The prevailing model for anteroposterior (AP) axis formation during vertebrate embryo genesis implies distinct organizer regions inducing head and trunk structures, respectively. A gradient of posteriorizing activity (transformer) which regulates AP patterning of the neuraxis has been suggested on the other hand by classical studies using amphibian embryos. Here, we will review the roles of Wnt signaling during head formation in various vertebrate model organisms. Early in vertebrate embryogenesis, organizer (and thus head) formation depends on a Wnt-type b-catenin-mediated signal. During gastrulation, posteriorizing Wnt/b-catenin signaling antagonizes the head organizer whose distinguishing feature is the secretion of Wnt inhibitors. The interplay between head organizer-derived Wnt inhibitors and posteriorizing Wnts establishes a gradient of Wnt/b-catenin activity that regulates AP patterning of the vertebrate gastrula. Thus, the concepts of head and trunk organizer and of the transformer gradient are reconciled in a dualistic view of AP axis formation.

Introduction

In vertebrates, the two main body axes, anteroposterior (AP) and dorsoventral (DV), are progressively established during early embryogenesis. The anterior extremity of a vertebrate is defined by its head, a complex structure formed from derivatives of all germ layers. The vertebrate head is characterized by an elaborate segmented brain, sensory organs and structural components such as skull bones, teeth and facial muscles. These features distinguish vertebrates even from their closest nonvertebrate relatives, the cephalochordates.¹ Here, we will review the different and sometimes opposing roles played by growth factors of the Wnt family during head formation with special emphasis on the emerging notion that inhibition of Wnt/ β-catenin signaling is crucially required for anterior specification. As we will discuss below, the seemingly different organizer and gradient models for AP axis formation are reconciled in light of the emerging underlying molecular mechanism.

The Vertebrate Organizer

Fundamental progress in understanding embryonic axis formation was made in the 1920s when the embryologists Hilde Mangold and Hans Spemann demonstrated the extraordinary inductive potency of the amphibian dorsal blastopore lip by performing transplantation experiments on newt embryos. They explanted the comparably small dorsal lip region from a donor gastrula and grafted it to the ventral side of a host. This treatment induced a complete secondary embryonic axis, including a well-patterned head, within the host embryo. Only few structures of the secondary axis were derived from the transplanted tissue while most of the ectopic tissues were derived from the host. Mangold and Spemann concluded that the transplanted cells had induced and organized the surrounding host tissue nonautonomously to become part of the secondary axis.²

Tissues corresponding to Spemann's organizer have since been identified in chick, fish, rabbit and mouse.³⁷ The organizer regions of all these vertebrates show comparable tissue fates and are able to induce twinning when transplanted to ectopic locations in host embryos.^35,710 Thus, the vertebrate organizer appears to be an evolutionary conserved structure with a primary function in embryonic axis formation.

Classical Models for Anteroposterior Axis Formation

At least three different tissues derived from the organizer are distinguishable at neurula stages (from anterior to posterior): (1) the anterior endoderm (AE) which will give rise to the liver at later stages, (2) the prechordal mesendoderm (PME) which will differentiate into head mesenchyme, head and eye muscles and foregut and (3) the chordamesoderm (CM) which will give rise to the notochord. Together, these organizer derivatives are referred to as axial mesendoderm.

In the 1930s, Spemann's colleague Otto Mangold dissected the archenteron roof (containing the axial mesendoderm) of early newt neurulae into four transversal segments and implanted these pieces into the blastocoel of early gastrulae. As in the Mangold-Spemann experiment, the grafts were able to induce ectopic structures in the host embryos. Their inductive potential, however, seemed to be more restricted compared with early organizer grafts: The anteriormost grafts (containing AE) only induced ectopic balancers and parts of the oral apparatus while grafts containing PME and anterior CM frequently induced ectopic head structures such as balancers, eyes, otic vesicles, fore- and midbrain; grafts containing medial CM induced ectopic trunks including hindbrain and spinal cord tissue and the posteriormost type of grafts (containing posterior CM) induced ectopic tails (Fig. 1A). Mangold concluded that derivatives of the organizer differ not only in fate but also in inducing potential, with anterior organizer inducing head structures and posterior organizer inducing trunk and tail structures.¹¹ Similarily, Einsteck assays using early gastrula lips resulted in the formation of complete secondary axes including heads while late gastrula lips only induced secondary trunks (Fig. 1B). Taken together, these results suggested that the organizer consists of distinct head- and trunkorganizing regions and that the region between PME and anterior CM harbors the strongest head-inducing potential.

Figure 1

Regionally specific induction by Spemann's organizer. (A) Mangold's experiment demonstrating regionally specific induction by axial mesendoderm. Transversal segments of the archenteron roof of newt neurulae (left) were implanted into the blastocoel of (more...)

An alternative model for AP axis formation was introduced in the 1950s by the “Dutch school” of embryologists represented by Pieter Nieuwkoop and colleagues.¹² Nieuwkoop implanted folds of competent ectoderm into prospective neural plates of amphibian embryos at different AP levels. Analysis of the grafts revealed that folds implanted at the forebrain level had differentiated exclusively into anterior neural structures (such as nasal pits, pineal gland, eyes and forebrain) while folds implanted at the hindbrain level had differentiated into hindbrain at their base and into forebrain distally. Folds implanted at the spinal cord level had differentiated into spinal cord at their base, into hindbrain medially and into forebrain distally (Fig. 2A). Comparable results were obtained using flattened explants of ectoderm juxtaposed with fragments of CM.¹³ Based on these observations Nieuwkoop proposed the sequential activity of two signals (Fig. 2B): First, a signal from the dorsal mesoderm (organizer) induces the overlying ectoderm to become neuroectoderm of anterior character (activation step). Second, a signal which is absent from the anterior of the embryo and becomes increasingly stronger posteriorly confers progressively posterior identity to the corresponding part of the neural plate (transformation step).

Figure 2

Classical models for AP patterning. In all schemes, anterior points to the left, dorsal to the top. (A) Sagittal section of an early neurula newt embryo with ectodermal folds implanted at three different AP levels of the neural plate (1°,2°,3°). (more...)

Similarly, a model based on the activity of two opposing activities was devised by the “Finnish school” of embryologists. According to this model, AP axial patterning is regulated by the concentration ratio between an archencephalic (forebrain-type) and a spinocaudal (posteriorizing/mesodermalizing) inducer (Fig. 2C).^14,15 It is of note that both Nieuwkoops two-step and the two-inducer model predict gradients of posteriorizing factors. As we will discuss below, organizer and gradient models merely reflect different perspectives of the same phenomenon.

Wnt/β-catenin Signaling Antagonizes the Vertebrate Head Organizer

Gain- and loss-of-function approaches in Xenopus and zebrafish have revealed that Wnt/ β-catenin-type signaling is necessary and sufficient for organizer induction and thus, indirectly, for head formation during an early phase of embryogenesis (reviewed in chapter 4; refs. 16,17). The competence of Xenopus and zebrafish embryos to respond to Wnt signals changes dramatically after the midblastula transition (MBT) when zygotic transcription starts. Post-MBT Wnt/ β-catenin signaling does not have organizer-inducing properties anymore but it antagonizes the head organizer and confers posteriorizing activity: Overexpression of various Wnts, the Wnt transducers β-catenin or XTCF-3, or treatment with the artificial Wnt pathway activator Li+ after MBT lead to repression of anterior and concomittant induction of posterior neural markers and suppression of head development (Fig. 3).¹⁸³² Wnt/β-catenin signaling is able to counteract Xenopus head organizer activity at least until the end of gastrulation as revealed by experiments with embryos carrying an inducible XWnt-8 transgene.³³ A posteriorizing role for Wnts is also suggested by studies on transgenic mice ectopically expressing chick Wnt-8C and by Li treatment of gastrulating chick embryos.^34,35

Figure 3

Wnts posteriorize Xenopus embryos. Activation of the Wnt/β-catenin pathway (+Wnt) leads to headless tadpoles (lower panel); its inhibition (-Wnt) results in enlargement of anterior and concomittant reduction of trunk structures (upper panel). (more...)

Wnt/β-catenin signaling not only suppresses anterior but is also required for posterior development: Overexpression of Wnt antagonists in Xenopus and zebrafish, respectively, results in the formation of embryos with enlarged head structures and shortened trunks (Fig. 3).³⁶⁴⁶ Similarly, both overexpression of GSK3β in Xenopus anterior ectoderm and depletion of β-catenin from this region using morpholino antisense oligos elicits neural anteriorization.^47,48 Mice mutant for Wnt-3A, Wnt-5A, LEF-1 and TCF-1 -encoding nuclear transducers of Wnt signaling-, and LRP6 -encoding a Wnt coreceptor- show posterior truncations, providing genetic evidence for the requirement for Wnts in posteriorization.⁴⁹⁵² Pronounced trunk defects are also observed in zebrafish lacking Wnt-8 function.⁵³ Interestingly, allelic combinations of mouse Wnt-3A^- and Vestigial tail, a hypomorphic mutation of Wnt-3A, display dose-dependent posterior truncations suggesting a requirement for increasing levels of Wnt signaling to specify increasingly more posterior fates.⁵⁴ Likewise, posterior neural fates are progressively deleted in zebrafish by increasing doses of morpholino oligos.⁵⁵

Thus, Wnts elicit phenotypically opposing effects with early signaling promoting and late signaling counteracting head formation. This complicates the phenotypic interpretation of global gain- and loss-of-function experiments and advocates the importance of manipulating gene expression in defined time windows. The change of cellular competence to respond to Wnt signals (from organizer induction to head antagonism) does not seem to be caused by the employment of a different (noncanonical) Wnt pathway but by a change of nuclear cofactors. ^28,31

Tissues with Posteriorizing Activity Express Wnts

Nieuwkoop and colleagues have described graded transforming activity residing within posterior axial mesoderm (CM) and this has been confirmed by tissue recombination experiments in Xenopus, mouse and chick.⁵⁶⁵⁹ However, studies in Xenopus, chick and zebrafish have also revealed a posteriorizing function of nonaxial tissues.⁶⁰⁶⁴ All tissues implicated in posteriorization express at least one member of the Wnt family during gastrulation (Table 1).

Table 1

Wnt expression in vertebrate gastrulae.

The Two Inhibitor Model for Head Induction

How does the notion of separate head and trunk organizers relate to the posteriorizing activity of post-MBT Wnt signaling? The vertebrate organizer expresses a large number of secreted growth factors and growth factor antagonists which continue to be expressed in the axial mesendoderm in different patterns. It is well established that inhibitors of bone morphogenetic proteins (BMPs—a TGFβ subfamily) which are expressed all along the AP axis (Table 2) mediate the dorsalizing and neural inducing activity of Spemann's organizer in amphibians (reviewed in ref. 65). Ectopic expression of BMP antagonists on the ventral side of Xenopus embryos induces secondary trunks lacking anterior neural structures up to the hindbrain suggesting that trunk organizer function is mediated by BMP inhibition (Fig. 4A). A distinguishing feature of the anterior derivatives of the organizer (AE and PME) is the expression of Wnt inhibitors (Table 2; reviewed in refs. 66,67). Coexpression of BMP and Wnt antagonists induces complete secondary axes including heads with anterior neural structures (Fig. 4B).^38,42 Based on these observations, the “two inhibitor model” for head induction has been proposed: The head organizer functions by simultaneously antagonizing BMP and Wnt signaling while the trunk organizer only antagonizes BMP signaling (Fig. 4C).^38,66

Table 2

Regional-specific expression of Secreted Organizer Effectors in Xenopus.

Figure 4

The two inhibitor model for head induction. (A) Ectopic BMP antagonism results in trunk duplication, (B) a combination of BMP and Wnt antagonism induces heads. (C) Diagram representing the two inhibitor model.

In amphibians, heterotopic grafting studies have located the strongest head-inducing potential to PME and anterior CM and ablation studies have proven a requirement for this part of the organizer in head formation.^11,68 Grafts of corresponding regions in chick and zebrafish also result in anterior neural induction, emphasizing the instructive role of anterior axial mesendoderm, a major source of Wnt inhibitors, in head development (Fig. 5).^9,69

Figure 5

Comparative scheme of vertebrate gastrulae. Simplified schemes of midsagittal sections through (A) Xenopus, (B) zebrafish, (C) chick and (D) mouse gastrulae. Axial mesendodermal tissues are shown in dark grey; expression of Wnt inhibitors is indicated (more...)

Supporting the two inhibitor model, inhibition of Wnt signaling is necessary in vivo for formation of anterior neural structures in Xenopus, zebrafish and mouse: Inhibition of the secreted Wnt antagonist Dickkopf1 (Dkk-1) in Xenopus using neutralizing antibodies as well as genetic inactivation of the Wnt antagonists TCF-3 and axin1 in the zebrafish headless and masterblind mutants, respectively, all result in microcephalic embryos.^27,42,70,71 A targeted knockout of the Dkk-1 gene in mouse leads to embryos completely lacking rostral head structures.⁷²

Interestingly, a very recent study has revealed that instructive signaling by insulin-like growth factors (IGFs) is necessary and sufficient for anterior neural induction in Xenopus.⁷³ In line with the two inhibitor model, IGF signaling has been demonstrated to counteract the Wnt/β-catenin pathway. Epistatically, this inhibition seems to occur between GSK3β and β-catenin and one of the next important steps will certainly be to analyze the underlying biochemical mechanism. Furthermore, it remains to be investigated whether Wnt inhibition is the major effect of IGF signaling in head induction or whether IGFs perform other independent functions beyond Wnt inhibition—a likely possibility given that, in contrast to known Wnt inhibitors, IGFs are able to ectopically induce anterior structures such as eyes and cement glands.

Similar to the head, the vertebrate heart is derived from anterior regions of the gastrulating embryo. Although the definitive heart ends up in a ventral position, its precursor -the cardiogenic mesoderm- is located adjacent to the PME, in a region with low levels of Wnt/β- catenin signaling. Three recent studies using chick and Xenopus embryos have highlighted a central role of Wnt inhibition in heart specification, lending further support to the two inhibitor model (reviewed in chapter 11).⁷⁴⁷⁶ Notably, ectopic expression of Wnt-8C in transgenic mouse embryos results in reduced heart and foregut structures suggesting that low levels of Wnt signaling are required for anterior specification of all germ layers.³⁴

Anterior Organizing Centers Express Wnt Inhibitors

As discussed above, anterior axial mesendoderm functions as head organizer in amphibians, zebrafish and chick (Fig. 5A–C).^9,11,69 In the mouse, however, grafts of both late and early gastrula organizer regions only induce secondary embryonic axes lacking anterior neural structures. ^7,77 The murine anterior visceral endoderm (AVE), an extraembryonic structure derived from the distal tip of the early embryo, shifts anteriorly before the onset of gastrulation until it underlies the prospective anterior neural plate (Fig. 5D).⁷⁸⁸⁰ Remarkably, the AVE has been found to express many organizer genes involved in anterior specification and its surgical ablation results in anterior defects.^78,79 Analyses of chimeric mice in which the function of AVEexpressed genes is specifically disrupted in extraembryonic tissues has supported the notion that the AVE is essential for head formation (for reviews see refs. 79,81). Yet, AVE alone is not able to ectopically induce anterior neural markers when grafted heterotopically.⁷⁷ Recombination of ectodermal explants with AVE does not result in anterior neural induction although posterior markers become suppressed, suggesting a rather permissive role for AVE in anterior development.⁸² It is likely that this suppression of posterior fates is mediated by Wnt antagonism and, in line with this hypothesis, the AVE secretes the Wnt antagonists Cerberus-like (Cer-l) and Dkk-1.⁸³⁸⁶ As mentioned above, Dkk-1^-/- mice are headless but the loss of Cer-l function does not elicit head defects.^72,8791 Thus, if Wnt inhibition is an essential function of AVE it may be mediated redundantly.

An AVE-like role has been suggested for the anterior hypoblast of the chick embryo which is also a source of several Wnt inhibitors (Fig. 5C). This tissue is not able to stably induce forebrain identity in naïve chick epiblast, but it transiently induces early neural markers and directs epiblast movements.^92,93 A model has been proposed in which the role of the anterior hypoblast (and the murine AVE) is to protect presumptive anterior neuroectoderm from posteriorizing activities.^82,93 Yet, despite emerging similarities between the mammalian AVE and the chick anterior hypoblast (gene expression, pregastrulation movements), there are fundamental differences between these tissues with regard to their inducing abilities as revealed by heterospecies transplantations: Rabbit anterior hypoblast is able to stably induce anterior neural fates upon transplantation under chick epiblast, compared to only transient induction of some pre-neural markers by chick anterior hypoblast.^92,93 The molecular basis for these differences remains elusive.

If the role of the AVE in head induction is permissive, which structures do actually provide instructive head-inducing signals in the mouse embryo? The earlier observation that mouse organizer grafts fail to induce complete embryonic axes including anterior neural structures has been challenged recently: Transplantation of mouse or rabbit organizers into chick host embryos results in formation of complete secondary axes including forebrain structures, indicating that, in principle, the mammalian organizer harbors the capacity for anterior neural induction. ⁹⁴ Very recently, Tam and colleagues have described induction of complete secondary axes in mouse embryos by mid-gastrula stage organizers.¹⁰ Thus, the early gastrula organizer (EGO; see ref. 77) may not yet have acquired full head-inducing potential while the late gastrula organizer (see ref. 7) may have lost its head-inducing potential already. A similar situation is encountered in chick: Neither the anterior primitive streak of the early chick gastrula nor the node after the emigration of the head process are able to induce complete secondary embryonic axes.^95,96 Finally, it has been demonstrated by ablation that anterior axial mesendoderm is not only sufficient but also required for forebrain development in mice.⁹⁷

Ablation of a single row of cells from the ectoderm that borders the neural plate anteriorly in the zebrafish gastrula (row-1 cells) results in head defects (Fig. 5B), indicating the presence of another early anterior-inducing center.⁹⁸ Notably, a Wnt inhibitor of the sFRP (secreted Frizzled-related protein) class has been identified as mediator of row-1 function (see Note Added in Proof). Taken together, all of the tissues implicated in anterior neural induction in various species are sources of secreted Wnt inhibitors.

A Transforming Gradient of Wnt/β-catenin Activity Regulates AP Neural Patterning

While Spemann and coworkers suggested distinct head- and trunk-inducing regions within the embryo—and this view is in agreement with the two-inhibitor model for vertebrate head induction—the AP axis of the embryo is patterned by a posteriorizing gradient of a transformer according to the activation-transformation model proposed by Nieuwkoop and colleagues. Clearly, the two-inhibitor model—in its simple form—is not sufficient to explain regional AP patterning of the neuraxis like Nieuwkoop's model. To reconcile the two-inhibitor and the activation-transformation model we have investigated recently whether Wnts may constitute the transforming signal.³² We found that in Xenopus (1) Wnts posteriorize neuroectoderm dose-dependently, (2) Wnt/β-catenin signaling is required for AP ectodermal patterning during gastrulation and (3) Wnts are able to signal directly and over a distance within neuroectoderm, conferring polar AP neural pattern to neuralized ectodermal explants and in vivo. Importantly, an endogenous AP gradient of Wnt/β-catenin signaling was detected in the presumptive neural plate of the Xenopus gastrula. These data support a model in which a posteriorizing activity gradient of Wnt/β-catenin signaling is established during gastrulation by the interplay of head organizer-derived Wnt inhibitors and posteriorly expressed Wnts (Fig. 6). Furthermore, they suggest that Wnts may act as morphogens in vertebrates. However, a recent study describes nonautonomous induction of posterior neural markers following overexpression of β-catenin in Xenopus ectodermal explants.²⁹ Differences in the experimental setup may explain this apparent discrepancy. In particular, we have used an earlier readout in our experimental approach.

Figure 6

Simplified model for AP patterning of neuroectoderm by a Wnt activity gradient. Wnts (black) and Wnt inhibitors are expressed in the axial mesendoderm underlying the neuroectoderm. The expression of Wnts and Wnt inhibitors in the neuroectoderm is not (more...)

In vivo, the expression domains of regional neural marker genes change following modulation of Wnt signaling: Forced activation of the Wnt/β-catenin pathway after the MBT results in a loss of anterior neural fates while, conversely, an expansion of anterior at the expense of posterior neural markers is observed upon overexpression of Wnt antagonists. Yet, the expression domains of these markers always respect certain boundaries and do never expand throughout the entire neural plate.³² This suggests that the competence of neural cells to respond to Wnts is regionally restricted. An organizer-independent ectodermal AP prepattern which depends on differential competence of the epiblast has been described in zebrafish.⁹⁹ Besides Wnts, other signaling molecules such as fibroblast growth factors (FGFs) and retinoic acid (RA) have been implicated in posteriorization which may regulate cellular competence (see refs. 100–102 for reviews). FGFs are potent posteriorizing agents (e. g. see ref. 103), yet, Wnt antagonists rescue FGF-induced posteriorization in Xenopus, suggesting that this effect is mediated —at least in part—indirectly through Wnts.^23,27 Interestingly, a recent study in chick suggests that FGFs are required during gastrulation to maintain a population of neural progenitors within Hensen's node.¹⁰⁴ The authors propose a model in which FGFs are not a transforming agent but define a niche for neural stem cells in the posteriorly regressing organizer where cells remain exposed to the true transformer (discussed in ref. 105). Obviously, Wnts are good candidates for this signal. Taken together, AP neural patterning is regulated by Wnt/β-catenin signaling while RA and FGFs may also contribute to this process, maybe by regulating the competence of neural cells to respond to posteriorizing Wnt signals.

Wnt Signaling and Gastrulation Movements

Zebrafish silberblick mutants display mild cyclopia and other head midline defects.¹⁰⁶ Interestingly, a mutation in the Wnt-11 locus has been identified as the cause of this phenotype.¹⁰⁷ Yet, the silberblick head defects are unlikely to result from a failure in head induction or AP patterning: Several recent studies in Xenopus and zebrafish have provided strong evidence for a noncanonical, β-catenin-independent Wnt pathway (involving Wnt-11 and Dishevelled) regulating the convergent-and-extension movements characteristic of vertebrate gastrulation (see Chapter 1).¹⁰⁷¹¹⁴ Impaired elongation of axial mesendoderm which is a source of midline patterning signals is the most probable explanation for the silberblick phenotype. Overexpression of the Wnt inhibitor Crescent/Frzb2 (Crs) in Xenopus elicits a comparable phenotype, suggesting that Crs inhibits Wnt-11-like signals.^115,116 Remarkably, the neural plate is anteriorized in Crs-expressing embryos.¹¹⁶ Thus, Crs may inhibit a spectrum of different Wnts implicated in canonical and noncanonical signaling. In conclusion, head defects may arise due to inhibition of noncanonical Wnt signaling.

Later Roles of Wnts

At later stages of vertebrate development, Wnts and Wnt inhibitors are expressed in complex patterns and perform various roles depending on their timing and location of expression. Almost all Wnts and many Wnt modulators are differentially expressed in the embryonic cen- tral nervous system.^50,117119 Not much is known concerning the precise functions of Wnt signaling at these stages. They have been implicated in pattern refinement and subregionalisation, as retrograde signals in axon remodeling and synaptic differentiation and in modulating apoptosis (refs. 120,121). Wnt-1 which is expressed in the mesencephalon is required for maintaining the midbrain-hindbrain boundary (isthmic) organizer and for the development of the cerebellum, an isthmus-derived structure (reviewed in refs. 122,123). A local Wnt-3A signal from the developing cerebral cortex promotes cell proliferation during hippocampus formation.^124,125

Gain- and loss-of-function analyses in all vertebrate model organisms have revealed that Wnt signals are crucial for formation and specification of the neural crest, a specialized cell population that is induced at the lateral edges of the neural plate and becomes localized to the dorsalmost region of the neural tube.^24,25,126129 Neural crest cells undergo an epithelial-mesenchymal transition, migrate to many different targets within the developing embryo and give rise to a variety of different cell types (reviewed in ref. 130). The cranial neural crest populates the branchial arches, gives rise to cranial ganglia and craniofacial cartilage and bone and is therefore of central importance for later events in head formation.¹³⁰¹³³ Many Wnts are predominantly expressed in dorsal regions of the neural tube (e. g. in Xenopus: XWnt-1—midbrain, dorsal neural tube; XWnt-2B—dorsal forebrain/midbrain boundary; XWnt-3A—dorsal neural tube; XWnt-7B—dorsal neural tube; XWnt-8B—dorsal di- and mesencephalon, forebrain/midbrain boundary).^25,134136 Mouse Wnt-1^-/-;Wnt-3A^-/- double mutants display, besides many other defects, a dramatic reduction of neural crest and severe craniofacial abnormalities. ¹²⁶ Interestingly, Wnt-5A^-/- mutant mice also have abnormally shaped heads. Wnt-5A—which is considered not to activate the canonical Wnt/β-catenin pathway because it belongs to a distinct class of Wnts—is expressed in the outgrowing first branchial arch and in outgrowing facial primordia. It has been suggested that a Wnt-5A pathway is generally involved in regulating the outgrowth of extending structures.⁵⁰

Taken together, Wnts play various roles during later stages of head development which may be positive as described for midbrain, hippocampus and neural crest development. However, the large number of Wnts and Wnt modulators expressed in a highly regionalized manner highlights the need for further detailed investigations.

Conclusions and Outlook

At least three phases of Wnt signaling can be distinguished during vertebrate head formation. First, early β-catenin-dependent signaling plays a central role in organizer induction which is a prerequisite for head formation. Second, Wnt/β-catenin signaling antagonizes the head organizer during gastrulation. Different anterior-organizing centers have been identified in vertebrate gastrulae and all of them secrete Wnt antagonists. The interplay of these antagonists with posteriorizing Wnts may establish a tranforming activity gradient of Wnt/β-catenin signaling which regulates AP neural patterning. Based on these molecular data, we propose that Spemann's concept of separate head and trunk organizers and Nieuwkoop's transforming gradient do not contradict each other but represent two different views of AP axis formation: Like an electron will appear as a wave or a particle, AP patterning appears as regulated by organizers or gradients, depending on the experimental approach.

While Wnt/β-catenin signaling regulates AP patterning, noncanonical Wnt-11-type signaling promotes axial elongation and interference with this pathway may affect head formation indirectly. In the third and least characterized phase of Wnt signaling, multiple Wnts are expressed in a highly regionalized fashion. Wnts play important roles in the regional patterning of the brain and in the specification of neural crest which is essential for later steps of craniofacial development.

In the future, it will be crucial to modulate Wnt signaling during defined intervals and in restricted regions in order to specifically target distinct Wnt functions. First steps in this direc- tion have been made by using inducible transgenes and tissue transplantation approaches.^{28,29,31 33} Furthermore, the identification of transcriptional targets of Wnt signaling will be of great interest. In particular, the identification of genes activated by later (e.g. posteriorizing) Wnt/β-catenin signaling is in its very beginnings.²⁶

Acknowledgements

We apologize to all researchers whose work we could not cite due to space constraints. We thank Dr. Gary Davidson for helpful comments on the manuscript.

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