movements. 89 Consequently, at the beginning of gastrulation, the blastoporal lip of ascidian embryos that will involute to give rise to the notochord is defined as “anterior” and the opposite lip as “posterior.”

Ascidian Wnt, Notch, and brachyury genes are not expressed around the blastopore as they are in other deuterostomes. Consequently, the expression of these genes differs from that of their homologs in other deuterostomes. For example, from gastrulation through neurulation, ascidian Wnt-7 is expressed in a subset of neural precursor cells and subsequently, at the tailbud stage, transcription is detectable exclusively in the dorsal and ventral ependymal cells of the nervous system.16 For ascidian Wnt-5, the early maternal expression is only slightly reminiscent of expression around the blastopore in sea urchins, amphioxus and vertebrates13,22,92 in that maternal transcripts are initially posterior, being localized first in the myoplasm and then, at the 64-cell stage, in the vegetal blastomeres.15 However, zygotic Wnt-5 transcripts, although initially present in cells giving rise to endoderm, notochord, and muscle, subsequently appear in all the animal blastomeres at the 110-cell stage (when gastrulation begins). Expression is very weak during the gastrula and, during the neurula stage, becomes restricted to the notochord and endodermal strand.15 Moreover, in ascidians, nuclear β-catenin appears to function in endoderm specification and not in axial patterning.93 Thus, if β-catenin is experimentally down-regulated, endoderm does not differentiate and the notochord does not form. Conversely, during early cleavage, treatments up-regulating the Wnt/β-catenin pathway (such as exposure to lithium or injection of β-catenin mRNA) transform notochord cells into endoderm, 94 and result in shortened tails. In addition, brachyury expression in mesoderm around the blastopore has evidently been lost in ascidians. Expression is limited to the notochord95 as in other chordates, although a divergent T-box gene (AsT2) is expressed at the tip of the tail.96 Similarly, posterior expression of Notch during the gastrula stage appears to have been lost in ascidians, as has all mesodermal expression. During cleavage stages, maternal (Notch is concentrated in animal ectodermal cells. Only the relatively late (Notch expression, which begins at the neural plate stage in the neural folds,97 is probably evolutionarily related to the neural plate expression of amphioxus and vertebrate Notch genes.98,99

Amphioxus

In amphioxus embryos, there is no evidence for maternal expression of Wnt genes. However, zygotic expression of amphioxus Wnt genes suggests that, as in sea urchins, there is an early posterior Wnt signaling center. The earliest zygotic expression of Wnt genes is that of AmphiWnt-8—the only Wnt-8 gene in amphioxus.21,23 Expression of AmphiWnt-8 is first detectable at the onset of gastrulation beginning at the same time and in the same pattern as brachyury100—in a ring around the equator of the late blastula corresponding to the future blastoporal lip (Fig. 3A). However, AmphiWnt-8 is also expressed somewhat more weakly throughout the mesendoderm.23 By the early gastrula, expression is down-regulated in the dorsal lip of the blastopore and anterior mesendoderm, and, by the mid-gastrula, the only remaining expression is in the presomitic mesoderm and the posterior ventral endoderm (Fig. 3B, C).21,23 This pattern persists until the mid-late neurula. The expression pattern of AmphiWnt-8 around the blastopore and in the presumptive mesendoderm before the completion of invagination tentatively suggests that Wnt signals may be involved in restricting expression of genes such as and AmphiDll101 and AmphiDRAL102 (DRAL has been shown to modulate β-catenin-dependent transcription)103 to the ectoderm.

Figure 3. Developmental expression of Wnt genes in the cephalochordate amphioxus.

Figure 3

Developmental expression of Wnt genes in the cephalochordate amphioxus. The diagrams are not to scale. (A) Zygotic expression of AmphiWnt-8 and AmphiWnt-11 begins around the blastopore during the early gastrula stage. (B) AmphiWnt-1 expression begins (more...)

Transplantation of the dorsal lip of an amphioxus embryo into the blastocoel of a second embryo produced a secondary axis, suggesting that the dorsal blastoporal lip functions as an organizer in amphioxus as it does in vertebrates.104 Early expression of AmphiWnt-8 around the blastopore supports this idea, since in Xenopus both transplantation of the blastoporal lip and injection of XWnt-8 or other Wnt genes signaling through the Wnt/β-catenin pathway induce a secondary axis (see below). However, transplantation experiments in amphioxus must be viewed with caution. Because of the small size of the embryos (about 150μm in diameter), the transplants may have contained more than just the blastoporal lip. Moreover, because the embryos were not marked, it is not certain whether the secondary axes obtained were derived from the transplant or the recipient. Injection of Wnt mRNA into amphioxus embryos has not yet been performed, but should help to resolve this question.

In the mid-late gastrula, all the other amphioxus Wnt genes described to date are also expressed around the blastopore (Fig. 3AC). Thus, it seems likely that, as in sea urchins and vertebrates, Wnt signals from the blastoporal region are involved in specifying posterior identity in the neural plate and other tissues. Following AmphiWnt-8, AmphiWnt-11 expression starts around the blastoporal lip in the very early gastrula, when invagination is half-completed (expression is more intense dorsally) (Fig. 3A).20 Next, just as invagination is finished, AmphiWnt-1 turns on uniformly around the blastoporal lip (Fig. 3B).18 By the mid-gastrula, four other amphioxus Wnt genes (AmphiWnt-3, AmphiWnt-4, AmphiWnt-5, and AmphiWnt-6), together with Notch, start to be expressed uniformly around the blastopore.19,22,99 At the same time, AmphiWnt-7 transcription commences throughout the mesendoderm (Fig. 3C).19 AmphiWnt-4, although most intensely expressed around the blastopore, is also expressed weakly throughout the mesendoderm.19 In addition, AmphiWnt-4, AmphiWnt-7, and AmphiWnt-11 are expressed in the ectoderm of the gastrula - AmphiWnt-11 predominantly in presumptive neuroectoderm,20 AmphiWnt-4 and AmphiWnt-7 most conspicuously in the dorsoposterior ectoderm. It has been proposed that Wnt genes signaling via the Wnt/Ca2+ pathway may antagonize signals from Wnt genes signaling via the Wnt/β-catenin pathway.4,105 Thus, since Wnt-11 has been proposed to signal through the Wnt/Ca2+ as well as the Wnt/JNK pathway, AmphiWnt-11 might be antagonizing AmphiWnt-1, AmphiWnt-3, and AmphiWnt-8 to allow expression of neural markers.

In amphioxus, AmphiWnt-8 is the only Wnt gene expressed before the specification of the neuroectoderm as shown by down-regulation of AmphiDll and concomitant up-regulation of the pan-neural marker AmphiSox1/2/3 in the dorsal ectoderm of the early gastrula.21,23,101,106 Subsequent down-regulation of β-catenin in the dorsal ectoderm of the late gastrula and in the early neural plate (our unpublished data) suggests that, as in vertebrates (see below), down-regulation of signaling via the Wnt/β-catenin pathway is necessary for the maintenance of the neuroectoderm and the specification of anterior neural identity. This idea is supported by the finding that up-regulation of the Wnt/β-catenin pathway by a pulse of lithium delivered at the late blastula stage eliminates expression of AmphiSox1/2/3 in the neural plate (our unpublished data).

Vertebrates

In developing vertebrates, such as the amphibian Xenopus, the canonical Wnt pathway is involved first in establishing the dorsoventral axis and later in patterning the anteroposterior axis. The first of these roles probably has no counterpart in other organisms, except perhaps for amphioxus (see above). Establishment of the dorsoventral axis involves preferential localization of maternal β-catenin to nuclei of cells on the future dorsal side of the embryo.107 This nuclear localization of β-catenin, which sends a transient dorsal signal to neighboring cells to establish dorsoventral identity,108 disappears briefly about the time the blastopore begins to form.109111 Thus, treatments which stimulate signaling via β-catenin (such as a pulse of lithium, injection of dominant negative forms of GSK3 or injection of XWnt-8) when applied before the mid-blastula transition, result in embryos that are dorsoanteriorized.112 Conversely, treatments inhibiting β-catenin signaling block the formation of the dorsoventral axis.108,113,114 Although several Xenopus Wnt genes (XWnt-5a, XWnt-7b, XWnt-8b, and XWnt-11) are maternally expressed (Fig. 4A),115,116 Wnt genes are not required for the dorsal β-catenin signal. However, induction of secondary axes by injection of Wnt genes signaling through the Wnt/β-catenin pathway into ventral blastomeres at the 4-cell stage116118 suggests that transcripts of Wnt genes such as XWnt-8b, which are enriched in animal cells of the blastula (Fig. 4B, C),115 may be involved in maintenance of the dorsal β-catenin signal. This signal is essential for the expression of the early pan-neural markers chordin, Zicr-1 and Sox-2. Expression of these genes shows that specification of the presumptive neuroectoderm occurs before the onset of gastrulation in Xenopus119,120 and does not require the activities of Spemann's organizer.121 Similarly, in the chick, signals beginning before gastrulation induce transient expression of the early anterior neural markers Sox-3 and Otx2, although signals from the organizer and its derivatives such as the prechordal mesendoderm are subsequently required for maintenance of expression of these genes.122 It is unlikely that either Notch or brachyury functions together with β-catenin signaling in this early role of the Wnt/β-catenin pathway in establishing dorsoventral polarity. The brachyury gene is not expressed maternally, and although Xenopus Notch1 (Xotch) is expressed maternally, its expression is uniform until the early gastrula, when it becomes slightly enriched in the dorsal mesoderm.123

Figure 4. Expression of Wnt genes in the vertebrate Xenopus laevis.

Figure 4

Expression of Wnt genes in the vertebrate Xenopus laevis. The diagrams are not to scale. (A) Maternal XWnt-5a and XWnt-8b are uniformly distributed throughout the oocyte shortly after fertilization, whereas XWnt-11 mRNA is vegetally localized. In addition, (more...)

The early role of the Wnt/β-catenin pathway in establishing the dorsoventral axis in vertebrates appears to be quite distinct from its subsequent role in anteroposterior patterning. It is this later role, which begins in Xenopus at the mid-blastula transition,114 that appears to be broadly conserved in evolution. There are several models for how Xenopus embryos are patterned along the anteroposterior axis. In recent years, Nieuwkoop's two-signal model involving an anterior activator released from presumptive prechordal plate mesoderm and a transformer released from the notochord, which confers posterior identity, has been modified into a two-inhibitor model.124 In this model, there is a posterior Wnt signaling center producing an anteroposterior gradient of Wnt activity in the neuroectoderm. This Wnt signal, mediated by members of the Wnt/β-catenin class, posteriorizes neuroectoderm.125127

There is considerable evidence to support such a model for anteroposterior patterning. In Xenopus, a posterior Wnt signaling center is established during the late blastula/early gastrula stage. The marginal zone and the blastoporal region express Wnt genes of both the Wnt/β-catenin (XWnt-3a, XWnt-8, and XWnt-8b) and the Wnt/JNK and Wnt/Ca2+ classes (XWnt-11) (Fig. 4D, E).115,128138 In accord with a change in the role of Wnt/β-catenin signaling at the mid-blastula transition, a new domain of intense nuclear β-catenin expression appears during the late blastula in a ring around the marginal zone, i.e., the future mesoderm.111 As gastrulation begins, this ring becomes complete around the marginal zone, except for a small region in the dorsal midline; this zone of nuclear β-catenin approximately coincides with expression of XWnt-8. Later, nuclear β-catenin becomes concentrated at the dorsal and ventral lips of the blastopore. This pattern is similar to that of expression of Xbra and XWnt-11.111

Posterior Wnt signaling by the Wnt/β-catenin pathway probably is responsible for establishing and/or maintaining posterior identity. First, injection of XWnt-8113,115,135 after the mid-blastula transition antagonizes signals from the organizer, inhibiting dorsoanterior development and resulting in embryos without head or notochord, but with enlarged somitic muscle. This contrasts with injection of XWnt-8 before the mid-blastula transition, which promotes dorsoanterior development. Similarly, lithium applied after the mid-blastula transition, posteriorizes embryos, which develop with malformations of anterior structures.139,140 Conversely, injection of a dominant negative XWnt-8 anteriorizes embryos. Moreover, when either XWnt-8 or XWnt-8b is injected into 2- to 4-cell zebrafish embryos, there are dorsoanterior defects including suppression of eye and midbrain-hindbrain boundary formation. This is quite different from the effect of injection of XWnt-8 genes into embryos of Xenopus itself, and suggests that the primary role of Wnt signaling in zebrafish is in anteroposterior patterning, and that a role for Wnt signaling in establishing the dorsoventral axis as in Xenopus may not be general among vertebrates.141

Wnt-3 genes, which signal through the same pathway as Wnt-8, have been shown to play a major role in early anteroposterior patterning. Expression of Xenopus XWnt-3a begins in the dorsal marginal zone, chiefly in the ectoderm (Fig. 4D).116,132 However, as the blastopore closes, the dorsal lip becomes dorsoposterior and the previously dorsal expression of XWnt-3a in the blastopore lip becomes progressively more posterior (Fig. 4E). XWnt-3a appears to play a dominant role in anteroposterior patterning of the central nervous system.136,142,143 At the early neurula stage, expression of XWnt-3a begins along the neural folds and in a diffuse ring around the blastopore. Subsequently, during neurulation, the expression around the blastopore and in the posterior neural folds becomes very strong (Fig. 4E, F).136 Several lines of experimental evidence support roles for XWnt-3a in anteroposterior patterning of the neuroectoderm. Overexpression of XWnt-3a or β-catenin suppresses the expression of anterior neural genes and promotes the expression of posterior neural genes, suggesting that XWnt-3a functions in establishing and/or maintaining posterior identity in the nerve cord.132,136,144 The roles of XWnt-8 and XWnt-3a may be interchangeable, because in other vertebrates, the roles of XWnt-3a in Xenopus appear to be performed by Wnt-8 genes: for example, the phenotype of transgenic mice expressing chicken CWnt-8c is similar to that of Xenopus overexpressing XWnt-3a.145 Conversely, expression of a dominant negative Wnt-8 reduces the expression of posterior neural genes and increases that of anterior neural genes.136 In addition, zebrafish Wnt-8 has been shown to be required for the posteriorization of the early neuroectoderm.146148

In vertebrates, Wnt signals are kept low anteriorly by secretion of Wnt and BMP antagonists by the anterior endoderm and the prechordal plate (in Xenopus and zebrafish) or by the anterior visceral endoderm (in mice).107,148,149 Thus, injection of secreted Wnt inhibitors (e.g., frzb-1, dickkopf-1, cerberus, or crescent) anteriorizes the neuroectoderm.150154 These results are in agreement with inhibition of the development of dorsoanterior structures, like the head and the notochord by injection of XWnt-8 after the mid-blastula transition.115,135 Anterior suppression of Wnt signaling is also necessary for proper anteroposterior patterning of the mesoderm and endoderm. Mouse mutants have shown that Wnt-3a is important for both anteroposterior patterning of the vertebral column155 and posterior somite formation.83,156 Moreover, in chicken as well as mice, the Wnt/β-catenin signaling pathway is implicated in patterning along the anteroposterior axis of the mesoderm and endoderm as well as the nerve cord.157,158 Thus for heart formation, inhibition of Wnt/β-catenin signaling is required anteriorly in the cardiac mesoderm in the chick158 and overexpression of the Wnt antagonists Dickkopf-1 and Crescent can induce heart formation in explants of the ventral marginal zone in Xenopus.159

As in amphioxus and sea urchins, in Xenopus, Wnt, Notch, and Brachyury probably act together to induce posterior structures.160,161 Starting at the gastrula stage, Xenopus Notch and delta-1 are expressed in mesodermal cells posterior to the neural plate. Increasing amounts of Dishevelled, a component of both the Notch and Wnt signaling pathways, posteriorizes neural tissues and activates posterior markers such as brachyury.162 Conversely, in mice, loss of either Wnt-3a or Notch1 function results in posterior defects.163,164 Expression of XWnt-3a together with active Notch (the Notch intracellular domain) induces animal cap explants grafted at any anteroposterior level onto the neural plate to form ectopic tails with neural tubes, but without notochords. Active Notch alone only induces ectopic tails in animal caps grafted onto the posterior neural plate near the zone where XWnt-3a is expressed.160,161 These ectopic tails induced by active Notch express brachyury (Xbra), XWnt-3a, caudal (Xcad), FGF-8, Xdelta-1, and lunatic fringe (lfng). The brachyury and caudal genes are downstream targets of XWnt-3a.83,155 Brachyury is expressed around the blastopore in early Xenopus embryos, and expression continues in the tail bud.165,166

Evolutionary Conservation of Wnt Gene Involvement in Axial Patterning of Protostomes, Deuterostomes, and Cnidarians

In insects (Drosophila, grasshoppers, and beetles), the first expression of wingless, the homolog of Wnt-1, is in a ring around the posterior end of the early embryo.39,45,167 Brachyury has a similar pattern.168170 Similarly, in Hydra, genes of the Wnt/β-catenin pathway are expressed together with brachyury at the apical tip of the body around the single gut opening.54,171,172 Co-expression of Wnt and brachyury at one end of the embryo in protostomes, deuterostomes, and cnidarians suggests that the activation of brachyury by Wnt genes of the Wnt/β-catenin class is a component of a highly conserved signaling network mediating axial patterning, which evolved very early during metazoan evolution.

There are additional similarities in the roles of Wnt genes in anteroposterior patterning between Drosophila and deuterostomes. The teashirt gene encodes a zinc-finger transcription factor, which associates with and requires Armadillo (i.e., Drosophila β-catenin) for its function. 173,174 Expression is limited to the trunk, where it is required to maintain wingless expression, presumably in a feedback loop. Manipulation of teashirt expression shows that it is required for specification of trunk identity. When teashirt expression is lost in the trunk of the embryo, the ventral part of the trunk takes on anterior head identity.175,176 Similarly, the two orthologs of teashirt in mice, mtsh1 and mtsh2, are expressed in the neural tube and the somites of the trunk and the tail.177 Thus, it seems likely that in vertebrates as well as Drosophila, teashirt may be acting to maintain trunk and tail expression of Wnt genes of the Wnt/β-catenin class.

Wnt Genes and Body Elongation From the Chordate Tail Bud

During amphioxus and Xenopus development, much of the tail bud is derived from the blastoporal lip. From the neurula stage on, the tail bud is responsible for much of the elongation of the embryo and larva.22,166,178 In contrast, in ascidians, the tail elongates by rearrangement of preexisting cells,179 and a definitive tail bud is lacking. The vertebrate tail bud is subdivided into a mosaic of different regions that originate during gastrulation from the blastoporal lip and are subsequently incorporated into the tail bud. In vertebrates, cells from particular tail bud regions can be traced to specific differentiated structures in the tail. Cell tracing experiments and gene expression patterns have shown that the arrangement of tissues in the tail bud is similar in Xenopus and the chick, even though the chick tail bud has a much higher proportion of mesenchyme than that of Xenopus.166,180,181 At the neurula stage, the blastopore-associated expression of XWnt-3a, XWnt-5a, XWnt-8, and XWnt-11 is carried over into the tail bud and maintained during subsequent elongation of the tail (Fig. 4E-G). Wnt, Notch, and brachyury genes are expressed in specific regions of the tail bud. For example, XWnt-3a and XWnt-5a are both expressed in the most dorsoposterior region of the neural tube and in the overlying ectoderm.160,161 Brachyury is expressed in the chordoneural hinge, the posterior part of the notochord, the posterior wall of the neurenteric canal, and the roof plate of the spinal cord, and Notch and its ligand delta-1/2 in the distal tip of the tail bud and the posterior wall of the neurenteric canal.160,165,166 Posteriorly expressed Wnt genes function in elongation of the tail bud as well as in anteroposterior patterning (discussed above).182 In mice, loss of Wnt-3a leads to a complete loss of the tail bud and the caudal somites,83,163 while Wnt-5a deficiency leads to a reduced extension of the primary body axis.183 In zebrafish, both Wnt-5 and Wnt-8 are required for tail outgrowth.147,148,184 Wnt genes signaling through the Wnt/Ca2+ pathway and/or the Wnt/JNK pathway (e.g., Wnt-5 and Wnt-11) have been implicated in convergent extension movements in both Xenopus and zebrafish (see below).9,82 In Xenopus, at the gastrula and early neurula stages, the expression patterns of XWnt-11 and brachyury are congruent, with XWnt-11 being a target of brachyury,9 and it seems likely that they may function together in elongation of the body axis.

A recent analysis of the amphioxus tail bud has revealed both similarities and differences between the amphioxus and vertebrate tail buds.22 As in vertebrates, the amphioxus tail bud represents a developmental continuation of the blastoporal lip, provides cells for the caudal outgrowth of other tissues, and is subdivided into specific regions. However, in contrast to vertebrates, the amphioxus tail bud does not have any mesenchymal component. Moreover, the specific Wnt genes expressed in the tail bud and the regions of the tail bud where they are expressed are similar in some respect but differ in others between amphioxus and Xenopus. Similarities include the expression of both Wnt-8 and Wnt-11 in the tail bud (Figs. 3D, E; 4F, G). In addition, the expression of mouse Wnt-5b in the posterior hindgut and notochord92 resembles the tail bud-associated expression of amphioxus AmphiWnt-5. However, while Wnt-3 and Wnt-5 are expressed in partially overlapping domains in the tail bud of both amphioxus and Xenopus,22,160,161 their expression domains are not identical in the two organisms. Both amphioxus AmphiWnt-3 and Xenopus XWnt-3a are expressed in the posterior neural tube and in the dorsoposterior ectoderm. However, the expression of AmphiWnt-3 in the posterior wall of the neurenteric canal has no counterpart in Xenopus. Moreover, while AmphiWnt-5 is expressed in the chordoneural hinge and the posterior wall of the neurenteric canal, Xenopus XWnt-5a is expressed in the posterior ectoderm and posterior neural tube. In addition, in amphioxus, AmphiWnt-1 is expressed in the blastoporal lip and tail bud (Fig. 3BG), but XWnt-1 is not expressed at all in either region. Similarly, Wnt-4 and Wnt-7 are expressed in the amphioxus tail bud (Fig. 3D, E), but not in that of Xenopus. Other genes expressed in the tail buds of both amphioxus and Xenopus include brachyury, Notch, BMP-2/4, caudal, Hox3, and hedgehog. 99,100,160,166,185188 Although the localization of expression of these genes within the amphioxus tail bud has not been studied in as much detail as for Wnt genes, in general, the expression seems comparable to that of their vertebrate homologs. Thus, a combinatorial code of developmental gene expression, which patterns the tail bud and generates specific tissues during posterior elongation, seems to be conserved between amphioxus and vertebrates and was therefore probably already present in the last invertebrate ancestor of the vertebrates.

Conservation of Wnt Signaling in the Paraxial Mesoderm in Amphioxus and Vertebrates

In amphioxus and the vertebrates, the axial and the paraxial mesoderm develops into the notochord and somites, respectively. In vertebrates, the Wnt genes with roles in formation and patterning of the axial and paraxial mesoderm are Wnt-1, Wnt-3a, Wnt-7a, Wnt-8, and Wnt-11. In Xenopus, XWnt-8 and XWnt-11 are expressed, respectively, in the ventral and lateral mesoderm and the somites (Fig. 4F, G). In Xenopus and the zebrafish, loss of Wnt-8 leads to a loss of posterior and ventral structures.147,148 The role of Wnt-11 in paraxial mesoderm patterning is more difficult to assess since in null mutants, gastrulation movements are disrupted and consequently the mesoderm does not form properly.9,189

The roles of Wnt-1, Wnt-3a, and Wnt-7a in mesodermal patterning have been better studied than that of Wnt-11. Wnt-3a plays an early role in formation of the posterior mesoderm, while Wnt-1 and Wnt-7a act later in patterning of the somites. Wnt-3a is expressed in the tail bud in cells that will give rise to paraxial mesoderm190,191 and is essential for the formation of caudal somites. In a mouse mutant for Wnt-3a, neither tail bud, notochord, nor caudal somites form, although the anterior somites are relatively normal.83,142,156,163,192 Similar results are obtained by injection of WIF-1 (a secreted inhibitor of Wnt proteins of the Wnt/β-catenin class) into 4-cell Xenopus embryos, which antagonizes the formation of posterior presomitic mesoderm.193 Conversely, co-injection of XWnt-3a or XWnt-8 with noggin into Xenopus animal caps induces dorsal mesodermal markers.194

Wnt signaling also acts later in development in the segmentation of the paraxial mesoderm and in the patterning of the somites.195 Components of the Wnt/β-catenin pathway are expressed in the paraxial mesoderm before the onset of expression of the myogenic factor MyoD,196 and have been shown to function in patterning of the somites. In homozygous LEF-1/TCF-1 mouse mutants, expression of several regional marker genes in the somites is abnormal, and expression of Notch1 and Wnt-5a in the presomitic mesoderm is eliminated.197 In addition, up-regulation of the Wnt/β-catenin pathway with lithium results in supernumerary and malformed somites, especially in anterior regions of the paraxial mesoderm.198 Experimental studies have demonstrated that Wnt-1 can induce myogenesis in explants of paraxial mesoderm.199 Additional evidence for a role of Wnt genes in somitogenesis is that six Frizzled (Fz) genes (Frizzled 1, 3, 6, 7, 8, and 9), which encode Wnt receptors, are expressed in the forming and differentiating somites.200 Moreover, injection of the Wnt antagonist, WIF-1, which is normally expressed in paraxial mesoderm in vertebrates, into Xenopus neurulae results in disorganized somites with impaired segmentation.193 A model has been presented for the chick, whereby Wnt-1, secreted by the neural tube, binds to Fz-1 and, acting via β-catenin, activates transcription of the myogenic factor Myf5, while Wnt-7a, secreted by the ectoderm overlying the somites binds to Fz-7 and, acting through a β-catenin independent pathway, leads to the activation of myogenesis.199203 In Xenopus, expression of XWnt-7a appears to be restricted to the neural tube,204 but the gene could be playing a similar role in somitogenesis. In addition, Wnt-11 may play a role in the regionalization of the somite. In the mouse, Wnt-11 is expressed at the junction of the dermatome and myotome,205 while in the zebrafish, expression in the somites is limited to the dermatome.82

Although, it seems likely that Wnt signaling plays similar roles in the formation and patterning of the paraxial mesoderm in amphioxus and vertebrates, there are some differences between them in the formation of the mesoderm and in the Wnt genes that are mesodermally expressed. The first major difference in mesoderm formation is in the origin of ventral mesoderm. In amphioxus, the ventral mesoderm (also called lateral plate mesoderm) arises relatively late in development (at the mid-neurula stage) as ventral outgrowths from the somites. In contrast, in vertebrates, the ventral mesoderm arises earlier (at the gastrula stage) about the same time the paraxial mesoderm initially forms. Nevertheless, in spite of this offset in developmental timing, Vent gene expression marks nascent ventral mesoderm in both amphioxus and vertebrates.206 The second major difference in mesoderm formation is that in amphioxus, unlike vertebrates, the somites extend to the anterior tip of the animal. The anteriormost eight somites pinch off enterocoelically from grooves of the dorsolateral mesendoderm during the late gastrula, whereas the posterior somites are formed from the tail bud, as are vertebrate somites. Nevertheless, the fate of the anterior and posterior somites is the same: they all form muscles used in undulatory swimming. Thus, while some genes (e.g., engrailed and Wnt-8) are expressed early in formation of the anteriormost eight somites, but not in the more posterior somites, for the most part, gene expression in the anterior and posterior somites is the same.

Experiments blocking Wnt signaling have not been done in amphioxus. However, the expression patterns of amphioxus Wnt genes suggest that, as in vertebrates, Wnt proteins are required for formation of the axial and paraxial mesoderm as well as for somitogenesis. As noted above, during the gastrula stage, all of the amphioxus Wnt genes are expressed around the blastopore, and AmphiWnt-4, AmphiWnt-5, AmphiWnt-7, and AmphiWnt-8 are expressed in the dorsal mesoderm and/or ectoderm as well. In addition, several Wnt genes continue to be expressed in the tail bud as somites are budded off from it. These include AmphiWnt-1, AmphiWnt-3, AmphiWnt-4, AmphiWnt-5, AmphiWnt-6, and AmphiWnt-11 (Fig. 3D, E).1820,22 Of these, AmphiWnt-5 and AmphiWnt-6 are expressed in the posterior mesoderm. The signaling pathway of Wnt-6 genes has not been determined; however, in phylogenetic analyses, Wnt-6 genes group with Wnt-8 genes,22 which signal via β-catenin. Thus, it is likely that Wnt-6 signals via the Wnt/β-catenin pathway and that blocking this pathway at the tailbud stage would prevent the formation of new somites in amphioxus as it does in vertebrates.

Expression of Wnt genes during somitogenesis in amphioxus suggests that, as in vertebrates, Wnt genes have roles in patterning both the anterior and posterior somites. However, in amphioxus, Wnt-1 cannot be the Wnt/β-catenin-class gene that performs this function. Rather, Wnt-6 and Wnt-8 may function in somitogenesis in amphioxus as Wnt-1 does in vertebrates. AmphiWnt-8 is expressed in the anterior presomitic (early paraxial) mesoderm, which gives rise to somites two through eight that form by enterocoely. It is possible that in the presomitic mesoderm, AmphiWnt-8 is interacting with engrailed (AmphiEn), which is expressed in the posterior portion of each of the first eight somites before there is overt segmentation.207 Segmentation and patterning of the more posterior somites may be mediated by AmphiWnt-6 and AmphiWnt-7, secreted by the nerve cord, as is known for Wnt-1 and Wnt-7a in vertebrates. As in vertebrates, AmphiWnt-7 is expressed in the dorsal mesoderm and ectoderm at the gastrula stage, in the edges of the neural plate at the neurula stage, and in much of the neural tube in the larva. AmphiWnt-6, which, as discussed above, may signal via the Wnt/β-catenin pathway, is expressed in the nerve cord from the neurula stage on and could be performing the same function as Wnt-1 does in vertebrate somitogenesis. AmphiWnt-4 and AmphiWnt-11 are expressed in the somites in amphioxus; AmphiWnt-11 in the myotomal compartment (there is no subdivision into dermatome and myotome in amphioxus) and AmphiWnt-4 in the dorsal part of the somite.19,20 Thus, as in vertebrates, AmphiWnt-11 may be involved in regionalization of the somite; however, the expression of AmphiWnt-4 in amphioxus somites has no counterpart in vertebrates.

In addition to Wnt genes, the expression of Notch genes in the tail bud and in forming somites is similar in amphioxus and the vertebrates. In vertebrates, Notch expression is required for normal somitogenesis and segmentation of vertebrate embryos.208210 Although a direct link between the Notch and Wnt signaling pathways in vertebrate somitogenesis and segmentation has not been shown, the absence of Notch1 expression in the presomitic mesoderm in homozygous LEF-1/TCF-1 mouse mutants suggests a possible interaction between the two pathways.197 Moreover, Notch signaling through hairy-related genes appears to specifically regulate expression of MyoD, a gene, which has also been shown to be downstream of Wnt/β-catenin signaling.211 In amphioxus, Notch is expressed in the posterior mesoderm in the gastrula and neurula, the tail bud and the dorsoposterior portion of all somites,99 suggesting that its role in somitogenesis may be similar to that in vertebrates. In both vertebrates and amphioxus, brachyury is also expressed around the blastopore and in the tail bud. In vertebrates, brachyury is a target of Wnt-3a,83 but is upstream of Wnt-11.9 Co-expression of these two Wnt genes and brachyury in amphioxus suggests that a similar relation between those genes may exist in amphioxus and vertebrates.

Formation of the muscle in ascidians differs considerably from that in amphioxus. While ascidian larvae have a notochord, the tail muscle is not organized into somites, and there is no tail bud. Instead, the tail muscle lineage is determined shortly after fertilization, and the muscle cell lineage is partitioned into the tail. Nevertheless, ascidian Wnt-7, the only ascidian homolog cloned to date of a Wnt gene involved in vertebrate (and possibly amphioxus) somitogenesis and segmentation, is expressed exclusively in cells of the ascidian nervous system,16 and it could be acting to pattern the paraxial mesoderm in ascidians as in other chordates. Thus, even though there is no tail bud in ascidians and the tail musculature is unsegmented, a role of Wnt-7 in patterning the paraxial musculature might be conserved among chordates.

The Roles of Wnt Genes in Patterning the Chordate Notochord

Zebrafish Wnt-11 is the only Wnt gene described to date that is expressed in the notochord, and the expression of this gene depends on that of brachyury.82 Wnt-11 signals via the Wnt/JNK pathway, which has been shown to mediate convergent extension movements.7,9 In other vertebrates, Wnt genes are not expressed in the notochord and a down-regulation of signaling via the Wnt/β-catenin pathway is necessary for specification of the notochord.212 In agreement with this idea, ectopic expression of XWnt-8 inhibits expression of the notochord marker Xnot138 and can transform notochord into somitic muscle.130 Moreover, the Wnt antagonist frzb is expressed in the Xenopus notochord during the gastrula stage.212 In amphioxus, the only Wnt gene expressed in the notochord is AmphiWnt-5, which is in the Wnt/Ca2+ class. AmphiWnt-5 is expressed in the anterior and posterior ends of the notochord.22 This suggests that, even though the amphioxus notochord is a special type of muscle, as in vertebrates, the Wnt/β-catenin pathway must be repressed for notochord formation.

Is There an Evolutionary Conservation of Wnt Signaling in Segmentation Between Chordates and Protostomes?

As discussed above, in amphioxus, engrailed and Wnt-8 are expressed in the anteriormost eight somites.21,207 The expression of engrailed in the posterior half of each nascent somite is reminiscent of the pattern of engrailed in the Drosophila ectoderm. During the segmentation of the Drosophila embryo, engrailed cooperates with wingless, cubitus interruptus, hedgehog and other gap/pair rule and segment polarity genes.213 The expression of several segmentation genes in similar patterns in other insects, such as grasshoppers and beetles, suggests that the genetic basis of segmentation of the ectoderm is conserved among all insects.45,167,214 However, it is now clear that amphioxus homologs of many of the genes, which cooperate with engrailed in segmentation of the Drosophila ectoderm (e.g., Wnt-1, hh, gli)18,188,215 (SM Shimeld pers. com.), although they are expressed in the amphioxus mesoderm, are not expressed together with AmphiEn in a pattern that suggests an evolutionary conservation of the mechanisms for segmenting the ectoderm in Drosophila and the mesoderm in amphioxus. Indeed, whether, a genetic mechanism involving engrailed and Wnt genes of the Wnt/β-catenin class acts in segmentation of the ectoderm in any protostomes other than arthropods is controversial. Engrailed is expressed in the ectoderm of embryos and regenerates of the annelid Platynereis dumerilii in a pattern suggesting a role in segmentation,216 but it is not similarly expressed in the ectoderm of other annelids217220 or of the onychophoran Peripatus,221 which has led to the suggestion that a role for engrailed in ectodermal segmentation is limited to arthropods.222

However, evolutionary conservation of the roles of engrailed and wingless in mesodermal segmentation remains a possibility. As in amphioxus, engrailed is segmentally expressed in the Drosophila mesoderm.223,224 The genetic networks controlling segmental expression of engrailed in the Drosophila mesoderm are not entirely understood, but do not appear to be identical to those for segmenting the ectoderm.225 In the current models, segmental wingless (wg) expression in the ectoderm induces striped expression of downstream targets (e.g., the forkhead gene sloppy paired) in the mesoderm. Expression of sloppy paired is required for specification of somatic muscle. Engrailed is downstream of sloppy paired.226 Although the wingless signal for mesodermal segmentation appears to come from the ectoderm, wingless is also expressed in the invaginating mesoderm up to stage 9 in a rather patchy pair-rule pattern.227 The roles of this wingless expression have not been precisely determined in Drosophila. However, because overexpression of wingless throughout the mesoderm up-regulates and prolongs the striped mesodermal expression of engrailed, it has been suggested that mesodermal wingless may act to maintain mesodermal engrailed expression.228 Since amphioxus AmphiWnt-8, which, like wingless, signals via β-catenin, is uniformly expressed in the presomitic and somitic mesoderm of somites two through eight,21 it could be that there is a similar mechanism for segmenting the anterior mesoderm of amphioxus and the mesoderm of Drosophila, involving Wnt and engrailed genes. Whether this mechanism is evolutionarily conserved in arthropods and amphioxus awaits a thorough understanding of the interactions between Wnt/wingless in mesodermal segmentation in the anterior somites of amphioxus and the somatic mesoderm of Drosophila.

Evolutionary Conservation of the Roles of Wnt Genes in Convergent Extension and Planar Polarity Signaling

Convergent extension movements lead to a narrowing and lengthening of tissues mainly by compaction and intercalation of cells. Among the chordates, the mechanism of convergent extension has been well studied only in vertebrates. In amphioxus, cell tracing and time-lapse analyses have not been done to address the question of convergent extension during elongation of the body axis. In ascidian tunicates, elongation of the tail is solely due to convergent extension. Wnt-5 may play a role in this process. It is expressed in the notochord and, when overexpressed, the anteroposterior body axis of the embryo is shortened due to the abnormal movement of notochord cells. Thus, ascidian Wnt-5 may act to limit the extent of convergent extension.229 The possible roles of other Wnt genes in convergent extension have not been studied in ascidians.

In vertebrates, convergent extension is important in gastrulation and in elongation of the body axis.230 Wnt signaling, both through the two Dishevelled-mediated pathways (JNK/SAPK and β-catenin) and through the Wnt/Ca2+ pathway is important in convergent extension.4 Wnt-1, signaling through the β-catenin pathway, promotes cell adhesion and enhances convergent extension due to regulation of the nodal gene Xnr3.4 In addition, Wnt-11, which acts via the Wnt/JNK pathway, potentiates convergent extension movements in the neuroectoderm and the mesoderm.9,189,231234 In contrast, XWnt-5a, signaling through the Wnt/Ca2+ pathway decreases cell adhesion and depresses convergent extension. It has been suggested that signaling via this pathway functions to balance signaling between the two Dishevelled-mediated pathways. 4 In Xenopus, defects in convergent extension of the mesoderm can be rescued by Dishevelled, but not by molecules that specifically activate the Wnt/β-catenin pathway.235,236 In addition, time-lapse analyses of cell movements in Xenopus have shown that Dishevelled controls the polarity of the lamellipodia driving convergent extension, which is consistent with a role for planar polarity signaling in convergent cell extension.233 Experiments with deletions of Xenopus Dishevelled suggest that the Wnt-11 pathway controlling convergent extension movements in vertebrates may be similar to the Drosophila planar cell polarity cascade.9,233 In Drosophila, the planar cell polarity cascade controls the organization of cells within the plane of an epithelium, for example the coordinated orientation of hairs and bristles in the ectoderm. The transduction of the planar polarity signal in Drosophila is mediated, among other factors, by Frizzled and Dishevelled.237 In Drosophila, separate functional domains of the Dishevelled protein transduce the signal for the Wnt/β-catenin and the planar polarity cascade, respectively,238,239 which is comparable to the function of the Xenopus Dishevelled protein.9,233 Thus, although the Wnt/JNK pathway has not been as well characterized as the Wnt/β-catenin pathway, it is possible that the former pathway may have mediated convergent extension movements in the ancestral bilaterian.

The Involvement of Wnt Genes in Patterning the Developing Central Nervous System

Vertebrate Wnt genes are involved in patterning several regions of the central nervous system. Of these regions, the midbrain-hindbrain boundary region (MHB), which expresses Wnt-1, together with engrailed genes, Pax2 and Pax5, has received the most attention.116,132,137,240242 Loss of murine Wnt-1 leads to loss of the midbrain and anterior hindbrain and to a complete ablation of engrailed expression in this region of the central nervous system.243246 These defects can be rescued by overexpression of engrailed-2.246 In addition, the Xenopus engrailed-2 promoter contains three binding sites for the transcription factors TCF/LEF in the Wnt/β-catenin signaling pathway, suggesting that engrailed is a direct target of Wnt/β-catenin signaling.247

In amphioxus, Wnt genes of the Wnt/β-catenin class, engrailed, and Pax2/5/8 are not expressed in the nerve cord in patterns consistent with there being a homolog of the MHB.18,207,248,249 Thus, AmphiWnt-1 is not expressed at all in the amphioxus nerve cord,18 while engrailed is expressed in two domains in the amphioxus nerve cord, one in the equivalent of rhombomere 5, and the second in the equivalent of the forebrain.207 The latter domain is within that of AmphiWnt-3 in the forebrain and close to that of AmphiWnt-8.21,22,207 Thus, the functional relationship between engrailed and Wnt/β-catenin Wnt genes may be conserved between amphioxus, vertebrates and Drosophila, even though the structures being patterned are not homologous.

It is controversial whether the absence of a MHB in amphioxus represents a loss or is an indication that the MHB first evolved within the vertebrate lineage. In ascidians, Pax-2/5/8 is expressed in two cells just posterior to the Otx domain and just anterior to the Hox1 domain, where a MHB would be expected to be if there were one.250 The expression of Pax-2/5/8 has led to the suggestion that ascidians have a homolog of the MHB and that amphioxus must have evolutionarily lost it. However, expression of Wnt-1 and engrailed has not yet been determined in ascidian tunicates, and, therefore, the possibility that the Pax-2/5/8 domain corresponds to hindbrain cannot be ruled out.

In vertebrates, Wnt genes are also involved in the induction of neural crest. In Xenopus, XWnt-7b is expressed in the dorsal marginal zone in the early gastrula and later in the dorsal midline of the neural tube and the MHB.137 When injected into embryos or when co-injected with the neural inducer noggin into ectodermal explants, XWnt-7b induces the neural crest markers Xslug and Xtwist.137 Moreover, blocking XWnt-8 inhibits the expression of neural crest markers.251 In addition to the MHB region, Xenopus XWnt-1 is transcribed in the dorsal midline of midbrain, hindbrain, and anterior spinal cord,132,137,241 and loss of both Wnt-1 and Wnt-3a in mice leads to deficiencies in neural crest derivatives.252 The Wnt receptor Frizzled 3, which is expressed in the dorsal part of the neural tube, interacts with XWnt-1 and can induce neural crest in embryos and explants.253 Thus, Wnt genes of the Wnt/β-catenin class probably function in induction and/or maintenance and/or proliferation of neural crest. In addition, Wnt genes play several roles in later neural patterning. For example, Wnt-3a is necessary for the development of the murine hippocampus,143 and Wnt-7a is required for axonal remodeling and synaptic differentiation in the cerebellum.254 Loss of both Wnt-1 and Wnt-3a in mice leads to a reduction of neural precursors within the neural tube as well as defective neural crest.252

In Drosophila, wingless is expressed in all three regions of the brain, most conspicuously in the protocerebrum. In wingless null mutants, approximately half the protocerebrum is missing.255 It has been proposed that wingless functions in the determination of neural cell fate in the brain and in other areas of the central nervous system where it is expressed.255258 Wingless is also expressed in the eye, where it appears to act in specification of the dorsoventral axis.259 Ectopic expression of another Drosophila Wnt gene, DWnt-3, which is expressed along the ventral midline of the nerve cord, results in abnormal commissural axon tracts in the developing central nervous system.42,43,46 In addition, DWnt-4 and DWnt-10 are expressed in the Drosophila central nervous system;47,50 however, their function in the development of the central nervous system has not been studied.

The expression of Wnt genes of the Wnt/β-catenin class in specific subsets of developing neurons in both chordates and Drosophila might reflect a conserved evolutionary role in neuroblast specification. However, since these roles have not been studied in detail in either chordates or Drosophila, it remains possible that Wnt genes have been repeatedly co-opted for roles in neuroblast specification.

Conclusions

Sequence comparisons and genome analyses suggest that the last common ancestor of protostomes and deuterostomes had at least six Wnt genes (Wnt-1, Wnt-3, Wnt-5, Wnt-6, Wnt-7, and Wnt-10). However, this number could be an underestimate, since phylogenetic analyses do not adequately resolve the relationships between Wnt subfamilies. Specific Wnt genes signal preferentially through one of three major pathways: (1) Wnt/Ca2+, (2) Wnt/JNK, and (3) Wnt/β-catenin.

The Wnt/β-catenin pathway is the best studied of the three Wnt pathways, and its major conserved role in metazoan development appears to be in patterning the longitudinal axis of the body. In Hydra, Wnt-3 and other components of this pathway are expressed around the common gut opening at the oral end of the animal, which corresponds to the posterior end of the larva. In both deuterostomes and protostomes, one or more of the Wnt genes signaling through this pathway (wingless/Wnt-1, Wnt-3, and/or Wnt-8) are expressed at the posterior/ vegetal end of the early embryo. Experimental evidence from deuterostomes indicates that this posterior Wnt signaling center creates an anteroposterior gradient of Wnt activity that is down-regulated anteriorly to allow for normal anterior development. Notch genes and brachyury are typically co-expressed with Wnt genes in this posterior signaling center, and there is evidence to demonstrate that they all interact. Another conserved role of the Wnt/β-catenin pathway appears to be in boundary formation. Such boundaries include segment boundaries in Drosophila and the midbrain-hindbrain boundary in vertebrates.

The Wnt/Ca2+ pathway, through which Wnt-4 and Wnt-5 preferentially signal, appears to antagonize the effects of the Wnt/β-catenin pathway. During deuterostome development, Wnt genes signaling through the Wnt/β-catenin and the Wnt/Ca2+ pathway are often expressed in the same or contiguous cells, as would be expected if they had balancing effects on a given developmental process. The Wnt/JNK pathway appears to play a major role in convergent extension. It has been suggested that the role of Wnt genes in convergent extension may be evolutionarily related to the role of Wnt genes in the planar cell polarity pathway in Drosophila.

Wnt genes signaling through all three pathways function in patterning numerous structures in developing embryos. Such functions include specification of particular areas of the brain and neuroblast identity in both protostomes and deuterostomes and roles in somitogenesis and induction of neural crest in chordates. However, a role of the Wnt/β-catenin pathway in establishing the dorsoventral axis of the frog Xenopus appears to have no counterpart in invertebrate chordates, and may have arisen only during vertebrate evolution.

Acknowledgements

The authors would like to thank ND Holland for comments and advice on the manuscript. In addition, we are indebted to V Laudet and the members of his laboratory for supporting M Schubert while writing this book chapter at the Ecole Normale Supérieure de Lyon. This work was supported by a fellowship within the PostDoc-Program of the German Academic Exchange Service (DAAD) to M Schubert and by NSF grant number IBN00-78599 to LZ Holland and ND Holland.

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