The Wnt Gene Family and the Evolutionary Conservation of Wnt Expression

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

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.

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 4^th 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 vertebrates^13,22,92 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.

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. 3A–C). 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 Xenopus^119,120 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

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-8^113,115,135 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. 3B–G), 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 mesoderm^190,191 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.

References

1.

Christian JL. BMP, Wnt and Hedgehog signals: How far can they go? Curr Opin Cell Biol. 2000;12:244–249. [PubMed: 10819541]

2.

Hamilton FS, Wheeler GN, Hoppler S. Difference in XTCF-3 dependency accounts for change in response to β-catenin-mediated Wnt signaling in Xenopus blastula. Development. 2001;128:2063–2073. [PubMed: 11493528]

3.

Huelsken J, Birchmeier W. New aspects of Wnt signaling pathways in higher vertebrates. Curr Opin Genet Dev. 2001;11:547–553. [PubMed: 11532397]

4.

Kühl M, Geis K, Sheldahl LC. et al. Antagonistic regulation of convergent extension movements in Xenopus by Wnt/β-catenin and Wnt/Ca²⁺ signaling. Mech Dev. 2001;106:61–76. [PubMed: 11472835]

5.

McEwen DG, Peifer M. Wnt signaling: the naked truth? Curr Biol. 2001;11:R524–R526. [PubMed: 11470426]

6.

Niehrs C. Solving a sticky problem. Nature. 2001;413:787–788. [PubMed: 11677588]

7.

Winklbauer R, Medina A, Swain RK. et al. Frizzled-7 signalling controls tissue separation during Xenopus gastrulation. Nature. 2001;413:856–860. [PubMed: 11677610]

8.

Landesman Y, Sokol SY. Xwnt-2b is a novel axis-inducing Xenopus Wnt, which is expressed in embryonic brain. Mech Dev. 1997;63:199–209. [PubMed: 9203142]

9.

Tada M, Smith JC. Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development. 2000;127:2227–2238. [PubMed: 10769246]

10.

Kühl M, Sheldahl LC, Malbon CC. et al. Ca²⁺/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J Biol Chem. 2000;275:12701–12711. [PubMed: 10777564]

11.

Ferkowicz MJ, Stander MC, Raff RA. Phylogenetic relationships and developmental expression of three sea urchin Wnt genes. Mol Biol Evol. 1998;15:809–819. [PubMed: 9656482]

12.

Angerer LM, Angerer RC. Animal-vegetal axis patterning mechanisms in the early sea urchin embryo. Dev Biol. 2000;218:1–12. [PubMed: 10644406]

13.

Ferkowicz MJ, Raff RA. Wnt gene expression in sea urchin development: heterochronies associated with the evolution of developmental mode. Evol Dev. 2001;3:24–33. [PubMed: 11256431]

14.

Zhu X, Mahairas G, Illies M. et al. A large-scale analysis of mRNAs expressed by primary mesenchyme cells of the sea urchin embryo. Development. 2001;128:2615–2627. [PubMed: 11493577]

15.

Sasakura Y, Ogasawara M, Makabe KW. HrWnt-5: a maternally expressed ascidian Wnt gene with posterior localization in the embryos. Int J Dev Biol. 1998;42:573–580. [PubMed: 9694628]

16.

Sasakura Y, Makabe KW. Ascidian Wnt-7 gene is expressed exclusively in the tail neural tube of tailbud embryos. Dev Genes Evol. 2000;210:641–643. [PubMed: 11151302]

17.

Makabe KW, Kawashima T, Kawashima S. et al. Large-scale cDNA analysis of the maternal genetic information in the egg of Halocynthia roretzi for a gene expression catalog of ascidian development. Development. 2001;128:2555–2567. [PubMed: 11493572]

18.

Holland LZ, Holland ND, Schubert M. Developmental expression of AmphiWnt1, an amphioxus gene in the Wnt1/wingless subfamily. Dev Genes Evol. 2000a;210:522–524. [PubMed: 11180802]

19.

Schubert M, Holland LZ, Holland ND. Characterization of two amphioxus Wnt genes (AmphiWnt4 and AmphiWnt7b) with early expression in the developing central nervous system. Dev Dyn. 2000a;217:205–217. [PubMed: 10706144]

20.

Schubert M, Holland LZ, Holland ND. Characterization of an amphioxus Wnt gene, AmphiWnt11, with possible roles in myogenesis and tail outgrowth. Genesis. 2000b;27:1–5. [PubMed: 10862149]

21.

Schubert M, Holland LZ, Panopoulou GD. et al. Characterization of amphioxus AmphiWnt8: insights into the evolution of patterning of the embryonic dorsoventral axis. Evol Dev. 2000c;2:85–92. [PubMed: 11258394]

22.

Schubert M, Holland LZ, Stokes MD. et al. Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: the evolution of somitogenesis in chordates. Dev Biol. 2001;240:262–273. [PubMed: 11784062]

23.

Yasui K, Saiga H, Zhang PJ. et al. Early expressed genes showing a dichotomous developing pattern in the lancelet embryo. Dev Growth Differ. 2001;43:185–194. [PubMed: 11284968]

24.

Llimargas M, Lawrence PA. Seven Wnt homologues in Drosophila: A case study of the developing tracheae. Proc Natl Acad Sci USA. 2001;98:14487–14492. [PMC free article: PMC64708] [PubMed: 11717401]

25.

Kamb A, Weir M, Rudy B. et al. Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification. Proc Natl Acad Sci USA. 1989;86:4372–4376. [PMC free article: PMC287271] [PubMed: 2734290]

26.

Shackleford GM, Shivakumar S, Shiue L. et al. Two Wnt genes in Caenorhabditis elegans. Oncogene. 1993;8:1857–1864. [PubMed: 8510930]

27.

Herman MA, Horvitz HR. The Caenorhabditis elegans gene lin-44 controls the polarity of asymmetric cell divisions. Development. 1994;120:1035–1047. [PubMed: 8026318]

28.

Herman MA, Vassilieva LL, Horvitz HR. et al. The C. elegans gene lin-44, which controls the polarity of certain asymmetric cell divisions, encodes a Wnt protein and acts cell nonautonomously. Cell. 1995;83:101–110. [PubMed: 7553861]

29.

Harris J, Honigberg L, Robinson N. et al. Neuronal cell migration in C. elegans: regulation of Hox gene expression and cell position. Development. 1996;122:3117–3131. [PubMed: 8898225]

30.

Cadigan KM, Nusse R. Wnt signaling: a common theme in animal development. Genes Dev. 1997;11:3286–3305. [PubMed: 9407023]

31.

Han M. Gut reaction to Wnt signaling in worms. Cell. 1997;90:581–584. [PubMed: 9288737]

32.

Rocheleau CE, Downs WD, Lin R. et al. Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos. Cell. 1997;90:707–716. [PubMed: 9288750]

33.

Thorpe CJ, Schlesinger A, Carter JC. et al. Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm. Cell. 1997;90:695–705. [PubMed: 9288749]

34.

Hawkins N, Garriga G. Asymmetric cell division: from A to Z. Genes Dev. 1998;12:3625–3638. [PubMed: 9851969]

35.

Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol. 1998;14:59–88. [PubMed: 9891778]

36.

Whangbo J, Kenyon C. A Wnt signaling system that specifies two patterns of cell migration in C. elegans. Mol Cell. 1999;4:851–858. [PubMed: 10619031]

37.

Whangbo J, Harris J, Kenyon C. Multiple levels of regulation specify the polarity of an asymmetric cell division in C. elegans. Development. 2000;127:4587–4598. [PubMed: 11023862]

38.

Baker NE. Molecular cloning of sequences from wingless, a segment-polarity gene in Drosophila; the spatial distribution of a transcript in embryos. EMBO J. 1987;6:1765–1773. [PMC free article: PMC553553] [PubMed: 16453776]

39.

Baker NE. Localization of transcripts from the wingless gene in whole Drosophila embryos. Development. 1988;103:289–298. [PubMed: 3224555]

40.

Ingham PW, Baker NE, Arias AM. The products of the ftz and eve genes act as positive and negative regulators of engrailed and wingless expression in the Drosophila blastoderm. Nature. 1988;331:73–75. [PubMed: 2893285]

41.

Doe CQ. Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system. Development. 1992;116:855–863. [PubMed: 1295739]

42.

Eisenberg LM, Ingham PW, Brown AMC. Cloning and characterization of a novel Drosophila Wnt gene, DWnt-5, a putative downstream target of the homeobox gene Distal-less. Dev Biol. 1992;154:73–83. [PubMed: 1358729]

43.

Russell J, Gennissen A, Nusse R. Isolation and expression of two novel Wnt/wingless gene homologues in Drosophila. Development. 1992;115:475–485. [PubMed: 1425336]

44.

Chu-LaGraff Q, Doe CQ. Neuroblast specification and formation regulated by wingless in the Drosophila CNS. Science. 1993;261:1594–1597. [PubMed: 8372355]

45.

Nagy LM, Carroll S. Conservation of wingless patterning functions in the short-germ embryos of Tribolium castaneum. Nature. 1994;367:460–463. [PubMed: 8107804]

46.

Fradkin LG, Noordermeer JN, Nusse R. The Drosophila Wnt protein DWnt-3 is a secreted glycoprotein localized on the axon tracts of the embryonic CNS. Dev Biol. 1995;168:202–213. [PubMed: 7883074]

47.

Graba Y, Gieseler K, Aragnol D. et al. Dwnt-4, a novel Drosophila Wnt gene acts downstream of homeotic complex genes in the visceral mesoderm. Development. 1995;121:209–218. [PubMed: 7867502]

48.

Kozopas KM, Samos CH, Nusse R. DWnt-2, a Drosophila Wnt gene required for the development of the male reproductive tract, specifies a sexually dimorphic cell fate. Genes Dev. 1998;12:1155–1165. [PMC free article: PMC316722] [PubMed: 9553045]

49.

Buratovich MA, Anderson S, Gieseler K. et al. DWnt-4 and wingless have distinct activities in the Drosophila dorsal epidermis. Dev Genes Evol. 2000;210:111–119. [PubMed: 11180811]

50.

Janson K, Cohen ED, Wilder EL. Expression of DWnt6, DWnt10, and DFz-4 during Drosophila development. Mech Dev. 2001;103:117–120. [PubMed: 11335117]

51.

Holland PWH, Williams NA, Lanfear J. Cloning of segment polarity gene homologues from the unsegmented brachiopod Terebratulina retusa (Linnaeus). FEBS Lett. 1991;291:211–213. [PubMed: 1682161]

52.

Kostriken R, Weisblat DA. Expression of a Wnt gene in embryonic epithelium of the leech. Dev Biol. 1992;151:225–241. [PubMed: 1577189]

53.

Huang FZ, Bely AE, Weisblat DA. Stochastic WNT signaling between nonequivalent cells regulates adhesion but not fate in the two-cell leech embryo. Curr Biol. 2001;11:1–7. [PubMed: 11166173]

54.

Hobmayer B, Rentzsch F, Kuhn K. et al. WNT signaling molecules act in axis formation in the dipoblastic metazoan Hydra. Nature. 2000;407:186–189. [PubMed: 11001056]

55.

Hoshiyama D, Suga H, Iwabe N. et al. Sponge Pax cDNA related to Pax-2/5/8 and ancient gene duplications in the Pax family. J Mol Evol. 1998;47:640–648. [PubMed: 9847404]

56.

Escriva H, Delaunay F, Laudet V. Ligand binding and nuclear receptor evolution. BioEssays. 2000;22:717–727. [PubMed: 10918302]

57.

Gauchat D, Mazet F, Berney C. et al. Evolution of Antp-class genes and differential expression of Hydra Hox/paraHox genes in anterior patterning. Proc Natl Acad Sci USA. 2000;97:4493–4498. [PMC free article: PMC18262] [PubMed: 10781050]

58.

Kourakis MJ, Martindale MQ. Combined-method phylogenetic analysis of Hox and ParaHox genes of the Metazoa. J Exp Zool. 2000;288:175–191. [PubMed: 10931500]

59.

Blaxter ML, De Ley P, Garey JR. et al. A molecular evolutionary framework for the phylum Nematoda. Nature. 1998;392:71–75. [PubMed: 9510248]

60.

Giribet G, Edgecombe GD, Wheeler WC. Arthropod phylogeny based on eight molecular loci and morphology. Nature. 2001;413:157–161. [PubMed: 11557979]

61.

Hwang UW, Friedrich M, Tautz D. et al. Mitochondrial protein phylogeny joins myriapods with chelicerates. Nature. 2001;413:154–157. [PubMed: 11557978]

62.

Aguinaldo AMA, Turbeville JM, Linford LS. et al. Evidence for a clade of nematodes, arthropods and other moulting animals. Nature. 1997;387:489–493. [PubMed: 9168109]

63.

Schubert M, Holland LZ, Holland ND. et al. A phylogenetic tree of the Wnt genes based on all available full-length sequences, including five from the cephalochordate amphioxus. Mol Biol Evol. 2000d;17:1896–1903. [PubMed: 11110906]

64.

Jockusch EL, Ober KA. Phylogenetic analysis of the Wnt gene family and discovery of an arthropod Wnt-10 orthologue. J Exp Zool. 2000;288:105–119. [PubMed: 10931495]

65.

Holland PWH, Garcia-Fernàndez J, Williams NA. et al. Gene duplications and the origins of vertebrate development. Development. 1994;120S:125–133. [PubMed: 7579513]

66.

Sidow A. Gen(om)e duplications in the evolution of early vertebrates. Curr Opin Genet Dev. 1996;6:715–722. [PubMed: 8994842]

67.

Sidow A. Diversification of the Wnt gene family on the ancestral lineage of vertebrates. Proc Natl Acad Sci USA. 1992;89:5098–5102. [PMC free article: PMC49236] [PubMed: 1534411]

68.

Nusse R. An ancient cluster of Wnt paralogues. TIG. 2001;17:443. [PubMed: 11491115]

69.

Kenny AP, Kozlowski DJ, Oleksyn DW. et al. SpSoxB1, a maternally encoded transcription factor asymmetrically distributed among early sea urchin blastomeres. Development. 1999;126:5473–5483. [PubMed: 10556071]

70.

Ghiglione C, Lhomond G, Lepage T. et al. Cell-autonomous expression and position-dependent repression by Li of two zygotic genes during sea urchin early development. EMBO J. 1993;12:87–96. [PMC free article: PMC413178] [PubMed: 7679074]

71.

Emily-Fenouil F, Ghiglione C, Lhomond G. et al. GSK3β/shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo. Development. 1998;125:2489–2498. [PubMed: 9609832]

72.

Wikramanayake AH, Huang L, Klein WH. β-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo. Proc Natl Acad Sci USA. 1998;95:9343–9348. [PMC free article: PMC21340] [PubMed: 9689082]

73.

Logan CY, Miller JR, Ferkowicz MJ. et al. Nuclear β-catenin is required to specify vegetal cell fates in the sea urchin embryo. Development. 1999;126:345–357. [PubMed: 9847248]

74.

Huang L, Li X, El-Hodiri HM. et al. Involvement of TCF/Lef in establishing cell types along the animal-vegetal axis of sea urchins. Dev Genes Evol. 2000;210:73–81. [PubMed: 10664150]

75.

McClay DR, Peterson RE, Range RC. et al. A micromere induction signal is activated by β-catenin and acts through Notch to initiate specification of secondary mesenchyme cells in the sea urchin embryo. Development. 2000;127:5113–5122. [PubMed: 11060237]

76.

Vonica A, Weng W, Gumbiner BM. et al. TCF is the nuclear effector of the β-catenin signal that patterns the sea urchin animal-vegetal axis. Dev Biol. 2000;217:230–243. [PubMed: 10625549]

77.

Howard EW, Newman LA, Oleksyn DW. et al. SpKrl1: a direct target of β-catenin regulation required for endoderm differentiation in sea urchin embryos. Development. 2001;128:365–375. [PubMed: 11152635]

78.

Sweet HC, Hodor PG, Ettensohn CA. The role of micromere signaling in Notch activation and mesoderm specification during sea urchin embryogenesis. Development. 1999;126:5255–5265. [PubMed: 10556051]

79.

Sherwood DR, McClay DR. Identification and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation. Development. 1997;124:3363–3374. [PubMed: 9310331]

80.

Sherwood DR, McClay DR. LvNotch signaling mediates secondary mesenchyme specification in the sea urchin embryo. Development. 1999;126:1703–1713. [PubMed: 10079232]

81.

Sherwood DR, McClay DR. LvNotch signaling plays a dual role in regulating the position of the ectoderm-endoderm boundary in the sea urchin embryo. Development. 2001;128:2221–2232. [PubMed: 11493542]

82.

Makita R, Mizuno T, Koshida S. et al. Zebrafish wnt11: pattern and regulation of the expression by the yolk cell and No tail activity. Mech Dev. 1998;71:165–176. [PubMed: 9507106]

83.

Yamaguchi TP, Takada S, Yoshikawa Y. et al. T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev. 1999a;13:3185–3190. [PMC free article: PMC317203] [PubMed: 10617567]

84.

Harada Y, Yasuo H, Satoh N. A sea urchin homologue of the chordate Brachyury (T) gene is expressed in the secondary mesenchyme founder cells. Development. 1995;121:2747–2754. [PubMed: 7555703]

85.

Peterson KJ, Harada Y, Cameron RA. et al. Expression pattern of Brachyury and Not in the sea urchin: comparative implications for the origins of mesoderm in basal deuterostomes. Dev Biol. 1999;207:419–431. [PubMed: 10068473]

86.

Gross JM, McClay DR. The role of Brachyury (T) during gastrulation movements in the sea urchin Lytechinus variegatus. Dev Biol. 2001;239:132–147. [PubMed: 11784024]

87.

Tagawa K, Humphreys T, Satoh N. Novel pattern of Brachyury gene expression in hemichordate embryos. Mech Dev. 1998;75:139–143. [PubMed: 9739128]

88.

Shoguchi E, Satoh N, Maruyama YK. Pattern of Brachyury gene expression in starfish embryos resembles that of hemichordate embryos but not of sea urchin embryos. Mech Dev. 1999;82:185–189. [PubMed: 10354483]

89.

Nishida H. Cell fate specification by localized cytoplasmic determinants and cell interactions in ascidian embryos. Int Rev Cytol. 1997;176:245–305. [PubMed: 9394921]

90.

Nishida H. Localization of determinants for formation of the anterior-posterior axis in eggs of the ascidian Halocynthia roretzi. Development. 1994;120:3093–3104.

91.

Conklin EG. The organization and cell-lineage of the ascidian egg J Acad Nat Sci Phila 1905135–119.+ plates I-XI .

92.

Gofflot F, Hall M, Morriss-Kay GM. Genetic patterning of the developing mouse tail at the time of posterior neuropore closure. Dev Dyn. 1997;210:431–445. [PubMed: 9415428]

93.

Imai K, Takada N, Satoh N. et al. β-catenin mediates the specification of endoderm cells in ascidian embryos. Development. 2000;127:3009–3020. [PubMed: 10862739]

94.

Yoshida S, Marikawa Y, Satoh N. Regulation of the trunk-tail patterning in the ascidian embryo: a possible interaction of cascades between lithium/β-catenin and localized maternal factor pem. Dev Biol. 1998;202:264–279. [PubMed: 9769178]

95.

Corbo JC, Fujiwara S, Levine M. et al. Suppressor of hairless activates brachyury expression in the Ciona embryo. Dev Biol. 1998;203:358–368. [PubMed: 9808786]

96.

Yasuo H, Kobayashi M, Shimauchi Y. et al. The ascidian genome contains another T-domain gene that is expressed in differentiating muscle and the tip of the tail of the embryo. Dev Biol. 1996;180:773–779. [PubMed: 8954744]

97.

Hori S, Saitoh T, Matsumoto M. et al. Notch homologue from Halocynthia roretzi is preferentially expressed in the central nervous system during ascidian embryogenesis. Dev Genes Evol. 1997;207:371–380. [PubMed: 27747436]

98.

Coffman CR, Skoglund P, Harris WA. et al. Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell. 1993;73:659–671. [PubMed: 8500162]

99.

Holland LZ, Abi Rached L, Tamme R. et al. Characterization and developmental expression of the amphioxus homolog of Notch (AmphiNotch): evolutionary conservation of multiple expression domains in amphioxus and vertebrates. Dev Biol. 2001;232:493–507. [PubMed: 11401408]

100.

Holland PWH, Koschorz B, Holland LZ. et al. Conservation of Brachyury (T) genes in amphioxus and vertebrates: developmental and evolutionary implications. Development. 1995;121:4283–4291. [PubMed: 8575328]

101.

Holland ND, Panganiban G, Henyey EL. et al. Sequence and developmental expression of AmphiDll, an amphioxus Distal-less gene transcribed in the ectoderm, epidermis and nervous system: insights into the evolution of craniate forebrain and neural crest. Development. 1996;122:2911–2920. [PubMed: 8787764]

102.

Schubert M, Holland ND, Holland LZ. Amphioxus AmphiDRAL encoding a LIM-domain protein: expression in the epidermis but not in the presumptive neuroectoderm. Mech Dev. 1998;76:203–205. [PubMed: 9767167]

103.

Martin B, Schneider R, Starzinski-Powitz A. FHL2/MDRAL interacts with β-catenin and represses TCF/β-catenin dependent transcription. Biol Cell. 2001;93:203.

104.

Tung TC, Wu SC, Tung YYF. Experimental studies on the neural induction in amphioxus. Sci Sin. 1962;11:805–820.

105.

Torres MA, Yang-Snyder JA, Purcell SM. et al. Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development. J Cell Biol. 1996;133:1123–1137. [PMC free article: PMC2120849] [PubMed: 8655584]

106.

Holland LZ, Schubert M, Holland ND. et al. Evolutionary conservation of the presumptive neural plate markers AmphiSox1/2/3 and AmphiNeurogenin in the invertebrate chordate amphioxus. Dev Biol. 2000b;226:18–33. [PubMed: 10993671]

107.

De Robertis EM, Larraín J, Oelgeschläger M. et al. The establishment of Spemann's organizer and patterning of the vertebrate embryo. Nat Rev Genet. 2001;1:171–181. [PMC free article: PMC2291143] [PubMed: 11252746]

108.

Wylie C, Kofron M, Payne C. et al. Maternal β-catenin establishes a ‘dorsal signal’ in early Xenopus embryos. Development. 1996;122:2987–2996. [PubMed: 8898213]

109.

Schneider S, Steinbeisser H, Warga RM. et al. β-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mech Dev. 1996;57:191–198. [PubMed: 8843396]

110.

Larabell CA, Torres M, Rowning BA. et al. Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in β-catenin that are modulated by the Wnt signaling pathway. J Cell Biol. 1997;136:1123–1136. [PMC free article: PMC2132470] [PubMed: 9060476]

111.

Schohl A, Fagotto F. β-catenin, MAPK, and Smad signaling during early Xenopus development. Development. 2002;129:37–52. [PubMed: 11782399]

112.

Ryu S-L, Fujii R, Yamanaka Y. et al. Regulation of dharma/bozozok by the Wnt pathway. Dev Biol. 2001;231:397–409. [PubMed: 11237468]

113.

Kao KR, Elinson RP. The legacy of lithium effects on development. Biol Cell. 1998;90:585–589. [PubMed: 10069003]

114.

Heasman J, Kofron M, Wylie C. β-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev Biol. 2000;222:124–134. [PubMed: 10885751]

115.

Cui Y, Brown JD, Moon RT. et al. Xwnt-8b: a maternally expressed Xenopus Wnt gene with a potential role in establishing the dorsoventral axis. Development. 1995;121:2177–2186. [PubMed: 7635061]

116.

Gradl D, Kühl M, Wedlich D. Keeping a close eye on Wnt-1/wg signaling in Xenopus. Mech Dev. 1999;86:3–15. [PubMed: 10446261]

117.

Moon RT, Kimelman D. From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus. BioEssays. 1998;20:536–545. [PubMed: 9723002]

118.

Sokol SY. Wnt signaling and dorso-ventral axis specification in vertebrates. Curr Opin Genet Dev. 1999;9:405–410. [PubMed: 10449345]

119.

Sakai Y, Hiraoka Y, Konishi M. et al. Isolation and characterization of Xenopus laevis xSox-B1 cDNA. Arch Biochem Biophys. 1997;346:1–6. [PubMed: 9328277]

120.

Mizuseki K, Kishi M, Matsui M. et al. Xenopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinct activities in the initiation of neural induction. Development. 1998;125:579–587. [PubMed: 9435279]

121.

Wessely O, Agius E, Oelgeschläger M. et al. Neural induction in the absence of mesoderm: β-catenin-dependent expression of secreted BMP antagonists at the blastula stage in Xenopus. Dev Biol. 2001;234:161–173. [PMC free article: PMC3039525] [PubMed: 11356027]

122.

Foley AC, Skromne I, Stern CD. Reconciling different models of forebrain induction and patterning: a dual role for the hypoblast. Development. 2000;127:3839–3854. [PubMed: 10934028]

123.

Coffman C, Harris W, Kintner C. Xotch, the Xenopus homolog of Drosophila Notch. Science. 1990;249:1438–1441. [PubMed: 2402639]

124.

Niehrs C. Head in the WNT: The molecular nature of Spemann's head organizer. TIG. 1999;15:314–319. [PubMed: 10431193]

125.

Gamse JT, Sive H. Early anteroposterior division of the presumptive neuroectoderm in Xenopus. Mech Dev. 2001;104:21–36. [PubMed: 11404077]

126.

Kiecker C, Niehrs C. A morphogen gradient of Wnt/β-catenin signalling regulates anteroposterior neural patterning in Xenopus. Development. 2001;128:4189–4201. [PubMed: 11684656]

127.

Villanueva S, Glavic A, Ruiz P. et al. Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction. Dev Biol. 2002;241:289–301. [PubMed: 11784112]

128.

Christian JL, McMahon JA, McMahon AP. et al. Xwnt-8, a Xenopus Wnt-1/int-1-related gene responsive to mesoderm-inducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis. Development. 1991;111:1045–1055. [PubMed: 1879349]

129.

Smith WC, Harland RM. Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center. Cell. 1991;67:753–765. [PubMed: 1657405]

130.

Christian JL, Moon RT. Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus. Genes Dev. 1993;7:13–28. [PubMed: 8422982]

131.

Ku M, Melton DA. Xwnt-11: a maternally expressed Xenopus Wnt gene. Development. 1993;119:1161–1173. [PubMed: 8306880]

132.

Wolda SL, Moody CJ, Moon RT. Overlapping expression of Xwnt-3A and Xwnt-1 in neural tissue of Xenopus laevis embryos. Dev Biol. 1993;155:46–57. [PubMed: 8416844]

133.

Kloc M, Etkin LD. Two distinct pathways for the localization of RNAs at the vegetal cortex in Xenopus oocytes. Development. 1995;121:287–297. [PubMed: 7539356]

134.

Glinka A, Delius H, Blumenstock C. et al. Combinatorial signalling by Xwnt-11 and Xnr3 in the organizer epithelium. Mech Dev. 1996;60:221–231. [PubMed: 9025074]

135.

Fredieu JR, Cui Y, Maier D. et al. Xwnt-8 and lithium can act upon either dorsal mesodermal or neuroectodermal cells to cause a loss of forebrain in Xenopus embryos. Dev Biol. 1997;186:100–114. [PubMed: 9188756]

136.

McGrew LL, Hoppler S, Moon RT. Wnt and FGF pathways cooperatively pattern anteroposterior neural ectoderm in Xenopus. Mech Dev. 1997;69:105–114. [PubMed: 9486534]

137.

Chang C, Hemmati-Brivanlou A. Neural crest induction by Xwnt7b in Xenopus. Dev Biol. 1998;194:129–134. [PubMed: 9473337]

138.

Hoppler S, Moon RT. BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm. Mech Dev. 1998;71:119–129. [PubMed: 9507084]

139.

Yamaguchi Y, Shinagawa A. Marked alteration at midblastula transition in the effect of lithium on formation of the larval body pattern of Xenopus laevis. Dev Growth Differ. 1989;31:531–541.

140.

Kinoshita K, Asashima M. Effect of activin and lithium on isolated Xenopus animal blastomeres and response alteration at the midblastula transition. Development. 1995;121:1581–1589. [PubMed: 7600976]

141.

Kelly GM, Greenstein P, Erezyilmaz DF. et al. Zebrafish wnt8 and wnt8b share a common activity but are involved in distinct developmental pathways. Development. 1995;121:1787–99. [PubMed: 7600994]

142.

Liu P, Wakamiya M, Shea MJ. et al. Requirement for Wnt3 in vertebrate axis formation. Nature Genet. 1999;22:361–365. [PubMed: 10431240]

143.

Lee SMK, Tole S, Grove E. et al. A local Wnt-3a signal is required for development of the mammalian hippocampus. Development. 2000;127:457–467. [PubMed: 10631167]

144.

Gamse J, Sive H. Vertebrate anteroposterior patterning: the Xenopus neuroectoderm as a paradigm. BioEssays. 2000;22:976–986. [PubMed: 11056474]

145.

Pöpperl H, Schmidt C, Wilson V. et al. Misexpression of Cwnt8c in the mouse induces an ectopic embryonic axis and causes a truncation of the anterior neuroectoderm. Development. 1997;124:2997–3005. [PubMed: 9247341]

146.

Erter CE, Wilm TP, Basler N. et al. Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo. Development. 2001;128:3571–3583. [PubMed: 11566861]

147.

Lekven AC, Thorpe CJ, Waxman JS. et al. Zebrafish wnt8 encodes two Wnt8 proteins on a bicistronic transcript and is required for mesoderm and neuroectoderm patterning. Dev Cell. 2001;1:103–114. [PubMed: 11703928]

148.

Schier AF. Axis formation and patterning in zebrafish. Curr Opin Genet Dev. 2001;11:393–404. [PubMed: 11448625]

149.

Yamaguchi TP. Heads or tails: Wnts and anterior-posterior patterning. Curr Biol. 2001;11:R713–R724. [PubMed: 11553348]

150.

Bouwmeester T, Kim SH, Sasai Y. et al. Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature. 1996;382:595–601. [PubMed: 8757128]

151.

Leyns L, Bouwmeester T, Kim SH. et al. Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer. Cell. 1997;88:747–756. [PMC free article: PMC3061830] [PubMed: 9118218]

152.

Wang S, Krinks M, Lin K. et al. Frzb, a secreted protein expressed in the Spemann organizer, binds and inhibits Wnt-8. Cell. 1997;88:757–766. [PubMed: 9118219]

153.

Glinka A, Wu W, Delius H. et al. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature. 1998;391:357–362. [PubMed: 9450748]

154.

Pera EM, De Robertis EM. A direct screen for secreted proteins in Xenopus embryos identifies distinct activities for the Wnt antagonists Crescent and Frzb-1. Mech Dev. 2000;96:183–195. [PubMed: 10960783]

155.

Ikeya M, Takada S. Wnt-3a is required for somite specification along the anteroposterior axis of the mouse embryo and for regulation of cdx-1 expression. Mech Dev. 2001;103:27–33. [PubMed: 11335109]

156.

Yoshikawa Y, Fujimori T, McMahon AP. et al. Evidence that absence of Wnt-3a signaling promotes neuralization instead of paraxial mesoderm development in the mouse. Dev Biol. 1997;183:234–242. [PubMed: 9126297]

157.

Huelsken J, Vogel R, Brinkmann V. et al. Requirement for β-catenin in anterior-posterior axis formation in mice. J Cell Biol. 2000;148:567–578. [PMC free article: PMC2174807] [PubMed: 10662781]

158.

Marvin MJ, Di Rocco G, Gardiner A. et al. Inhibition of Wnt activity induces heart formation from posterior mesoderm. Genes Dev. 2001;15:316–327. [PMC free article: PMC312622] [PubMed: 11159912]

159.

160.

Beck CW, Slack JMW. Analysis of the developing Xenopus tail bud reveals separate phases of gene expression during determination and outgrowth. Mech Dev. 1998;72:41–52. [PubMed: 9533951]

161.

Beck CW, Slack JMW. A developmental pathway controlling outgrowth of the Xenopus tail bud. Development. 1999;126:1611–1620. [PubMed: 10079224]

162.

Itoh K, Sokol SY. Graded amounts of Xenopus dishevelled specify discrete anterioposterior cell fates in prospective ectoderm. Mech Dev. 1997;61:113–125. [PubMed: 9076682]

163.

Takada S, Stark KL, Shea MJ. et al. Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes Dev. 1994;8:174–189. [PubMed: 8299937]

164.

Conlon RA, Reaume AG, Rossant J. Notch1 is required for the coordinate segmentation of somites. Development. 1995;121:1533–1545. [PubMed: 7789282]

165.

Smith JC, Price BMJ, Green JBA. et al. Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell. 1991;67:79–87. [PubMed: 1717160]

166.

Gont LK, Steinbeisser H, Blumberg B. et al. Tail formation as a continuation of gastrulation: the multiple cell populations of the Xenopus tailbud derive from the late blastopore lip. Development. 1993;119:991–1004. [PubMed: 7916680]

167.

Dearden PK, Akam M. Early embryo patterning in the grasshopper, Schistocerca gregaria: wingless, decapentaplegic and caudal expression. Development. 2001;128:3435–3444. [PubMed: 11566850]

168.

Kispert A, Herrmann BG, Leptin M. et al. Homologs of the mouse Brachyury gene are involved in the specification of posterior terminal structures in Drosophila, Tribolium, and Locusta. Genes Dev. 1994;8:2137–2150. [PubMed: 7958884]

169.

Reuter R. The T-related gene (Trg), a Brachyury homologue in insects. Sem Dev Biol. 1995;6:427–435.

170.

Wu LH, Lengyel JA. Role of caudal in hindgut specification and gastrulation suggests homology between Drosophila amnioproctodeal invagination and vertebrate blastopore. Development. 1998;125:2433–2442. [PubMed: 9609826]

171.

Technau U, Bode HR. HyBra1, a Brachyury homologue, acts during head formation in Hydra. Development. 1999;126:999–1010. [PubMed: 9927600]

172.

Technau U. Brachyury, the blastopore and the evolution of the mesoderm. BioEssays. 2001;23:788–794. [PubMed: 11536291]

173.

Gallet A, Erkner A, Charroux B. et al. Trunk-specific modulation of Wingless signalling in Drosophila by Teashirt binding to Armadillo. Curr Biol. 1998;8:893–902. [PubMed: 9707400]

174.

Gallet A, Angelats C, Erkner A. et al. The C-terminal domain of Armadillo binds to hypophosphorylated Teashirt to modulate Wingless signalling in Drosophila. EMBO J. 1999;18:2208–2217. [PMC free article: PMC1171304] [PubMed: 10205174]

175.

Fasano L, Röder L, Coré N. et al. The gene teashirt is required for the development of Drosophila embryonic trunk segments and encodes a protein with widely spaced zinc finger motifs. Cell. 1991;64:63–79. [PubMed: 1846092]

176.

Röder L, Vola C, Kerridge S. The role of the teashirt gene in trunk segmental identity in Drosophila. Development. 1992;115:1017–1033. [PubMed: 1360402]

177.

Caubit X, Coré N, Boned A. et al. Vertebrate orthologues of the Drosophila region-specific patterning gene teashirt. Mech Dev. 2000;91:445–448. [PubMed: 10704881]

178.

Davis RL, Kirschner MW. The fate of cells in the tailbud of Xenopus laevis. Development. 2000;127:255–267. [PubMed: 10603344]

179.

Katsuyama Y, Sato Y, Wada S. et al. Ascidian tail formation requires caudal function. Dev Biol. 1999;213:257–268. [PubMed: 10479446]

180.

Catala M, Teillet MA, Le Douarin NM. Organization of the tail bud analyzed with quail-chick chimaera system. Mech Dev. 1995;51:51–65. [PubMed: 7669693]

181.

Catala M, Teillet MA, De Robertis EM. et al. A spinal cord fate map in the avian embryo: while regressing, Hensen's node lays down the notochord and floor plate thus joining the spinal cord lateral walls. Development. 1996;122:2599–2610. [PubMed: 8787735]

182.

Prinos P, Joseph S, Oh K. et al. Multiple pathways governing Cdx1 expression during murine development. Dev Biol. 2001;239:257–269. [PubMed: 11784033]

183.

Yamaguchi TP, Bradley A, McMahon AP. et al. A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development. 1999b;126:1211–1223. [PubMed: 10021340]

184.

Rauch GJ, Hammerschmidt M, Blader P. et al. Wnt5 is required for tail formation in the zebrafish embryo. Cold Spring Harbor Symp Quant Biol. 1997;62:227–234. [PubMed: 9598355]

185.

Holland PWH, Holland LZ, Williams NA. et al. An amphioxus homeobox gene: sequence conservation, spatial expression during development and insights into vertebrate evolution. Development. 1992;116:653–661. [PubMed: 1363226]

186.

Brooke NM, Garcia-Fernàndez J, Holland PWH. The ParaHox gene cluster is an evolutionary sister of the Hox gene cluster. Nature. 1998;392:920–922. [PubMed: 9582071]

187.

Panopoulou GD, Clark MD, Holland LZ. et al. AmphiBMP2/4, an amphioxus bone morphogenetic protein closely related to Drosophila decapentaplegic and vertebrate BMP2 and BMP4: insights into evolution of dorsoventral axis specification. Dev Dyn. 1998;213:130–139. [PubMed: 9733108]

188.

Shimeld SM. The evolution of the hedgehog gene family in chordates: insights from amphioxus hedgehog. Dev Genes Evol. 1999a;209:40–47. [PubMed: 9914417]

189.

Heisenberg CP, Tada M, Rauch GJ. et al. Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature. 2000;405:76–81. [PubMed: 10811221]

190.

Lawson KA, Meneses JJ, Pedersen RA. Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. Development. 1991;113:891–911. [PubMed: 1821858]

191.

Parameswaran M, Tam PP. Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation. Dev Genet. 1995;17:16–28. [PubMed: 7554492]

192.

Greco TL, Takada S, Newhouse MM. et al. Analysis of the vestigial tail mutation demonstrates that Wnt-3a gene dosage regulates mouse axial development. Genes Dev. 1996;10:313–324. [PubMed: 8595882]

193.

Hsieh JC, Kodjabachian L, Rebbert ML. et al. A new secreted protein that binds to Wnt proteins and inhibits their activities. Nature. 1999;398:431–436. [PubMed: 10201374]

194.

Domingos PM, Itasaki N, Jones CM. et al. The Wnt/β-Catenin pathway posteriorizes neural tissue in Xenopus by an indirect mechanism requiring FGF signalling. Dev Biol. 2001;239:148–160. [PubMed: 11784025]

195.

Spörle R. Epaxial-adaxial-hypaxial regionalisation of the vertebrate somite: evidence for a somitic organiser and a mirror-image duplication. Dev Genes Evol. 2001;211:198–217. [PubMed: 11455436]

196.

Schmidt M, Tanaka M, Münsterberg A. Expression of β-catenin in the developing chick myotome is regulated by myogenic signals. Development. 2000;127:4105–4113. [PubMed: 10976043]

197.

Galceran J, Fari'as I, Depew MJ. et al. Wnt3a^-/-like phenotype and limb deficiency in LEF-1^-/-TCF-1^-/- mice. Genes Dev. 1999;13:709–717. [PMC free article: PMC316557] [PubMed: 10090727]

198.

Linask KK, Ludwig C, Han MD. et al. N-cadherin/catenin-mediated morphoregulation of somite formation. Dev Biol. 1998;202:85–102. [PubMed: 9758705]

199.

Stern HM, Brown AMC, Hauschka SD. Myogenesis in paraxial mesoderm: preferential induction by dorsal neural tube and by cells expressing Wnt-1. Development. 1995;121:3675–3686. [PubMed: 8582280]

200.

Borello U, Buffa V, Sonnino C. et al. Differential expression of the Wnt putative receptors Frizzled during mouse somitogenesis. Mech Dev. 1999;89:173–177. [PubMed: 10559494]

201.

Münsterberg AE, Kitajewski J, Bumcrot DA. et al. Combinatorial signaling by Sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite. Genes Dev. 1995;9:2911–2922. [PubMed: 7498788]

202.

Tajbakhsh S, Borello U, Vivarelli E. et al. Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development. 1998;125:4155–4162. [PubMed: 9753670]

203.

204.

Wolda SL, Moon RT. Cloning and developmental expression in Xenopus laevis of seven additional members of the Wnt family. Oncogene. 1992;7:1941–1947. [PubMed: 1408135]

205.

Christiansen JH, Dennis CL, Wicking CA. et al. Murine Wnt-11 and Wnt-12 have temporally and spatially restricted expression patterns during embryonic development. Mech Dev. 1995;51:341–350. [PubMed: 7547479]

206.

Kozmik Z, Holland LZ, Schubert M. et al. Characterization of amphioxus AmphiVent, an evolutionarily conserved marker for chordate ventral mesoderm. Genesis. 2001;29:172–179. [PubMed: 11309850]

207.

Holland LZ, Kene M, Williams NA. et al. Sequence and embryonic expression of the amphioxus engrailed gene (AmphiEn): the metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development. 1997;124:1723–1732. [PubMed: 9165120]

208.

Aulehla A, Johnson RL. Dynamic expression of lunatic fringe suggests a link between notch signaling and an autonomous cellular oscillator driving somite segmentation. Dev Biol. 1999;207:49–61. [PubMed: 10049564]

209.

Takke C, Campos-Ortega JA. her1, a zebrafish pair-rule like gene, acts downstream of notch signalling to control somite development. Development. 1999;126:3005–3014. [PubMed: 10357943]

210.

Jouve C, Palmeirim I, Henrique D. et al. Notch signalling is required for cyclic expression of the hairy-like gene HES1 in the presomitic mesoderm. Development. 2000;127:1421–1429. [PubMed: 10704388]

211.

Münsterberg AE, Lassar AB. Combinatorial signals from the neural tube, floor plate and notochord induce myogenic bHLH gene expression in the somite. Development. 1995;121:651–660. [PubMed: 7720573]

212.

Yasuo H, Lemaire P. Role of Goosecoid, Xnot and Wnt antagonists in the maintenance of the notochord genetic programme in Xenopus gastrulae. Development. 2001;128:3783–3793. [PubMed: 11585804]

213.

Klingensmith J, Nusse R. Signaling by wingless in Drosophila. Dev Biol. 1994;166:396–414. [PubMed: 7813765]

214.

Patel NH. The evolution of arthropod segmentation: insights from comparisons of gene expression patterns. Development. 1994;120S:201–207.

215.

Shimeld SM. The evolution of dorsoventral pattern formation in the chordate neural tube. Am Zool. 1999b;39:641–649.

216.

Prud'homme B, De Rosa R, Julien JF. et al. Is segmentation ancestral? Looking for conserved segmentation genes in annelids. Abst Ann Meeting SICB. 2002;2002:351.

217.

Wedeen CJ, Weisblat DA. Segmental expression of an engrailed-class gene during early development and neurogenesis in an annelid. Development. 1991;113:805–814. [PubMed: 1687984]

218.

Lans D, Wedeen CJ, Weisblat DA. Cell lineage analysis of the expression of an engrailed homolog in leech embryos. Development. 1993;117:857–871. [PubMed: 8325242]

219.

Bely AE, Wray GA. Evolution of regeneration and fission in annelids: insights from engrailed- and orthodenticle-class gene expression. Development. 2001;128:2781–2791. [PubMed: 11526083]

220.

Seaver EC, Paulson DA, Irvine SQ. et al. Variation in segment formation in polychaetes and its relationship to life history characteristics. Abst Ann Meeting SICB. 2002;2002:380.

221.

Wedeen CJ, Kostriken RG, Leach D. et al. Segmentally iterated expression of an engrailed-class gene in the embryo of an australian onychophoran. Dev Genes Evol. 1997;270:282–286. [PubMed: 27747425]

222.

Budd GE. Why are arthropods segmented? Evol Dev. 2001;3:332–342. [PubMed: 11710765]

223.

Lawrence PA, Johnston P. On the role of the engrailed+ gene in the internal organs of Drosophila. EMBO J. 1984;3:2839–2844. [PMC free article: PMC557774] [PubMed: 6441704]

224.

Baylies MK, Martinez Arias A, Bate M. wingless is required for the formation of a subset of muscle founder cells during Drosophila embryogenesis. Development. 1995;121:3829–3837. [PubMed: 8582292]

225.

Frasch M, Nguyen HT. Genetic control of mesoderm patterning and differentiation during Drosophila embryogenesis. Adv Dev Biochem. 1999;5:1–47.

226.

Riechmann V, Irion U, Wilson R. et al. Control of cell fates and segmentation in the Drosophila mesoderm. Development. 1997;124:2915–2922. [PubMed: 9247334]

227.

Bilder D, Scott MP. Hedgehog and wingless induce metameric pattern in the Drosophila visceral mesoderm. Dev Biol. 1998;201:43–56. [PubMed: 9733572]

228.

Lawrence PA, Johnston P, Vincent JP. Wingless can bring about a mesoderm-to-ectoderm induction in Drosophila embryos. Development. 1994;120:3355–3359. [PubMed: 7821207]

229.

Sasakura Y, Makabe KW. Ascidian Wnt-5 gene is involved in the morphogenetic movement of notochord cells. Dev Growth Differ. 2001;43:573–582. [PubMed: 11576174]

230.

Locascio A, Nieto MA. Cell movements during vertebrate development: integrated tissue behaviour versus individual cell migration. Curr Opin Genet Dev. 2001;11:464–469. [PubMed: 11448634]

231.

Moon RT, Campbell RM, Christian JL. et al. Xwnt-5a: A maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis. Development. 1993;119:97–111. [PubMed: 8275867]

232.

Sokol SY. Analysis of Dishevelled signalling pathways during Xenopus development. Curr Biol. 1996;6:1456–1467. [PubMed: 8939601]

233.

Wallingford JB, Rowning BA, Vogeli KM. et al. Dishevelled controls cell polarity during Xenopus gastrulation. Nature. 2000;405:81–85. [PubMed: 10811222]

234.

Wallingford JB, Harland RM. Xenopus Dishevelled signaling regulates both neural and mesodermal convergent extension: parallel forces elongating the body axis. Development. 2001;128:2581–2592. [PubMed: 11493574]

235.

Djiane A, Riou J, Umbhauer M. et al. Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis. Development. 2000;127:3091–3100. [PubMed: 10862746]

236.

Wallingford JB, Vogeli KM, Harland RM. Regulation of convergent extension in Xenopus by Wnt5a and Frizzled-8 is independent of the canonical Wnt pathway. Int J Dev Biol. 2001;45:225–227. [PubMed: 11291850]

237.

Strapps WR, Tomlinson A. Transducing properties of Drosophila Frizzled proteins. Development. 2001;128:4829–4835. [PubMed: 11731462]

238.

Axelrod JD, Miller JR, Shulman JM. et al. Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev. 1998;12:2610–2622. [PMC free article: PMC317102] [PubMed: 9716412]

239.

Boutros M, Mihaly J, Bouwmeester T. et al. Signaling specificity by Frizzled receptors in Drosophila. Science. 2000;288:1825–1828. [PubMed: 10846164]

240.

Joyner AL, Hanks M. The engrailed genes: Evolution of function. Sem Dev Biol. 1991;2:435–445.

241.

McGrew LL, Otte AP, Moon RT. Analysis of Xwnt-4 in embryos of Xenopus laevis: a Wnt family member expressed in the brain and floor plate. Development. 1992;115:463–473. [PubMed: 1425335]

242.

Liu A, Joyner AL. Early anterior/posterior patterning of the midbrain and cerebellum. Annu Rev Neurosci. 2001;24:869–896. [PubMed: 11520921]

243.

McMahon AP, Bradley A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell. 1990;62:1073–1085. [PubMed: 2205396]

244.

Thomas KR, Capecchi MR. Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature. 1990;346:847–850. [PubMed: 2202907]

245.

McMahon AP, Joyner AL, Bradley A. et al. The midbrain-hindbrain phenotype of Wnt-1^-/Wnt-1^- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell. 1992;69:581–595. [PubMed: 1534034]

246.

Danielian PS, McMahon AP. Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate midbrain development. Nature. 1996;383:332–334. [PubMed: 8848044]

247.

McGrew LL, Takemaru KI, Bates R. et al. Direct regulation of the Xenopus engrailed-2 promoter by the Wnt signaling pathway, and a molecular screen for Wnt-responsive genes, confirm a role for Wnt signaling during neural patterning in Xenopus. Mech Dev. 1999;87:21–32. [PubMed: 10495268]

248.

Holland LZ, Holland ND. Chordate origins of the vertebrate central nervous system. Curr Opin Neurobiol. 1999;9:596–602. [PubMed: 10508734]

249.

Kozmik Z, Holland ND, Kalousova A. et al. Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region. Development. 1999;126:1295–1304. [PubMed: 10021347]

250.

Wada H, Saiga H, Satoh N. et al. Tripartite organization of the ancestral chordate brain and the antiquity of placodes: insights from ascidian Pax-2/5/8, Hox and Otx genes. Development. 1998;125:1113–1122. [PubMed: 9463358]

251.

LaBonne C, Bronner-Fraser M. Neural crest induction in Xenopus: evidence for a two-signal model. Development. 1998;125:2403–2414. [PubMed: 9609823]

252.

Ikeya M, Lee SMK, Johnson JE. et al. Wnt signalling required for expansion of neural crest and CNS progenitors. Nature. 1997;389:966–970. [PubMed: 9353119]

253.

Deardorff MA, Tan C, Saint-Jeannet JP. et al. A role for frizzled 3 in neural crest development. Development. 2001;128:3655–3663. [PubMed: 11585792]

254.

Hall AC, Lucas FR, Salinas PC. Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling. Cell. 2000;100:525–535. [PubMed: 10721990]

255.

Richter S, Hartmann B, Reichert H. The wingless gene is required for embryonic brain development in Drosophila. Dev Genes Evol. 1998;208:37–45. [PubMed: 9518523]

256.

Bhat KM, van Beers EH, Bhat P. Sloppy paired acts as the downstream target of Wingless in the Drosophila CNS and interaction between sloppy paired and gooseberry inhibits sloppy paired during neurogenesis. Development. 2000;127:655–665. [PubMed: 10631185]

257.

Song Y, Chung S, Kunes S. Combgap relays wingless signal reception to the determination of cortical cell fate in the Drosophila visual system. Mol Cell. 2000;6:1143–1154. [PubMed: 11106753]

258.

Deshpande N, Dittrich R, Technau GM. et al. Successive specification of Drosophila neuroblasts NB 6-4 and NB 7-3 depends on interaction of the segment polarity genes wingless, gooseberry and naked cuticle. Development. 2001;128:3253–3261. [PubMed: 11546742]

259.

Kaphingst K, Kunes S. Pattern formation in the visual centers of the Drosophila brain: wingless acts via decapentaplegic to specify the dorsoventral axis. Cell. 1994;78:437–448. [PubMed: 8062386]

260.

Holland LZ. Body-plan evolution in the Bilateria: early antero-posterior patterning and the deuterostome-protostome dichotomy. Curr Opin Genet Dev. 2000;10:434–442. [PubMed: 10889057]

261.

Holland LZ. Heads or tails? Amphioxus and the evolution of anterior-posterior patterning in deuterostomes. Dev Biol. 2002;241:209–228. [PubMed: 11784106]