Table of contents

Wnts and epidermis

Wnt glycoproteins mediate short range intracellular communication that facilitates morphogenesis and, in some settings, promotes tumor formation. Although the involvement of the Drosophila homolog wingless in ectodermal patterning is well established, the role that Wnt genes play in mammalian skin biology is not defined. Wnt-4 and Wnt-10b mRNA are detected in adult murine epidermis. Normal murine keratinocytes and a melanocyte cell line (melan-A) propagated in vitro also contain Wnt-4 mRNA, whereas dermal fibroblasts and Langerhans cell-like dendritic cells do not. Because Wnt-4 mRNA is more abundant than Wnt-10b mRNA in epidermis and Wnt-10b transcripts are not detected in cells propagated in vitro, additional studies have emphasized Wnt-4 exclusively. Wnt-4 mRNA levels are increased in cultured keratinocytes as they approach confluence and are strikingly downregulated by mitogenic growth factors. Although Wnt-4 mRNA levels are not modulated during calcium-induced keratinocyte differentiation in vitro, assessment of Wnt-4 transcripts in keratinocyte cell lines suggests that loss of Wnt-4 gene expression is associated with a less differentiated, more malignant, phenotype. Despite this, epidermal abnormalities are not identified in newborn Wnt-4 null (-/-) skin, or in full-thickness -/- skin that has been engrafted to nude or athymic mice and allowed to mature for as long as 3 months. However, histologic examination of newborn Wnt-4 null skin reveals fibroplasia involving the dermis with increased accumulation of type I collagen fibrils. These results indicate that several Wnt genes are expressed in adult murine epidermis and suggest that Wnt-4 proteins may be involved in epidermal-dermal interactions in mammalian skin (Saitoh, 1998).

Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to an understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. The proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles. Its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding, resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle that lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways (Millar, 1999).

How do vertebrate epithelial appendages form from the flat epithelia? Following the formation of feather placodes, the previously radially symmetrical primordia become anterior-posterior (A-P) asymmetrical and develop a proximo-distal (P-D) axis. Analysis of the molecular heterogeneity reveals a surprising parallel of molecular profiles in the A-P feather buds and the ventral-dorsal (V-D) Drosophila appendage imaginal discs. The functional significance was tested with an in vitro feather reconstitution model. Wnt-7a expression initiates all over the feather tract epithelium, intensifying as it became restricted first to the primordia domain, then to an accentuated ring pattern within the primordia border, and finally to the posterior bud. In contrast, sonic hedgehog expression is induced later as a dot within the primordia. To further test the function of Wnt-7a in feather formation, Wnt-7a was overexpressed in reconstituted feather explants derived from stage 29 dorsal skin. Control skin forms normal elongated, slender buds with A-P orientation, but Wnt-7a overexpression leads to plateau-like skin appendages lacking an A-P axis. Feathers in the Wnt-7a overexpressing skin also have inhibited elongation of the P-D axes. This is not due to a lack of cell proliferation, which actually is increased although randomly distributed. While morphogenesis is perturbed, differentiation proceeds as indicated by the formation of barb ridges. Wnt-7a buds have reduced expression of anterior (Tenascin) bud markers. Middle (Notch-1) and posterior bud markers including Delta-1 and Serrate-1 are diffusely expressed. The results show that ectopic Wnt-7a expression enhances properties characteristic of the middle and posterior feather buds and suggest that P-D elongation of vertebrate skin appendages requires balanced interactions between the anterior and posterior buds (Widelitz, 1999).

Mutations in WNT effector genes perturb hair follicle morphogenesis, suggesting key roles for WNT proteins in this process. Expression of Wnts 10b and 10a is upregulated in placodes at the onset of follicle morphogenesis and in postnatal hair follicles beginning a new cycle of hair growth. The expression of additional Wnt genes is observed in follicles at later stages of differentiation. Among these, it has been found that Wnt5a is expressed in the developing dermal condensate of wild type but not Sonic hedgehog (Shh)-null embryos, indicating that Wnt5a is a target of SHH in hair follicle morphogenesis. These results identify candidates for several key follicular signals and suggest that WNT and SHH signaling pathways interact to regulate hair follicle morphogenesis (Reddy, 2001).

Although multiple Wnt genes are expressed in the surface epithelium of the embryo, transcripts for only two of these, Wnts 10a and 10b, become specifically localized to hair follicle placodes. Since WNT10b causes mammary tumors in mice, stabilization of ß-catenin in preadipocytes and partial axis duplication in Xenopus embryos, it may be classified as a Class I WNT. The signaling properties of WNT10a have not yet been determined, but at the sequence level, Wnts 10a and 10b encode closely related proteins, making it likely that WNT10a is also a Class I WNT. WNTs 10a and 10b are therefore strong candidates for the signals that cause accumulation of nuclear ß-catenin and activation of a LEF1/TCF responsive reporter, TOPGAL, in the epithelium and mesenchyme at early stages of follicular development. In the absence of epidermal ß-catenin, placodes fail to form, whereas expression of a stabilized form of ß-catenin in the epithelium causes the formation of ectopic hair follicles, indicating that the canonical WNT signaling pathway acts to promote formation of the placode and induction of the dermal condensate. WNTs10a and 10b may therefore comprise part of the 'first epithelial signal' operating in hair follicle morphogenesis. Wnt5a can function as a Class II Wnt although it is also capable of directing the canonical pathway in the presence of an appropriate Frizzled receptor. Wnt5a may therefore have functions in hair follicles other than or in addition to those suggested by TOPGAL expression and the effects of loss and gain of function mutations in Class I pathway genes. The TOPGAL expression pattern in developing hair follicles suggests that it is not involved in the initial positioning of follicles, but may form a component of the 'second dermal signal', directing the proliferation of overlying epithelial cells. Such a role would be consistent with the established properties of Wnt5a, which has been shown to be required for the proliferation of limb bud and snout progenitor cells (Reddy, 2001).

Several lines of evidence indicate that, in addition to roles in hair follicle morphogenesis and at anagen onset (onset of growth of an adult hair follicle), signaling by Class I WNTs may regulate differentiation of the hair shaft. (1) The few hairs that develop in Lef1-deficient mice appear to be incompletely keratinized. (2) The WNT-responsive TOPGAL reporter gene and the WNT effector protein DVL2 are expressed in hair shaft precursor cells. (3) Ectopic expression of Wnt3 in the follicular outer root sheath causes fragility of the hair shaft. The results presented here indicate that Wnt3 is expressed in the most differentiated hair shaft precursor cells during anagen, making it the strongest candidate for a regulator of hair shaft differentiation. However, the expression pattern of Wnt3 overlaps with that of Wnt4 in less differentiated hair shaft precursors and Wnts 10a, 10b and 3a are expressed in inner root sheath cells adjacent to hair shaft precursors, suggesting possible functional redundancy between these Wnts and Wnt3 in mature follicles (Reddy, 2001).

It has recently been demonstrated that during the anagen phase in mature follicles, epithelial progenitor cells in the outer root sheath migrate from the bulge region to the hair bulb where they proliferate and subsequently begin to differentiate into inner root sheath and hair shaft precursors. These cell movements are likely to be important for a normal rate of hair growth. Signals controlling cell movements have been studied in Xenopus and zebrafish embryos. These experiments reveal that cell migration during gastrulation is regulated by WNTs 5a and 11 via a non-canonical WNT signaling pathway. Signaling by WNT11 requires the WNT effector DVL, but not ß-catenin, and is similar to a pathway regulating planar polarity in Drosophila embryos. A characteristic of these pathways is that disruption of cell polarity and movement occurs as a result of either inhibiting or stimulating signaling. Wnt 5a and Wnt11 are expressed in outer layers of anagen hair follicles in cells that lie in the neck of the follicle between the bulge and the hair bulb. DVL2 is expressed in the outer root sheath in this same region of the follicle and overexpression of DVL2 in the outer root sheath causes a phenotype of short hair that is not the result of decreased follicle cell proliferation or altered control of the hair growth cycle. These results suggest that cell movements during anagen may be regulated by WNTs 5a and 11, via DVL2. Experiments in which the functions of these WNTs are increased or inhibited in the outer root sheath, combined with recently described methods for tracking cell movements in hair follicles, may be used to test this hypothesis (Reddy, 2001).

Although several factors capable of inducing a new cycle of hair growth in resting follicles have been identified, the endogenous signals regulating this process remain obscure at the molecular level. It has been proposed that the initiating signal for hair growth arises from the dermal papilla and instructs epithelial stem cells in the bulge region of the follicle to proliferate transiently. This hypothesis is supported by the observation that the first postnatal hair growth cycle is not initiated in the hairless (hr) mutant mouse, in which the dermal papilla loses contact with the epithelial portion of the follicle during catagen (regression). The transient expression of the TOPGAL reporter gene in bulge cells at anagen onset suggests that WNTs may be involved in triggering a new cycle of hair growth. In support of this hypothesis, onset of the first postnatal anagen does not occur in mice in which the ß- catenin gene is progressively deleted in the epidermis and follicular epithelium. Wnt10a is expressed in the dermal papilla at anagen onset, while Wnt10b is expressed in adjacent epithelial cells in the lower part of the follicle. These results suggest WNT10a as a possible component of an initiating signal from the dermal papilla and identify WNTs 10a and 10b as the strongest candidates for the WNTs that induce TOPGAL expression in the bulge. The timing and location of expression of Wnt10b correlate with the migration of progenitor cells to the lower part of the hair follicle, suggesting that WNT10b is expressed in these 'activated' progenitor cells. These results also identify WNT10b as the strongest candidate for the epithelial signal that maintains the inductive properties of follicular dermal cells (Reddy, 2001).

SHH is not required for the positioning of follicles, but plays essential roles in the regulation of follicular proliferation and formation of the dermal papilla. Previous data have indicated that expression of Shh in hair follicles is regulated by canonical WNT signaling and WNTs 10a and 10b are the most likely candidates for WNTs that control Shh expression in hair follicle morphogenesis. However, Wnt genes are also targets of SHH in several developmental systems and consistent with this observation, it has been found that expression of Wnt5a in developing hair follicles requires SHH. This result suggests that WNT5a may mediate some of the effects of SHH in hair follicle morphogenesis, a hypothesis supported by the fact that both WNT5a and SHH are capable of regulating proliferation. Since later stages of hair follicle morphogenesis are abnormal in Shh minus mutants, the question of whether expression of Wnt5a in mature hair follicles is also regulated by SHH could not be addressed; however, like Wnt5a, Shh is expressed in inner root sheath cells in anagen follicles (Reddy, 2001).

The finding that Wnt5a is a target of SHH signaling in hair follicles has important implications for the study of basal cell carcinoma (BCC), a human skin tumor that occurs with high frequency in Caucasian populations. BCC results from inappropriate activation of the SHH pathway in epidermal cells and is frequently associated with mutations in the gene encoding the SHH receptor PTC1. Like developing hair follicles, BCCs show elevated expression of PTC1 and GLI1, which encodes a transcriptional effector of SHH signaling. In addition to activation of the SHH signaling pathway, BCCs share many common characteristics with immature hair follicles, including similar histology, ultrastructure and patterns of keratin gene expression, suggesting that SHH activates the same downstream target genes in BCCs and hair follicles. BCC can be mimicked in transgenic mice by over-expression of Shh, Gli1 or Gli2 in the epidermis and Wnt gene expression is directly regulated by SHH via GLI transcription factors in Drosophila and zebrafish embryos. However, Wnt targets of the SHH pathway in BCC have not been identified. Given the similarity of BCC to immature hair follicles, the results presented here predict that Wnt5a is upregulated in BCC. Nuclear localization of ß-catenin is not observed in BCC consistent with classification of WNT5a as a Class II WNT (Reddy, 2001).

Hair follicle morphogenesis is initiated by a dermal signal that induces the development of placodes in the overlying epithelium. To determine whether WNT signals are required for initiation of follicular development, Dickkopf 1, a potent diffusible inhibitor of WNT action, was ectopically expressed in the skin of transgenic mice. This produced a complete failure of placode formation prior to morphological or molecular signs of differentiation, and blocked tooth and mammary gland development before the bud stage. This phenotype indicates that activation of WNT signaling in the skin precedes, and is required for, localized expression of regulatory genes and initiation of hair follicle placode formation (Andl, 2002).

The sensory nervous system in the vertebrate head arises from two different cell populations: neural crest and placodal cells. By contrast, in the trunk it originates from neural crest only. How do placode precursors become restricted exclusively to the head and how do multipotent ectodermal cells make the decision to become placodes or neural crest? At neural plate stages, future placode cells are confined to a narrow band in the head ectoderm, the pre-placodal region (PPR). The head mesoderm is identified as the source of PPR inducing signals, reinforced by factors from the neural plate. Several independent signals are needed: attenuation of BMP and WNT is required for PPR formation. Together with activation of the FGF pathway, BMP and WNT antagonists can induce the PPR in naive ectoderm. WNT signalling plays a crucial role in restricting placode formation to the head. Finally, the decision of multipotent cells to become placode or neural crest precursors is demonstrated to be mediated by WNT proteins: activation of the WNT pathway promotes the generation of neural crest at the expense of placodes. This mechanism explains how the placode territory becomes confined to the head, and how neural crest and placode fates diversify (Litsiou, 2005).

This study finds that FGF signalling cooperates with WNT and BMP antagonists to impart generic placode character to uncommitted ectoderm. In the chick, activation of the FGF pathway in naive ectoderm leads to rapid expression of pre-neural markers such as Sox3 and Erni, both of which are later co-expressed at the border of the neural plate. However, activation of the FGF pathway is not sufficient to specify cells (neural crest and placode precursors) that arise from this border. The observation that continued FGF signalling is not required for pre-placodal Six4 expression, but can directly induce Eya2, suggests that FGFs may play a dual role. Early FGF signalling may confer 'border character' to ectodermal cells to make them responsive to PPR and crest inducing signals. The finding that ectopic PPR induction occurs only in the presence of active FGF signalling supports this notion. Later, FGFs from the head mesoderm, probably FGF4, initiate the expression of Eya2 in the placode territory as a crucial step to activate downstream target genes (Litsiou, 2005).

Simultaneously, the head mesoderm provides both BMP and WNT antagonists, most likely DAN and Cerberus, to counteract the inhibitory effect of both factors on the generation of placode precursors. The results show that attenuation of either the BMP or WNT pathway leads to an expansion of the PPR into the adjacent ectoderm. However, while the expansion in response to BMP inhibition is limited to the head ectoderm, WNT antagonism also results in the expression of PPR specific genes in the trunk. This is in agreement with recent findings in Xenopus reporting that simultaneous overexpression of BMP and WNT antagonist expands Six1 expression posteriorly along the induced secondary axis. In the chick, Wnt8c (see Drosophila Wnt8) is expressed in trunk mesoderm and the mesoderm lateral to the heart primordium, whereas Wnt6 is found in trunk ectoderm. It is proposed that WNT activity from surrounding tissues is essential to restrict the placode territory to the head ectoderm next to the neural plate and thus ensure that sensory placodes are confined to the head. To allow placode formation, WNT antagonists in cooperation with FGF and anti-BMPs from the head mesoderm protect placode precursors from this inhibitory influence (Litsiou, 2005).

Wnt signaling is mediated through (1) the beta-catenin dependent canonical pathway and, (2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in mediating the non-canonical Wnt signaling have been well established. The role of Ror2 in canonical Wnt signaling is not fully understood. This study reports that Ror2 also positively modulates Wnt3a-activated canonical signaling in a lung carcinoma, H441 cell line. This activity of Ror2 is dependent on cooperative interactions with Fzd2 but not Fzd7. In addition, Ror2-mediated enhancement of canonical signaling requires the extracellular CRD, but not the intracellular PRD domain of Ror2. Evidence that the positive effect of Ror2 on canonical Wnt signaling is inhibited by Dkk1 and Krm1 suggesting that Ror2 enhances an Lrp-dependent STF response. The current study demonstrates the function of Ror2 in modulating canonical Wnt signaling. These findings support a functional scheme whereby regulation of Wnt signaling is achieved by cooperative functions of multiple mediators (Li, 2008; full text of article).

The activity of keratinocytes in the hair follicle is regulated by signals from a specialized mesenchymal niche at the base of the follicle, the dermal papilla (DP). In this study, mice expressing cre recombinase in the DP were developed to probe the interaction between follicular keratinocytes and the DP in vivo. Inactivation of theβ-catenin gene within DP of fully developed hair follicles results in dramatically reduced proliferation of the progenitors and their progeny that generate the hair shaft, and, subsequently, premature induction of the destructive phase of the hair cycle. It also prevents regeneration of the cycling follicle from stem cells. Gene expression analysis reveals that β-catenin activity in the DP regulates signaling pathways, including FGF and IGF, that can mediate the DP's inductive effects. This study reveals a signaling loop that employs Wnt/β-catenin signaling in both epithelial progenitor cells and their mesenchymal niche to govern and coordinate the interactions between these compartments to guide hair morphogenesis (Enshell-Seijffers, 2010).

Wnts and Hair Development

Progenitors oppositely polarize WNT activators and inhibitors to orchestrate tissue development

To spatially co-exist and differentially specify fates within developing tissues, morphogenetic cues must be correctly positioned and interpreted. This study investigated mouse hair follicle development to understand how morphogens operate within closely spaced, fate-diverging progenitors. Coupling transcriptomics with genetics, it was shown that emerging hair progenitors produce both WNTs and WNT inhibitors. Surprisingly, however, instead of generating a negative feedback loop, the signals oppositely polarize, establishing sharp boundaries and consequently a short-range morphogen gradient that was shown to be essential for three-dimensional pattern formation. By establishing a morphogen gradient at the cellular level, signals become constrained. The progenitor preserves its WNT signaling identity and maintains WNT signaling with underlying mesenchymal neighbors, while its overlying epithelial cells become WNT-restricted. The outcome guarantees emergence of adjacent distinct cell types to pattern the tissue (Matos, 2020).

Tissue regeneration relies on resident stem cells (SCs), whose activity and lineage choices are influenced by the microenvironment. Exploiting the synchronized, cyclical bouts of tissue regeneration in hair follicles (HFs), this study investigated how microenvironment dynamics shape the emergence of stem cell lineages. Employing epigenetic and ChIP-seq profiling, this study uncovered how signal-dependent transcription factors couple spatiotemporal cues to chromatin dynamics, thereby choreographing stem cell lineages. Using enhancer-driven reporters, mutagenesis, and genetics, this study showed that simultaneous BMP-inhibitory and WNT signals set the stage for lineage choices by establishing chromatin platforms permissive for diversification. Mechanistically, when binding of BMP effector pSMAD1 is relieved, enhancers driving HF-stem cell master regulators are silenced. Concomitantly, multipotent, lineage-fated enhancers silent in HF-stem cells become activated by exchanging WNT effectors TCF3/4 for LEF1. Throughout regeneration, lineage enhancers continue reliance upon LEF1 but then achieve specificity by accommodating additional incoming signaling effectors. Barriers to progenitor plasticity increase when diverse, signal-sensitive transcription factors shape LEF1-regulated enhancer dynamics (Matos, 2020).

Pattern formation plays near universal roles in tissue morphogenesis. The early developing skin is composed of a single layer of multipotent epithelial progenitor cells that will either stratify and develop into the skin's epidermal barrier or form epithelial placodes to launch hair follicle morphogenesis. Positional cues are important not only to specify the uniform distribution of hair follicles across the tissue, but also to differentiate the cells within each of these mini-organs. In uncovering the existence of an internal WNT morphogen gradient within the earliest progenitors of the hair placode, this study has begun to understand how WNT signals can be directionally distributed to neighboring cells to break symmetry and trigger the morphogenetic transition from the two-dimensional early placode to a three-dimensional mini-organ (Matos, 2020).

WNT signaling has long been known to be important broadly for regenerative and morphogenetic processes. The presence of inhibitors of the WNT signaling pathway has also long been recognized, and given the oft short-lived nature of WNT signals in development, it has always been assumed that inhibitors function in a negative feedback loop to turn off the signal for the next step in lineage specification. Although the existence of such feedback loops is well-established, such a mechanism did not reconcile how WNT signaling remains high in basal hair bud progenitors that also simultaneously express at least four different WNT inhibitors. An additional conundrum was how this WNT expressing, WNT signaling progenitor gives rise to only one daughter cell that retains this status, while the other daughter cell adopts a WNT-restricted state (Matos, 2020).

The findings of this study show that by differentially compartmentalizing WNTs and WNT inhibitors, basal placode progenitors not only maintain both positive and negative WNT morphogens simultaneously, but also directionally target the signals, providing the requisite positional cues to transition from two to three dimensional patterning within the developing tissue. By polarizing WNTs basally, progenitors are able to retain their own WNT signaling as well as that of their underlying mesenchymal neighbors to fuel hair follicle morphogenesis at the dermal-epidermal interface. Conversely, by polarizing WNT inhibitors apically, the same hair bud progenitors directionally orchestrate WNTloSOX9+ fate specification of their overlying neighbors to launch the diversification of the epithelial cells within the developing hair follicles (Matos, 2020).

During development, the formation of precise boundaries is fundamental to the specification of different cellular compartments. The current findings best fit a model whereby developing hair follicle progenitors use WNTs and WNT inhibitors to build local boundaries. By localizing WNT inhibitors apically, WNThigh progenitors limit the WNT response, preserving their own WNThigh signaling identify while simultaneously preventing their suprabasal daughter from responding to the WNT signal. A similar refining mechanism has been previously proposed in the Drosophila imaginal disks. In the wingless expressing cells of the wing margin, cells that are closer to the dorsoventral boundary are able to repress wingless expression in their juxtaposed neighbors through a self-refining mechanism. In this case, however, the mechanism appears to involve NOTCH, whose activity is required for wingless expression, which in turn appears to repress NOTCH activity. In the hair follicle, NOTCH signaling has not been detected in the WNT10B+ progenitors, but rather the WNTlow differentiating cells. Thus, while the mechanisms seem to be evolutionarily divergent, the functional output is similar and involves the establishment of a sharp boundary that enables the emergence of juxtaposed cell fates (Matos, 2020).

Although the current study focused on the existence of this single-cell length morphogen gradient and its functional significance, it will be interesting in the future to unravel how bidirectional targeting occurs. WNTs are known to be N-glycosylated, and studies in Drosophila suggest that WNTs can be secreted apically and then transported basally. Intriguingly, however, in Drosophila, N-glycosylation-deficient Wingless is secreted without consequence, and in mammalian cells, WNTs have been found to be more potent and bind extracellular matrix more robustly in the absence of N-glycosylation. Thus, although yhr tunicamycin results suggest a role for N-glycosylation in WNT-inhibitor apical secretion, it may be advantageous for cells such as hair bud progenitors that adhere to a basement membrane to secrete their WNTs basally. The ability of ECM to retain growth factors (Baeg et al. 2001), including WNT regulators, makes this hypothesis all the more attractive (Matos, 2020).

In closing, by establishing a morphogen gradient at the cellular level, signals are constrained such that two neighboring cell populations in direct physical contact can effectively receive different signaling inputs. Overall, the ability to directionally control rapid changes in daughter fates, and to establish sharp tissue boundaries without the need for direct competition and/or elimination, offers basic advantages to this mechanism that are likely to be broadly applicable in development (Matos, 2020).

Wnts and adipogenesis

The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of ß-catenin to the proteasome. In fact, sustained activation of ß-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. The mechanisms regulating the balance between ß-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes has been investigated. Specifically, it has been shown that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of ß-catenin, which depends on glycogen synthase kinase (GSK) 3ß activity. Mutation of serine 37 (a target of GSK3ß) to an alanine renders ß-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3ß phosphorylation-defective S37A-ß-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-ß-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant ß-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-ß-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKß is a key regulator of the balance between ß-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes (Liu, 2004).

WNTs and mammary gland morphogenesis

Mammary glands, like other skin appendages such as hair follicles and teeth, develop from the surface epithelium and underlying mesenchyme; however, the molecular controls of embryonic mammary development are largely unknown. Activation of the canonical WNT/ß-catenin signaling pathway in the embryonic mouse mammary region coincides with initiation of mammary morphogenesis, and WNT pathway activity subsequently localizes to mammary placodes and buds. Several Wnt genes are broadly expressed in the surface epithelium at the time of mammary initiation, and expression of additional Wnt and WNT pathway genes localizes to the mammary lines and placodes as they develop. Embryos cultured in medium containing WNT3A or the WNT pathway activator lithium chloride (LiCl) display accelerated formation of expanded placodes, and LiCl induces the formation of ectopic placode-like structures that show elevated expression of the placode marker Wnt10b. Conversely, expression of the secreted WNT inhibitor Dickkopf 1 in transgenic embryo surface epithelium in vivo completely blocks mammary placode formation and prevents localized expression of all mammary placode markers tested. These data indicate that WNT signaling promotes placode development and is required for initiation of mammary gland morphogenesis. WNT signals play similar roles in hair follicle formation and thus may be broadly required for induction of skin appendage morphogenesis (Chu, 2004).

WNTs and neural crest

Environmental signals are important in the development of neural crest, during which process multipotent progenitors must choose from several fates. However, the nature of these environmental signals is unknown. A fate map of zebrafish cranial neural crest shows that lineage-restricted clones of pigment cells arise from medial cells near the neural keel, and that clones of neurons arise from lateral cells farther from the neural keel. Wnt-1 and Wnt-3a are candidate genes for influencing neural crest fate, as they are expressed next to medial, but not lateral, crest cells. The role of Wnt signals in modulating the fate of neural crest has been determined by injecting messenger RNAs into single, premigratory neural crest cells of zebrafish. Lineage analysis of injected cells shows that activation of Wnt signaling by injection of mRNA encoding cytoplasmic beta-catenin promotes pigment-cell formation at the expense of neurons and glia. Conversely, inhibition of the Wnt pathway, by injection of mRNAs encoding either a truncated form of the transcription factor Tcf-3 or a dominant-negative Wnt, promotes neuronal fates at the expense of pigment cells. It is concluded that endogenous Wnt signaling normally promotes pigment-cell formation by medial crest cells and thereby contributes to the diversity of neural crest cell fates (Dorsky, 1998).

The molecular interactions underlying neural crest formation in Xenopus have been investigated. Neural crest induction requires a suppression of BMP-mediated epidermal fate. A simple model for induction of the neural crest, a cell type that arises at the junction between neural and non-neural ectoderm, would be that neural crest is specified at levels of BMP signaling intermediate to those that induce neural plate and epidermis. Using chordin overexpression to antagonize endogenous BMP signaling in whole embryos and explants, it is demonstrated that such inhibition alone is insufficient to account for neural crest induction in vivo. However, chordin-induced neural plate tissue can be induced to adopt neural crest fates by members of the FGF and Wnt families, growth factors that have previously been shown to posteriorize induced neural tissue. Overexpression of a dominant negative XWnt-8 inhibits the expression of neural crest markers, demonstrating the necessity for a Wnt signal during neural crest induction in vivo. The requirement for Wnt signaling during neural crest induction is shown to be direct, whereas FGF-mediated neural crest induction may be mediated by Wnt signals. Overexpression of the zinc finger transcription factor Slug, one of the earliest markers of neural crest formation, is insufficient for neural crest induction. Slug-expressing ectoderm will generate neural crest in the presence of Wnt or FGF-like signals, however, bypassing the need for BMP inhibition in this process. A two-step model for neural crest induction is proposed (LaBonne, 1998).

Wnts make up a large family of secreted molecules implicated in numerous developmental processes. Frizzled proteins are likely receptors for Wnts and are required for Wnt signaling in invertebrates. A large number of vertebrate frizzled genes have also been identified, but their roles in mediating specific responses to endogenous Wnts have not been well defined. Using a functional assay in Xenopus, a large screen was performed to identify potential interactions between Wnts and frizzleds. Signaling by Xwnt1, but not other Wnts, can be specifically enhanced by frizzled 3 (Xfz3). Since both Xfz3 and Xwnt1 are highly localized to dorsal neural tissues that give rise to neural crest, an examination was performed to see whether Xfz3 mediates Xwnt1 signaling in the formation of neural crest. Xfz3 specifically induces neural crest in ectodermal explants and in embryos, similar to Xwnt1, and at lower levels of expression, synergizes with Xwnt1 in neural crest induction. Furthermore, loss of Xfz3 function, either by depletion with a Xfz3-directed morpholino antisense oligonucleotide or by expression of an inhibitory form of Xfz3 (Nfz3), prevents Xwnt1-dependent neural crest induction in ectodermal explants and blocks neural crest formation in whole embryos. These results show that Xfz3 is required for Xwnt1 signaling in the formation of the neural crest in the developing vertebrate embryo (Deardorff, 2001).

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. The roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos, was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, is transformed into neural crest by posteriorizing signals. This transformation is mimicked by bFGF, Wnt-8, or retinoic acid treatment and is also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells is also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor has been shown to directly affect the ectoderm. A two-step model is proposed for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. A unifying model is discussed where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction (Villanueva, 2002).

It is suggested that at the early-gastrula stage, a gradient of BMP activity is established in the ectoderm, which specifies the neural plate, the neural plate border, and the epidermis at progressively higher concentrations of BMP. The neural plate border, induced at a precise location within the mediolateral axis of the ectoderm, has an anterior character. Later, between early and midgastrula stage, signals presumably originating from the ventrolateral mesoderm transform a region of the anterior neural plate border into prospective neural crest cells. A role for this mesoderm in neural crest induction has been shown. The spread of these molecules from the mesoderm into the ectoderm consequently locates them only in large animal caps, explaining why the neural crest was not induced when small animal caps were used. These signals could correspond to Wnt8 and eFGF, since it is known that they are expressed in the ventrolateral mesoderm, and could correspond to lateralizing signals. However, the neural crest is not specified at this stage; this does not occur until the end of gastrulation. Thus, additional signals are required for the final induction of the neural crest. Finally, as gastrulation proceeds, the ventrolateral mesoderm becomes localized to the posterior region of the embryo, where it continues to produce Wnt8, eFGF, and possibly retinoic acid, as well as another, as yet unknown, posteriorizing agent(s) that generates an anterior-posterior gradient of these morphogenes. This gradient would be required for the final specification of the neural crest in the most posterior region of the neural plate border. Thus, the lateral-posterior regions of the neural plate border receive the lateralizing/posteriorizing signals for an extended period of time, finally specifying them as neural crest. In contrast, the anterior neural plate border does not receive such signals or these are inhibited by other agents produced by the anterior regions of the embryo, such as cerberus or dkk1, two known Wnts inhibitors, and, as a consequence, this border region does not develop as neural crest cells. It is tempting to speculate that the anterior-posterior differences within the neural crest could be controlled by a similar mechanism (Villanueva, 2002).

While Wnt/ß-catenin signaling is known to be involved in the development of neural crest cells in zebrafish, it is unclear which Wnts are involved, and when they are required. To address these issues a zebrafish line that was transgenic for an inducible inhibitor of Wnt/ß-catenin signaling was used, and endogenous Wnt/ß-catenin signaling was inhibited at discrete times in development. Using this approach, a critical period for Wnt signaling in the initial induction of neural crest was defined that is distinct from the later period of development when pigment cells are specified from neural crest. Blocking Wnt signaling during this early period interferes with neural crest formation without blocking development of dorsal spinal neurons. Transplantation experiments suggest that neural crest precursors must directly transduce a Wnt signal. With regard to identifying which endogenous Wnt is responsible for this initial critical period, wnt8 was shown to be expressed in the appropriate time and place to participate in this process. Supporting a role for Wnt8, blocking its function with antisense morpholino oligonucleotides eliminates initial expression of neural crest markers. Taken together, these results demonstrate that Wnt signals are critical for the initial induction of zebrafish neural crest and suggest that this signaling pathway plays reiterated roles in its development (Lewis, 2004).

Delamination of premigratory neural crest cells depends on a balance between BMP/noggin and on successful G1/S transition. BMP regulates G1/S transition and consequent crest delamination through canonical Wnt signaling. Noggin overexpression inhibits G1/S transition and blocking G1/S abrogates BMP-induced delamination; moreover, transcription of Wnt1 is stimulated by BMP and by the developing somites, which concomitantly inhibit noggin production. Interfering with ß-catenin and LEF/TCF inhibits G1/S transition, neural crest delamination and transcription of various BMP-dependent genes, including Cad6B, Pax3 and Msx1, but transcription of Slug, Sox9 or FoxD3. Hence, it is proposed that developing somites inhibit noggin transcription in the dorsal tube, resulting in activation of BMP and consequent Wnt1 production. Canonical Wnt signaling in turn stimulates G1/S transition and generation of neural crest cell motility independently of its proposed role in earlier neural crest specification (Burstyn-Cohen, 2004).

While Wnt signaling is known to be involved in early steps of neural crest development, the mechanism remains unclear. Because Wnt signaling is able to posteriorize anterior neural tissues, neural crest induction by Wnts has been proposed to be an indirect consequence of posteriorization of neural tissues rather than a direct effect of Wnt signaling. To address the relationship between posteriorization and neural crest induction by Wnt signaling, gain of function and loss of function approaches in Xenopus have been used to modulate the level of Wnt signaling at multiple points in the pathway. Modulating the level of Wnt signaling allows separation of neural crest induction from the effects of Wnts on anterior-posterior neural patterning. It was also found that activation of Wnt signaling induces ectopic neural crest in the anterior region without posteriorizing anterior neural tissues. In addition, Wnt signaling induces neural crest when its posteriorizing activity is blocked by inhibition of FGF signaling in neuralized explants. Finally, depletion of ß-catenin confirms that the canonical Wnt pathway is required for initial neural crest induction. While these observations do not exclude a role for posteriorizing signals in neural crest induction, these data, together with previous observations, strongly suggest that canonical Wnt signaling plays an essential and direct role in neural crest induction (Wu, 2005).

FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, the role of the transcription factors Msx1 and Pax3 was analyzed. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, it has been show that Msx1 and Pax3 are both required for neural crest formation, display overlapping but nonidentical activities, and that Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction (Monsoro-Burq, 2004).

The neural crest is induced by a combination of secreted signals. Although previous models of neural crest induction have proposed a step-wise activation of these signals, the actual spatial and temporal requirement has not been analysed. Through analysing the role of the mesoderm this study shows that specification of neural crest requires two temporally and chemically different steps: first, an induction at the gastrula stage dependent on signals arising from the dorsolateral mesoderm; and second, a maintenance step at the neurula stage dependent on signals from tissues adjacent to the neural crest. By performing tissue recombination experiments and using specific inhibitors of different inductive signals, it was shown that the first inductive step requires Wnt activation and BMP inhibition, whereas the later maintenance step requires activation of both pathways. This change in BMP necessity from BMP inhibition at gastrula to BMP activation at neurula stages is further supported by the dynamic expression of BMP4 and its antagonists, and is confirmed by direct measurements of BMP activity in the neural crest cells. The demonstration that Wnt signals are required for neural crest induction by mesoderm solves an long-standing controversy. Finally, the results emphasise the importance of considering the order of exposure to signals during an inductive event (Steventon, 2009).

Ancient evolutionary origin of the neural crest gene regulatory network

The vertebrate neural crest migrates from its origin, the neural plate border, to form diverse derivatives. It has been hypothesized that a neural crest gene regulatory network (NC-GRN) guides neural crest formation. This study investigated when during evolution this hypothetical network emerged by analyzing neural crest formation in lamprey, a basal extant vertebrate. 50 NC-GRN homologs were identified and morpholinos were used to demonstrate a critical role for eight transcriptional regulators. The results reveal conservation in deployment of upstream factors, suggesting that proximal portions of the network arose early in vertebrate evolution and have been conserved for >500 million years. Biphasic expression was found of neural crest specifiers and differences in deployment of some specifiers and effectors expected to confer species-specific properties. By testing the collective expression and function of neural crest genes in a single, basal vertebrate, the ground state of the NC-GRN was revealed and ambiguities were resolved between model organisms (Sauka-Spengler, 2007).

A uniquely vertebrate innovation, the neural crest is defined by its origin at the neural plate border, migratory capability, multipotentiality, and combinatorial gene expression. As a basal jawless vertebrate, lamprey possesses neural crest cells that move along similar pathways and form many, but not all, neural-crest-derived structures found in jawed vertebrates. However, there is little or no information about early steps in neural crest specification in the lamprey. Analysis of a hypothetical NC-GRN in this basal vertebrate promises to inform on the general architecture and evolutionary history of an archetypical vertebrate gene regulatory network. As both a critical test of this putative network and a representation of its ground state, functional tests were performed involving multiple interactions within a single, basal vertebrate (Sauka-Spengler, 2007).

Fifty genes involved in neural crest formation in lamprey were identified. The findings are consistent with several features of a putative NC-GRN proposed to function in jawed vertebrates, particularly with respect to its proximal elements. Expression of signaling molecules and neural plate border specifiers is highly conserved, as are the functions of border specifiers tested in this study. BMP, Wnt, and Delta expression was found in similar patterns to those noted in frog and zebrafish, suggesting that signaling cues are present in lamprey at proper times and places to play analogous functions in neural crest specification to those in other vertebrates; e.g., Wnt8 is expressed in the nonneural ectoderm abutting the neural rod, much like chick Wnt6. Similarly, lamprey MsxA, ZicA, Dlx, and Pax3/7 are found within and adjacent to the neural plate border, implying that their combinatorial presence in the border is highly conserved across all vertebrate neurulae (Sauka-Spengler, 2007).

In contrast to these proximal steps, distal portions of the gene regulatory network exhibit both conserved and divergent features. The results suggest that neural crest specifiers are activated in two phases, with one set of transcription factors activated at the neural plate border of the early neurula and the other during a second later phase wherein the neural crest in the dorsal neural tube is forming. This differs from the previous formulation of the NC-GRN in which there was no discrimination in the timing of deployment of neural crest specifier genes into early (neural plate border) and late (bona fide neural crest precursor) categories. It is noted that the expression patterns and functions of late neural crest specifiers, like FoxD3 and SoxE family members, in lamprey resemble those observed in other vertebrates, whereas c-Myc, Id, AP2, and Snail are first deployed in the early neurula at the neural plate border rather than in nascent neural crest cells. These early-activated neural crest specifiers are expressed only slightly after the border specifiers, suggesting they may be their direct targets. Furthermore, these genes are involved in cell cycle control and therefore may play a role in maintaining multipotency of neural crest progenitors by acting as a cell cycle control switch between proliferation, cell death, and cell fate decisions (Sauka-Spengler, 2007).

The slow development of lamprey offers the advantage of allowing exquisite temporal resolution not possible in rapidly developing organisms like Xenopus and zebrafish. In jawed vertebrates, c-Myc and its direct target Id3 are expressed at the neural/nonneural ectoderm border prior to Snail1 and Sox8, but after expression of the border specifiers Msx1 and Pax3, whereas Snail2, Sox9, and FoxD3 are expressed by premigratory neural crest. However, the rapid development of Xenopus makes the exact timing of these expression patterns much more difficult to resolve. In amniotes like chick, Id family members are expressed at the neural plate border, together with proto-oncogenes c-Myc and n-Myc and bHLH transcription factor AP2a. In contrast, Sox9, FoxD3, and Snail2 are first expressed in the neural folds, while Sox10 is first expressed in delaminating neural crest. Thus, subdivision of lamprey neural crest specifiers into early- and late-acting categories may reflect either a lack of conservation or a previously unrecognized characteristic of the vertebrate neural crest network in general (Sauka-Spengler, 2007).

A difference in gene expression between lamprey and other species is that Snail is expressed earlier at the lamprey neural plate border, in contrast to its expression in premigratory neural crest in frogs, fish, and birds. Furthermore, the Snail homolog identified does not display a neural-crest-specific pattern at premigratory stages, but rather appears to be ubiquitous, similar to hagfish SnailA, and thus may represent an interesting regulatory difference between cyclostomes and gnathostomes. Similarly, the transcription factor Ets1 is expressed in premigratory, migrating, and postmigratory neural crest in Xenopus and chick and proposed to function in neural crest cell specification. In contrast, no lamprey Ets1 homologs are expressed in the neural crest cells during specification stages; rather, the first expression of both Ets1a and Ets1b is in populations of early differentiating neural crest within the branchial arches. In addition, Ets1b is expressed in hematopoietic and endothelial precursors, similar to its higher vertebrate ortholog implicated in hematopoiesis, vasculogenesis, and angiogenesis; this suggests that the lamprey gene functions hematopoetically while lacking early neural crest specifier function. Along the same lines, Twist is expressed in the premigratory crest in Xenopus, whereas lamprey Twist homologs appear to be expressed only in postmigratory crest cells lining branchial arches and persist in mesenchyme forming buccal cartilage. Extensive searches have yielded four Twist and two Ets1 homologs plus one Ets1-related factor. Given the current genome coverage (~95%), existence of another Twist or Ets1 homolog is unlikely, suggesting that the lamprey genes lack early neural crest specifier function. Intriguingly, these genes may have been co-opted to an earlier function in gnathostomes, lost early specification function in lampreys, or both. In contrast to this apparent lack of conservation, signaling receptors and adhesion and matrix molecules like Neuropilin2, Robo, and Col2a1 have similar expression patterns in gnathostomes and lamprey. An N-cadherin-like adhesion molecule, Cadherin IA, is expressed in neural tube and periocular region, but is absent from branchial arch neural crest population. A type II Cadherin homolog (Cad IIA), similar to Cad-6/7/10/11, shares similarities with all of its gnathostome counterparts and is found in premigratory (in the case of Cad-6) and early migrating (in the case of Cad-7 and Cad-11) neural crest, as well as in differentiating neurogenic derivatives (in the case of Cad-7 and Cad-10) (Sauka-Spengler, 2007).

The cumulative results suggest that lamprey possesses a NC-GRN which is a modified version of that hypothesized to function in gnathostomes (Sauka-Spengler, 2007).

Gain- and loss-of-function experiments performed in various jawed vertebrates give clues about the genetic interactions leading to neural crest specification; e.g., morpholino knockdown of neural plate border specifiers Msx1, Msx2, Pax3, and Zic1 in Xenopus, as well as Pax7 in chick, causes alterations in expression of neural crest specifiers Slug, FoxD3, Sox9, and Sox10. Concomitantly, inactivation of these neural plate border specifiers leads to the expansion of Pax3 and Zic1 and the neural marker Sox2. Inactivation of early (c-Myc, Ids, or AP2) and late (Sox8, Sox9, and Sox10) gnathostome neural crest specifiers affects expression of all neural crest specifiers. However, functional evidence for aspects of this putative network often conflicts between different jawed vertebrates; e.g, depletions of AP2, FoxD3, or Sox10 in Xenopus disrupts neural crest induction, while in zebrafish, their knockdown impinges on differentiation but has no apparent effect on induction. These differences may be due to the tetraploidy of zebrafish, compensation by redundant paralogs, or both. The emerging data suggest that the neural crest specifiers extensively cross-regulate to maintain their expression, though hierarchical relationships remain difficult to ascribe (Sauka-Spengler, 2007).

To better understand the network and obtain a more comprehensive picture of the relationships between its elements, the effects were examined of knockdown of three neural plate border and five neural crest specifier genes on more neural crest markers than has previously been done in any other vertebrate (SoxE1, SoxE2, FoxD-A, AP2, n-Myc, and Id. Although Snail is typically used as a crest marker in Xenopus, its quasi-ubiquitous presence during premigratory stages in lamprey obviated its usefulness in this study (Sauka-Spengler, 2007).

In comparing the current results with those previously described in gnathostomes, it was found that inactivation of border specifiers MsxA, Pax3/7, and ZicA results in depletion of neural crest specifier expression, consistent with observations in Xenopus. However, lamprey neural plate border specifiers do not appear to mutually coactivate. An expansion of the dorsal neural tube was observed and, correspondingly, of Pax3/7 expression therein, suggesting that inactivation of border specifiers may result in a fate conversion from neural crest to neural tube. Because many of the neural plate border specifiers are later expressed in the dorsal neural tube, they appear to have later and separate functions in the developing nervous system. These data show that inactivation of FoxD-A, n-Myc, or Id decreases expression of Pax3/7, ZicA, and SoxB1 in the roof plate, in agreement with findings in Xenopus that FoxD3 induces Zic1 and neural markers, whereas Sox9 is required for later expression of Pax3 and Msx1 (Sauka-Spengler, 2007).

Interestingly, rescue experiments using Xenopus Zic1, Msx1, AP2, and Sox9, as well as chick Pax3 mRNA, suggest that these heterospecific proteins can functionally compensate for the loss of their lamprey orthologs. These experiments imply that the protein structure of these transcription factors has been sufficiently conserved during vertebrate evolution to be interchangeable in the context of neural-crest-inducing function (Sauka-Spengler, 2007).

Traditionally the neural crest is considered an evolutionary innovation of vertebrates, since protochordates lack bona fide neural crest. In cephalochordates signaling molecules like BMP, Notch, and Wnt are expressed in a pattern closely resembling that of vertebrates, consistent with their conserved role in patterning the early ectoderm. Furthermore, in both Amphioxus and ascidians, homologs of neural plate border specifiers Msx, Zic, and Pax3/7 are present within the neural plate border territory in late gastrula/early neurula, suggesting that the initial steps of border patterning and specification are already present in protochordates. In contrast, no neural crest specifiers, with the exception of Snail, are deployed at the neural plate border. Recently, a large number of gene interactions were tested in the ascidian Ciona intestinalis using morpholino-mediated gene knockdown. While interactions related to later events in central nervous system formation appear to be conserved between urochordates and vertebrates, neural-crest-specific links are absent in Ciona, and only the activation of Snail by Zic is reminiscent of the vertebrate NC-GRN (Sauka-Spengler, 2007).

Evolution of neural crest was likely driven by changes at the gene-regulatory level that may include co-option of ancestral gene batteries to a new purpose, as well as recruitment of a supplementary transcription factor or factors into the regulatory cascade. While the proximal gene regulatory elements are highly conserved between lamprey and gnathostomes, the neural crest specifier portion can clearly be subdivided into two temporally separated subsets. More distal regulatory modules that involve deployment of intracellular and extracellular signaling cues and gene batteries responsible for migration and differentiation of neural crest cells are present, implying a high degree of evolutionary constraint. Differences between gnathostome models are likely to reflect lineage-specific alterations in expression of paralogous genes or slight alterations in degrees of cis-regulatory robustness (Sauka-Spengler, 2007).

This study shows that the molecular mechanisms guiding formation of neural crest are a vertebrate synapomorphy. As such, this conserved network fits the proposed criteria for defining gene regulatory networks functioning during development of animal body plans. The NC-GRN is composed of one or more 'kernels'. The neural plate border regulatory module is an evolutionarily inflexible unit that plays an essential upstream function in establishing the identity of the neural crest progenitor territory and is also found in protochordates that lack bona fide neural crest. It is likely that the incorporation of the neural crest specifier module into the network led to the vertebrate innovation of the neural crest kernel consisting of two interconnected parts -- the neural plate border and neural crest specifier modules. Other 'plug-ins' and 'switches' may have been co-opted into the circuit from existing developmental programs. Such plug-ins may provide signaling inputs (Wnts) or guidance cues (Npn/Sema ligand-receptor couple), whereas switches like Myc/Id, integrated at the specification level of the network, provide a mechanism of cell cycle control that alternates between neural crest cell proliferation and cell death (Sauka-Spengler, 2007).

Addition of neural crest modules to the network occurred prior to separation of jawed and jawless vertebrates, likely during the transition from protochordates to vertebrates. This reflects an ancient origin of the NC-GRN during the early Cambrian period within the estimated 200 million years between the divergence of cephalochordates and vertebrates. Furthermore, it is likely that 'differentiation' subcircuits may have been incorporated and co-opted to more proximal use in revising the NC-GRN from agnathans to gnathostomes. As an example, Ets1 and Twist are found to be deployed late in migratory and postmigratory lamprey neural crest, but exist more proximally in the gnathostome network. Thus, though a neural crest gene network was largely fixed at the base of vertebrates, there appears to be remodeling of individual subcircuits that may be responsible for species-specific traits. It is interesting to note that a recent paper reported the successful isolation of embryos from another agnathan, hagfish, for the first time after 100 years of known attempts throughout the literature. Intriguingly, the gene expression patterns for the neural crest markers reported in this study appear highly reminiscent of those of lamprey (Sauka-Spengler, 2007).

These findings are all the more significant when taking into account recent fossil finds suggesting that modern lampreys are 'living fossils,' with similar characteristics to the common ancestor with jawed vertebrates, thus reflecting the primitive vertebrate condition and occupying an important ancestral position. Prior to this study, only two genes were studied thoroughly in the context of early events in neural crest formation in the lamprey. By studying a large group of molecules, these observations couple the formation of the neural crest proper with the establishment of a NC-GRN at the dawn of vertebrates, pushing back the date that such a gene regulatory network was invented by at least 200 million years, and thus giving deep insight into the steps necessary for the creation of defining vertebrate features (Sauka-Spengler, 2007).

Wnt signalling is required for neural crest (NC) induction; however, the direct targets of the Wnt pathway during NC induction remain unknown. This study shows that the homeobox gene Gbx2 is essential in this process and is directly activated by Wnt/beta-catenin signalling. By ChIP and transgenesis analysis it was shown that Gbx2 regulatory elements that drive expression in the NC respond directly to Wnt/beta-catenin signalling. Gbx2 has previously been implicated in posteriorization of the neural plate. This study unveils a new role for this gene in neural fold patterning. Loss-of-function experiments using antisense morpholinos against Gbx2 inhibit NC and expand the preplacodal domain, whereas Gbx2 overexpression leads to transformation of the preplacodal domain into NC cells. The NC specifier activity of Gbx2 is dependent on the interaction with Zic1 and the inhibition of preplacodal genes such as Six1. In addition, that Gbx2 is upstream of the neural fold specifiers Pax3 and Msx1. These results place Gbx2 as the earliest factor in the NC genetic cascade being directly regulated by the inductive molecules, and support the notion that posteriorization of the neural folds is an essential step in NC specification. A new genetic cascade is proposed that operates in the distinction between anterior placodal and NC territories (Li, 2009).

WNTs and eye development

Eye development in both invertebrates and vertebrates is regulated by a network of highly conserved transcription factors. However, it is not known what controls the expression of these factors to regulate early eye formation and whether transmembrane signaling events are involved. A role for signaling via a member of the frizzled family of receptors has been established in regulating early eye development. Overexpression of Xenopus frizzled 3 (Xfz3), a receptor expressed during normal eye development, functions cell autonomously to promote ectopic eye formation and can perturb endogenous eye development. Ectopic eyes obtained with Xfz3 overexpression have a laminar organization similar to that of endogenous eyes and contain differentiated retinal cell types. Ectopic eye formation is preceded by ectopic expression of transcription factors involved in early eye development, including Pax6, Rx, and Otx2. Conversely, targeted overexpression of a dominant-negative form of Xfz3 (Nxfz3), consisting of the soluble extracellular domain of the receptor, results in suppression of endogenous Pax6, Rx, and Otx2 expression and suppression of endogenous eye development. This effect can be rescued by coexpression of Xfz3. Finally, overexpression of Kermit, a protein that interacts with the C-terminal intracellular domain of Xfz3, also blocks endogenous eye development, suggesting that signaling through Xfz3 or a related receptor is required for normal eye development. In summary, frizzled signaling is both necessary and sufficient to regulate eye development in Xenopus (Rasmussen, 2001).

The observation that Xfz3 can initiate ectopic eye formation identifies wnt signaling as the first identified extracellular signaling pathway that regulates eye formation. Several wnts, including Xwnt-1, Xwnt-3A, and Xwnt-8, are expressed in the anterior neural plate in a region that overlaps the eye fields. In addition, Xwnt-1 is much more potent than any other wnt ligand in synergizing with Xfz3 to promote both axis duplication and neural crest induction. However, given the relative promiscuity of binding and the number of wnts present in the developing nervous system, it remains to be determined which wnt regulates eye development in vivo (Rasmussen, 2001).

Frizzled activation can lead to signaling either through a canonical pathway involving ß-catenin or noncanonical pathways that regulate planar cell polarity in Drosophila and possibly vertebrates, as well as calcium mobilization and protein kinase C activation in Xenopus and zebrafish. The signaling pathway used by Xfz3 to promote eye development has not yet been defined, although limited evidence points to the noncanonical planar cell polarity pathway. Overexpression of Xfz3 alone (unlike that of Xfz8) does not lead to axis duplication, a phenotype linked to activation of the canonical signaling pathway, although coexpression of Xfz3 with Xwnt1 can promote efficient axis duplication. Expression of the closely related homolog Mfz3 in Xenopus embryos results in protein kinase C activation but not expression of siamois and Xnr3, which are downstream effectors in the canonical pathway. In addition, activation of the canonical wnt signaling pathway represses anterior neural development, arguing against mediation of the regulation of eye development by this pathway, although localized activation of the canonical pathway at later stages of development has not been examined (Rasmussen, 2001).

Eight-cell RNA injection was used to overexpress a truncated form of Dsh (Dsh-DeltaN), which preferentially activates the noncanonical planar cell polarity pathway. It was found that 28% of the embryos (31/110) had dense ectopic pigment at or near the midline in the region of the hindbrain, reminiscent of the phenotype observed with Xfz3 overexpression. Conversely, injection of RNA encoding a truncated form of Dsh (Dsh-DEP+), which preferentially inhibits the noncanonical planar cell polarity pathway, resulted in reduced or missing eyes in 51% of injected embryos (52/101), similar to what was observed with Nxfz3 overexpression. These findings implicate the noncanonical planar cell polarity signaling pathway in the regulation of eye development, although this has yet to be confirmed (Rasmussen, 2001).

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance. The expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, it has been demonstrated that the addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, the expression of several early lens placode-specific markers were examined in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway that is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. It is concluded that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation (Wawersik 1999).

Recent studies show that specification of some neural crest lineages occurs prior to or at the time of migration from the neural tube. Signaling events establishing the melanocyte lineage, which has been shown to migrate from the trunk neural tube after the neuronal and glial lineages, have been examined. Using in situ hybridization, it has been found that, although Wnts are expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, the Wnt inhibitor cfrzb-1 is expressed in the neuronal and glial precursors and not in melanoblasts. This expression pattern suggests that Wnt signaling may be involved in specifying the melanocyte lineage. Wnt-3a-conditioned medium dramatically increases the number of pigment cells in quail neural crest cultures while decreasing the number of neurons and glial cells, without affecting proliferation. Conversely, BMP-4 is expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, but is decreased coincident with the timing of melanoblast migration. This expression pattern suggests that BMP signaling may be involved in neural and glial cell differentiation or repression of melanogenesis. Purified BMP-4 reduces the number of pigment cells in culture while increasing the number of neurons and glial cells, also without affecting proliferation. These data suggest that Wnt signaling specifies melanocytes at the expense of the neuronal and glial lineages, and further, that Wnt and BMP signaling have antagonistic functions in the specification of the trunk neural crest (Jin, 2001).

The ciliary marginal zone of the vertebrate retina contains undifferentiated progenitor cells that continue to proliferate and add new neurons and glia peripherally during the embryonic stages -- even after the formation of a functional retina. To understand the molecular mechanism that controls the prolonged progenitor cell proliferation in the ciliary marginal zone, a candidate molecule approach was taken, focusing on Wnt2b (formerly know as Wnt13), which is expressed in the marginal most tip of the chicken retina. Frizzled 4 and 5, seven-pass transmembrane Wnt receptors, are expressed in the peripheral and central part of the retina, respectively. LEF1, a downstream Wnt signaling component, is expressed at high levels in the ciliary marginal zone with expression gradually decreasing towards the central retina. The LEF1-expressing region, which is where Wnt signaling is supposedly activated, expressed a set of molecular markers that are characteristic of the progenitor cells in the ciliary marginal zone. Overexpression of Wnt2b by use of in ovo electroporation in the central retina inhibits neuronal differentiation and induces the progenitor cell markers. Blocking of the Wnt downstream signaling pathway by a dominant-negative LEF1 inhibits proliferation of the cells in the marginal area, which results in their premature neuronal differentiation. The progenitor cells in the ciliary marginal zone differentiate into all the neuronal and glial cell types when cultured in vitro, and they proliferate for a longer period than do centrally located progenitor cells that undergo a limited number of cell divisions. In addition, the proliferation of these progenitor cells is promoted in the presence of Wnt2b. These results suggest that Wnt2b functions to maintain undifferentiated progenitor cells in the ciliary marginal zone, and thus serves as a putative stem cell factor in the retina (Kubo, 2003).

To study the molecular mechanism that controls the laminar organization of the retina, reaggregation cultures of dissociated retinal cells prepared from chicken embryos were used. These cells cannot generate laminated structures by themselves and, instead, form rosettes within the reaggregates. However, the dissociated cells can organize into a correctly laminated structure when cultured in the presence of a putative laminar inducing factor coming from particular tissue or cells, but its molecular identity of this factor has long remained elusive. In this study, it has been found that the anterior rim of the retina sends a signal to rearrange the rosette-forming cells into a neuroepithelial structure characteristic of the undifferentiated retinal layer. This activity of the anterior rim is mimicked by Wnt-2b expressed in this tissue, and is neutralized by a soluble form of Frizzled, which works as a Wnt antagonist. Furthermore, the neuroepithelial structure induced by Wnt-2b subsequently developed into correctly laminated retinal layers. These observations suggest that the anterior rim functions as a layer-organizing center in the retina, by producing Wnt-2b (Nakagawa, 2003).

The differentiation of epithelial cells and fiber cells from the anterior and posterior compartments of the lens vesicle, respectively, give the mammalian lens its distinctive polarity. While much progress has been made in understanding the molecular basis of fiber differentiation, little is known about factors that govern the differentiation of the epithelium. Members of the Wnt growth factor family appear to be key regulators of epithelial differentiation in various organ systems. Wnts are ligands for Frizzled receptors and can activate several signaling pathways, of which the best understood is the Wnt/ß-catenin pathway. The presence of LDL-related protein coreceptors (LRPs) 5 or 6 has been shown to be a requirement for Wnt signaling through the ß-catenin pathway. To access the role of this signaling pathway in the lens, mice were analyzed with a null mutation of lrp6. These mice have small eyes and aberrant lenses, characterized by an incompletely formed anterior epithelium resulting in extrusion of the lens fibers into the overlying corneal stroma. Multiple Wnts, including 5a, 5b, 7a, 7b, 8a, 8b, and Frizzled receptors 1, 2, 3, 4, and 6, are detected in the lens. Expression of these molecules is generally present throughout the lens epithelium and extended into the transitional zone, where early fiber elongation occurs. In addition to both LRP5 and LRP6, the expression was shown of other molecules involved in Wnt signaling and its regulation, including Dishevelleds, Dickkopfs, and secreted Frizzled-related proteins. Taken together, these results indicate a role for Wnt signaling in regulating the differentiation and behavior of lens cells (Stump, 2003).

Wnt signaling is implicated in many developmental processes, including cell fate changes. Several members of the Wnt family, as well as other molecules involved in Wnt signaling, including Frizzled receptors, LDL-related protein co-receptors, members of the Dishevelled and Dickkopf families, are known to be expressed in the lens during embryonic or postembryonic development. However, the function of Wnt signaling in lens fiber differentiation remains unknown. GSK-3ß kinase has been shown to be inactivated and ß-catenin accumulates during the early stages of lens fiber cell differentiation. In an explant culture system, Wnt conditioned medium (CM) induces the accumulation of ß-crystallin, a marker of fiber cell differentiation, without changing cell shape. In contrast, epithelial cells stimulated with Wnt after priming with FGF elongate, accumulate ß-crystallin, aquaporin-0, p57kip2, and alter their expression of cadherins. Treatment with lithium, which stabilizes ß-catenin, induces the accumulation of ß-crystallin, but explants treated with lithium after FGF priming do not elongate as they do after Wnt application. These results show that Wnts promote the morphological aspects of fiber cell differentiation in a process that requires FGF signaling, but is independent of ß-catenin. Wnt signaling may play an important role in lens epithelial-to-fiber differentiation (Lyu, 2004).

In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye (Masai, 2005).

During the development of the central nervous system, cell proliferation and differentiation are precisely regulated. In the vertebrate eye, progenitor cells located in the marginal-most region of the neural retina continue to proliferate for a much longer period compared to the ones in the central retina, thus showing stem-cell-like properties. Wnt2b is expressed in the anterior rim of the optic vesicles, and has been shown to control differentiation of the progenitor cells in the marginal retina. Stable overexpression of Wnt2b in retinal explants inhibits cellular differentiation and induces continuous growth of the tissue. Notably, Wnt2b maintained the undifferentiated progenitor cells in the explants even under the conditions where Notch signaling is blocked. Wnt2b downregulates the expression of multiple proneural bHLH genes as well as Notch. In addition, expression of Cath5 under the control of an exogenous promoter suppresses the negative effect of Wnt2b on neuronal differentiation. Importantly, Wnt2b inhibits neuronal differentiation independently of cell cycle progression. It is proposed that Wnt2b maintains the naive state of marginal progenitor cells by attenuating the expression of both proneural and neurogenic genes, thus preventing those cells from launching out into the differentiation cascade regulated by proneural genes and Notch (Kudo, 2005).

During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. Two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/ß-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/ß-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results suggest a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells (Cavodeassi, 2005).

These data add to the body of evidence that Wnt/β-catenin signaling regulates the regionalization of the forebrain. Overactivation of Wnt/β-catenin signaling promotes posterior diencephalic fates and suppresses anterior telencephalic and eye field identities. It is further shown that local suppression of Wnt/β-catenin signaling can expand eye field markers caudally into the posterior diencephalon. There are at least three Wnts potentially involved in this process: Wnt1, Wnt10b, and Wnt8b. However, a number of results argue in favor of Wnt8b being the one most likely involved in the regionalization of the forebrain. While wnt8b is expressed in the posterior diencephalon, wnt1 and wnt10b are expressed more posteriorly. Moreover, wnt1/wnt10b double mutants/morphants do not show an obvious patterning defect in the forebrain, and the slight posterior expansion of the eye field found in wnt8b morphants is not significantly enhanced in the wnt8b/wnt10b/wnt1 triple morphants (Cavodeassi, 2005).

The results strengthen the hypothesis that Fz8a is the receptor responsible for transducing the Wnt8b signal. fz8a is expressed in a broad domain within the ANP, consistent with the entire prospective forebrain being susceptible to reception of Wnt8b signals in a graded posterior/high to anterior/low fashion. Still, it is unclear whether Wnts can exert their action at a distance or can act only locally. A scenario is favored in which Wnt8b would be working as a short-range signal, since Wnt8b is required for the formation of diencephalon and midbrain, the main territories where it is expressed, and to establish the posterior boundary of the eye field, which is located no more than a few cell rows away from the anterior boundary of the wnt8b domain. Specification of the eye field more anteriorly requires local suppression of Wnt/β-catenin signaling, but as yet, there is no evidence that Wnts signaling through the β-catenin branch of the pathway significantly encroach throughout the eye field during gastrula stages of normal development (Cavodeassi, 2005).

Similar to the eye field, induction of the telencephalon also requires suppression of Wnt/β-catenin signals. What, then, might specify the difference between eye field and telencephalon? Slight differences in the level of Wnt signaling may be enough to effect the separation of these two territories. Alternatively, additional signals, such as those coming from the margin of the neural plate, may also be required for this patterning process. For instance, early-acting BMP signals promote telencephalic gene expression, but can suppress specification of eye field gene expression (Cavodeassi, 2005).

During formation of the eye, nascent eye field cells must be specified to acquire eye field identity and must undergo a program of morphogenesis quite distinct from that of adjacent forebrain territories. This study shows that a noncanonical Wnt pathway activated by Wnt11 in the eye field helps to coordinate these two events. Wnt11 function may direct the morphogenesis of the eye field by maintaining the coherence of this territory. Simultaneously, noncanonical Wnt activity would consolidate the extent of the territory defined as eye field by keeping it refractory to any residual Wnt/β-catenin signals encroaching from more posterior domains. Thus, through the coordinated antagonism of signals that suppress retinal identity and promotion of cell coherence, Wnt11 and Fz5 signaling would link induction and morphogenesis during the early stages of eye development (Cavodeassi, 2005).

Wnt signaling orchestrates multiple aspects of central nervous system development, including cell proliferation and cell fate choices. In this study, gene transfer was used to activate or inhibit canonical Wnt signaling in vivo in the developing eye. The expression of Wnt2b or constitutively active (CA) ß-catenin inhibited retinal progenitor gene (RPG) expression and the differentiation of retinal neurons. In addition, Wnt signal activation in the central retina is sufficient to induce the expression of markers of the ciliary body and iris, two tissues derived from the peripheral optic cup (OC). The expression of a dominant-negative (DN) allele of Lef1, or of a Lef1-engrailed fusion protein, leads to the inhibition of expression of peripheral genes and iris hypoplasia, suggesting that canonical Wnt signaling is required for peripheral eye development. It is proposed that canonical Wnt signaling in the developing optic vesicle (OV) and OC plays a crucial role in determining the identity of the ciliary body and iris. Because wingless (wg) plays a similar role in the induction of peripheral eye tissues of Drosophila, these findings indicate a possible conservation of the process that patterns the photoreceptive and support structures of the eye (Cho, 2006).

These findings provide an additional link between the development of the vertebrate and invertebrate eye. In Drosophila, photoreceptor cells are surrounded at the periphery with a non-neural cuticular structure. wg, the Drosophila homolog of the Wnt genes, is expressed in the margin of the eye imaginal disc, which is the anlage of peripheral eye tissues. Activation of wg, or armadillo, the Drosophila ß-catenin, in the eye imaginal disc promotes head cuticle formation at the expense of ommatidia, and has been proposed to act as a morphogen to pattern the peripheral structures (Cho, 2006).

Wnt signaling thus promotes the development of the non-neural, peripheral support structures in both Drosophila and chicks. The similarity of wg/Wnt expression and function in eye development provides an additional line of evidence that strengthens the proposed evolutionary conservation of the vertebrate and invertebrate eyes. The modern version of this model originated with the observation of a conserved expression and activity for the eyeless/Pax6 gene. The fact that wg/Wnt appears to play a role in patterning the central and peripheral eye structures suggests that the visual structure of the last common ancestor of flies and vertebrates had not only a photoreceptive component, but a support structure as well. A conserved unit of neural and non-neural eye tissues has also been suggested by the observation of a single-celled dinoflagellate that has several of the support structures of an eye, including pigment, a lens, a cornea and a photoreceptor. The fact that Pax6 plays a role in the development of not only the NR, but also the supporting tissues, such as the lens, cornea, iris and RPE, might also be seen as being in keeping with this model (Cho, 2006).

Accurate retinotectal axon pathfinding depends upon the correct establishment of dorsal-ventral retinal polarity. Dorsal retinal gene expression is regulated by Wnt signaling in the dorsal retinal pigment epithelium (RPE). A Wnt reporter transgene and Wnt pathway components are expressed in the dorsal RPE beginning at 14-16 hours post-fertilization. In the absence of Wnt signaling, tbx5 and Bmp genes initiate normal dorsal retinal expression but are not maintained. The expression of these genes is rescued by the downstream activation of Wnt signaling, and tbx5 is rescued by Bmp signaling. Furthermore, activation of Wnt signaling cannot rescue tbx5 in the absence of Bmp signaling, suggesting that Wnt signaling maintains dorsal retinal gene expression by regulating Bmp signaling. A model is presented in which dorsal RPE-derived Wnt activity maintains the expression of Bmp ligands in the dorsal retina, thus coordinating the patterning of these two ocular tissues (Veien, 2008).

This study has shown that Wnt signaling is required for the proper development of DV retinal polarity. Expression analysis suggests that Wnt signaling functions in the RPE, while Bmp ligands are expressed in both the RPE and retina. The results demonstrate that dorsal retinal genes initiate their expression normally at around 12 hpf in the absence of Wnt signaling, but soon thereafter require Wnt signaling for their maintained expression in the dorsal retinal domain. The expression of Bmp ligands in the dorsal retina is dependent on Wnt signaling, and following Wnt inhibition the loss of at least one Bmp ligand, gdf6a, can be rescued by activation of Wnt signaling. In addition, tbx5, an early marker of dorsal identity, is rescued by the activation of either Wnt or Bmp signaling following Wnt inhibition. By contrast, tbx5 cannot be rescued by the activation of Wnt signaling in the absence of Bmp signaling. These data together suggest a model for the maintenance of DV retinal identity in which Wnt signaling in the dorsal RPE transcriptionally maintains Bmp expression in the dorsal RPE and retina, which regulates the expression of downstream DV axis genes, including tbx5 and Ephrin B axon guidance molecules. This mechanism provides a conduit through which a Wnt signal in the RPE can exert its effects in the neural retina. It is likely that this mechanism functions to maintain the integrity of the dorsal retinal domain by coordinating its patterning with the dorsal RPE, but detailed fate-mapping in the developing retina and RPE is needed to show this conclusively (Veien, 2008).

In the vertebrate retina, stem cell-like progenitor cells are maintained in a distinct region called the ciliary marginal zone (CMZ). Canonical Wnt signaling regulates the maintenance of the progenitor cells in the CMZ. However, its downstream molecular mechanisms have remained largely unclear. This study shows that chick Hairy1, an established Notch signaling effector, mediates the Wnt-dependent maintenance of CMZ progenitor cells in chicken. Interestingly, unlike other developmental contexts in which Hes gene expression is regulated by Notch signaling, Hairy1 expression in the CMZ is regulated by Wnt signaling. Hairy1 is necessary and sufficient for the expression of a set of molecular markers characteristic of the CMZ, and Wnt2b fails to induce CMZ markers when Hairy1 activity is inhibited. Furthermore, microarray analysis identifies multiple Wnt-responsive transcription factors that activate Hairy1 expression. It is proposed that Hairy1 functions as a node downstream of Wnt signaling to maintain progenitor cells in the chick CMZ (Kubo, 2009).

Progenitor cells in the central nervous system must leave the cell cycle to become neurons and glia, but the signals that coordinate this transition remain largely unknown. Wnt signaling, acting through Sox2, promotes neural competence in the Xenopus retina by activating proneural gene expression. This study reports that Wnt and Sox2 inhibit neural differentiation through Notch activation. Independently of Sox2, Wnt stimulates retinal progenitor proliferation and this, when combined with the block on differentiation, maintains retinal progenitor fates. Feedback inhibition by Sox2 on Wnt signaling and by the proneural transcription factors on Sox2 mean that each element of the core pathway activates the next element and inhibits the previous one, providing a directional network that ensures retinal cells make the transition from progenitors to neurons and glia (Agathocleous, 2009).

Wnt/β-catenin signaling acting through Sox2 activates proneural gene expression in the frog retina. This study shows that Wnt and Sox2 inhibit proneural action through Notch, thereby blocking neuronal differentiation. In addition, Wnt signaling stimulates proliferation independently of Sox2, maintaining the progenitor fate, while Sox2 pushes retinal progenitors to Müller glial fates. Concurrent activation of Sox2 and the cell cycle can recapitulate the effects of Wnt in maintaining the retinal precursor cell (RPC) fate. Finally, inhibition of Wnt signaling by Sox2, and of Sox2 by the proneural transcription factors, facilitates a transition from proliferation to differentiation, thereby ensuring that progenitors progress forwards to a differentiated state (Agathocleous, 2009).

These results tie together disparate strands in the function of Wnt/β-catenin and Sox2 signaling as investigated in various vertebrate models. Sox2 both sets up neural potential and inhibits terminal neuronal differentiation. The present study shows that Sox2 plays a central role in suppressing retinal neurogenesis downstream of Wnt/β-catenin signaling, but it enhances Müller glial differentiation and does not maintain progenitors. Similarly, Sox2 overexpression increases Müller cells in mouse retinal explants and promotes the in vitro differentiation of neocortical progenitors into astroglial cells. Notch activation by Sox2 may be involved in this gliogenic effect, as activated Notch signaling promotes gliogenesis. Therefore, either the absence of proneural gene expression or the suppression of proneural activity allows retinal progenitors to adopt the glial fate (Agathocleous, 2009).

The Wnt pathway is activated in the peripheral retina near the ciliary marginal zone in other species besides Xenopus. Yet, Wnt activation in the chick causes cells to be blocked in a proneural-negative progenitor state and in the mouse they assume non-neuronal peripheral fates. In chick and mouse, Wnt signaling does not appear to regulate Sox gene expression; however, the suppression of neurogenesis via activation of Wnt/β-catenin is common to the frog, chick and mammalian retina (Agathocleous, 2009).

There is strong evidence for connections between Wnt/β-catenin, SoxB1 and proneural genes in the regulation of neural differentiation in other tissues. In the zebrafish hypothalamus, canonical Wnt signaling, acting via Sox3, is necessary for the expression of proneural and neurogenic genes. LRP mutant mice exhibit dramatic hypoplasia of the developing neocortex owing to a reduction in neurogenesis as well as in proliferation. Similarly, in the adult hippocampus, Wnt activation promotes both neurogenesis and stem cell proliferation in a dissociable manner, which fits with the explanation that Wnt/β-catenin signaling sets up neuronal potential but then suppresses differentiation and maintains progenitor cells (Agathocleous, 2009).

The results suggest two parallel aspects of the progenitor cell fate: the suppression of neuronal differentiation and the maintenance of proliferative ability, controlled by two branches of Wnt signaling, one of which is Sox2 dependent. This model fits with findings in the spinal cord that Wnt activates proliferation, whereas Sox2 does not. The parallel control of differentiation and proliferation might be a more general feature of Wnt signaling; for example, in the developing limb, Wnt/β-catenin signaling and Sox9 interact to couple proliferation and chondrocyte differentiation (Agathocleous, 2009).

If Sox2 is not mediating the proliferative effects of Wnt/β-catenin signaling, other effectors must be involved. Although exogenous Cyclin E1 was able to cooperate with Sox2 in progenitor maintenance, little or no change was detected in Cyclin E1 retinal expression after Wnt signaling perturbations, nor in the expression of other cell cycle activators including Cyclin D1, Cyclin A2, n-Myc and c-Myc, suggesting that these genes might not be transcriptional targets in the frog retina. Perhaps other genes might function as Wnt-dependent effectors of proliferation here, or perhaps proliferation is regulated through post-transcriptional mechanisms or by changing the mode of progenitor division (Agathocleous, 2009).

Müller cells are transcriptionally very similar to neuroepithelial progenitor cells. They can divide after injury or provision of growth factors, at which point they may return to a neuroepithelial-like state, perhaps through a Wnt-dependent mechanism. These and results therefore suggest that a crucial distinction between RPCs and Müller cells is a Wnt-mediated capacity to proliferate (Agathocleous, 2009).

For the progression from a progenitor to a neuronal fate, both Wnt/β-catenin signaling and Sox2 must be switched off, relieving the inhibition of proneural activity and stopping proliferation. The inhibition of Wnt by Sox2 is likely to take place during retinogenesis, as Sox2 injections do not result in early defects in the specification of retinal progenitor identity. This therefore suggests a negative-feedback mechanism of Sox2 on Wnt signaling. Interestingly, mutations in human SOX2 associated with anophthalmia have been mapped to the C-terminal domain, which normally interacts with β-catenin, resulting in an inability of Sox2 to inhibit canonical Wnt signaling in vitro (Agathocleous, 2009).

For neuronal differentiation to proceed, Sox2 must also be switched off to relieve the inhibition of proneural activity. In the Xenopus retina, it was found that the proneural bHLH transcription factor Xath5 induced a dramatic reduction of the Sox2 protein. In the cortex, a serine protease cleaves Sox2 specifically in neuronal but not glial precursors, thus relieving the block on neurogenesis. It will be interesting to see whether in the retina, proneural genes feed back on Sox2 through this mechanism or through transcriptional repression (Agathocleous, 2009).

Wnt, Sox2 and the proneural genes appear to form a modular circuit in which each step activates the subsequent step and is in turn inactivated by it, driving cells towards differentiation, while limiting the ability of an external proliferation signal, such as Wnt, to continue signaling indefinitely. The relative levels of Wnt, Sox2 and proneural genes determine where a cell lies along the pathway from proliferation to differentiation and whether it assumes a progenitor, glial or neuronal fate. Understanding fully the function of each interaction in the cascade must await a more quantitative analysis of the relationship between the participating factors. This mechanism of transition from one cell state to another by the integration of directional interactions and feedback loops resembles that reported in diverse systems; for example, during sporulation of Bacillus subtilis, where a circuit with five basic nodes displays successive hierarchical gene activations, coupled with negative-feedback loops that switch off the previous state. Further investigations will reveal whether general aspects of the mechanism that is described here are at work in other neural and non-neural tissues, and how this directional pathway integrates with other factors that help to coordinate neuronal proliferation and differentiation (Agathocleous, 2009).

RPE specification in the chick is mediated by surface ectoderm-derived BMP and Wnt signalling

The retinal pigment epithelium (RPE) is indispensable for vertebrate eye development and vision. In the classical model of optic vesicle patterning, the surface ectoderm produces fibroblast growth factors (FGFs) that specify the neural retina (NR) distally, whereas TGFbeta family members released from the proximal mesenchyme are involved in RPE specification. However, it was previously proposed that bone morphogenetic proteins (BMPs) released from the surface ectoderm are essential for RPE specification in chick. This study now shows that the BMP- and Wnt-expressing surface ectoderm is required for RPE specification. Wnt signalling from the overlying surface ectoderm is involved in restricting BMP-mediated RPE specification to the dorsal optic vesicle. Wnt2b is expressed in the dorsal surface ectoderm and subsequently in dorsal optic vesicle cells. Activation of Wnt signalling by implanting Wnt3a-soaked beads or inhibiting GSK3beta at optic vesicle stages inhibits NR development and converts the entire optic vesicle into RPE. Surface ectoderm removal at early optic vesicle stages or inhibition of Wnt, but not Wnt/beta-catenin, signalling prevents pigmentation and downregulates the RPE regulatory gene Mitf. Activation of BMP or Wnt signalling can replace the surface ectoderm to rescue MITF expression and optic cup formation. Evidence is provided that BMPs and Wnts cooperate via a GSK3beta-dependent but beta-catenin-independent pathway at the level of pSmad to ensure RPE specification in dorsal optic vesicle cells. A new dorsoventral model of optic vesicle patterning is proposed, whereby initially surface ectoderm-derived Wnt signalling directs dorsal optic vesicle cells to develop into RPE through a stabilising effect of BMP signalling (Steinfeld, 2013).

WNTs and ear development

Components of the Wnt signaling pathway are expressed in the developing inner ear. To explore their role in ear patterning, retroviral gene transfer was used to force the expression of an activated form of ß-catenin that should constitutively activate targets of the canonical Wnt signaling pathway. At embryonic day 9 (E9) and beyond, morphological defects were apparent in the otic capsule and the membranous labyrinth, including ectopic and fused sensory patches. Most notably, the basilar papilla, an auditory organ, contained infected sensory patches with a vestibular phenotype. Vestibular identity was based on: (1) stereociliary bundle morphology; (2) spacing of hair cells and supporting cells; (3) the presence of otoliths; (4) immunolabeling indicative of vestibular supporting cells; and (5) expression of Msx1, a marker of certain vestibular sensory organs. Retrovirus-mediated misexpression of Wnt3a also gave rise to ectopic vestibular patches in the cochlear duct. In situ hybridization revealed that genes for three Frizzled receptors, c-Fz1, c-Fz7, and c-Fz10, are expressed in and adjacent to sensory primordia, while Wnt4 is expressed in adjacent, nonsensory regions of the cochlear duct. It is hypothesized that Wnt/ß-catenin signaling specifies otic epithelium as macular and helps to define and maintain sensory/nonsensory boundaries in the cochlear duct (Stevens, 2003).

The development of the vertebrate inner ear is an emergent process. Its progression from a relatively simple disk of thickened epithelium within head ectoderm into a complex organ capable of sensing sound and balance is controlled by sequential molecular and cellular interactions. Fibroblast growth factor (FGF) and Wnt signals emanating from mesoderm and neural ectoderm have been shown to direct inner ear fate. However, the role of these multiple signals during inner ear induction is unclear. This study demonstrates that the action of the FGFs and Wnts is sequential, and that their roles support a model of hierarchical fate decisions that progressively restrict the developmental potential of the ectoderm until otic commitment. Signalling by Fgf3 and Fgf19 is required to initiate a proliferative progenitor region that is a precursor to both the inner ear and the neurogenic epibranchial placodes. Significantly, it was found that only after FGF action is attenuated can the subsequent action of Wnt signalling allow otic differentiation to proceed. In addition, gain and loss of function of Wnt-signalling components show a role for this signalling in repressing epibranchial fate. This interplay of signalling factors ensures the correct and ordered differentiation of both inner ear and epibranchial systems (Freter, 2009).

WNTs and tooth development

There has been rapid progress recently in the identification of signaling pathways regulating tooth development. It has become apparent that signaling networks involved in Drosophila development and the development of structures such as limbs are also used in tooth development. Teeth are epithelial appendages formed in the oral region of vertebrates; their early developmental anatomy resembles that of other strucures, such as hairs and glands. The neural crest origin of tooth mesenchyme has been confirmed and recent evidence suggests that specific combinations of homeobox genes expressed in the neural crest cells may regulate the types of teeth and their patterning. Signaling molecules in the Shh, FGF, BMP and Wnt families appear to regulate the early steps of tooth morphogenesis. Certain transcription factors associated with these pathways have been shown to be necessary for tooth development. Sonic hedgehog is expressed in dental epithelium as several stages starting in the early epithelial thickenings, then reappears in the enamel knot and subsequently is expressed in the ameloblast cell lineage. Lef-1, involved in the transduction of the Wnt signal, is expressed throughout tooth development: its expression is not restricted to either epithelial or mesenchymal tissues, although it is needed only in epithelium during early development. Lef-1 appears to be involved in the regulation of an epithelial signal acting on dental mesenchyme during the bud stage of tooth morphogenesis. Several Wnt genes are expressed during tooth development, including the Wnt-10 gene, which is expressed in early dental epithelium. Several of the conserved signals are also transiently expressed in the enamel knots in the dental epithelium. The enamel knots are associated with the characteristic epithelial folding morphogenesis, which is responsible for the development of tooth shape. It is currently believed that the enamel knots function as signaling centers, regulating the development of tooth shape. Enamel knots constitute a specific ectodermal cell lineage; it has been proposed that enamel knots determine the site of the first cusp of teeth and that they regulate the formation of other cusps in molar teeth (Thesleff, 1997).

Table of contents

wingless continued: Biological Overview | Transcriptional regulation |Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.