The maternal Xenopus beta-catenin signaling pathway, activated by frizzled homologs, induces goosecoid in a cell non-autonomous manner.

In spite of abundant evidence that Wnts play essential roles in embryonic induction and patterning, little is known about the expression or activities of Wnt receptors during embryogenesis. The isolation and expression of two maternal Xenopus frizzled genes, Xfrizzled-1 and Xfrizzled-7, is described. It is also demonstrated that both can activate the Wnt/beta-catenin signaling pathway as monitored by the induction of specific target genes. Activation of the beta-Catenin pathway has previously been shown to be necessary and sufficient for specifying the dorsal axis of Xenopus. beta-Catenin is thought to work through the cell-autonomous induction of the homeobox genes siamois and twin, that in turn bind to and activate the promoter of another homeobox gene, goosecoid. However, it was found that the beta-catenin pathway regulated the expression of both endogenous goosecoid, and a goosecoid promoter construct, in a cell non-autonomous manner. These data demonstrate that maternal Frizzleds can activate the Wnt/beta-catenin pathway in Xenopus embryos, and that induction of a known downstream gene can occur in a cell non-autonomous manner.

Direct regulation of the Xenopus engrailed-2 promoter by the Wnt signaling pathway, and a molecular screen for Wnt-responsive genes, confirm a role for Wnt signaling during neural patterning in Xenopus.

The co-activation of Wnt signaling and concomitant inhibition of BMP signaling has previously been implicated in vertebrate neural patterning, as evidenced by the combinatorial induction of engrailed-2 and krox-20 in Xenopus. However, screens have not previously been conducted to identify additional potential target genes. Using a PCR-based screening method we determined that XA-1, xCRISP, UVS.2, two UVS.2-related genes, and xONR1 are induced in response to Xwnt-3a and a BMP-antagonist, noggin. Two additional genes, connexin 30 and retinoic acid receptor gamma were induced by Xwnt-3a alone. To determine whether any of the induced genes are direct targets of Wnt signaling, we focussed on engrailed-2. In the present study we show that the Xenopus engrailed-2 promoter contains three consensus binding sites for LEF/TCF, which are HMG box transcription factors which bind to beta-catenin in response to activation of the Wnt- 1 signaling pathway. An engrailed-2 promoter luciferase reporter construct containing these LEF/TCF sites is induced in embryo explant assays by the combination of Xwnt-3a or beta-catenin and noggin. These LEF/TCF sites are required for expression of engrailed-2, as a dominant negative Xtcf-3 blocks expression of endogenous engrailed-2 as well as expression of the reporter construct. Moreover, mutation of these three LEF/TCF sites abrogates expression of the reporter construct in response to noggin and Xwnt-3a or beta-catenin. We conclude that the engrailed-2 gene is a direct target of the Wnt signaling pathway, and that Wnt signaling works with BMP antagonists to regulate gene expression during patterning of the developing nervous system of Xenopus.

Wnt and FGF pathways cooperatively pattern anteroposterior neural ectoderm in Xenopus.

Previous gain-of-function assays in Xenopus have demonstrated that Xwnt-3a can pattern neural tissue by reducing the expression of anterior neural genes, and elevating the expression of posterior neural genes. To date, no loss-of-function studies have been conducted in Xenopus to show a requirement of endogenous Wnt signaling for patterning of the neural ectoderm along the anteroposterior axis. We report that expression of a dominant negative Wnt in Xenopus embryos causes a reduction in the expression of posterior neural genes, and an elevation in the expression of anterior neural genes, thereby confirming the involvement of endogenous Wnt signaling in patterning the neural axis. We further demonstrate that the ability of Xwnt-3a to decrease expression of anterior neural genes in noggin-treated explants is dependent upon a functional FGF signaling pathway, while the elevation of expression of posterior neural genes does not require FGF signaling. The previously reported ability of FGF to elevate the expression of posterior neural genes in noggin-treated explants was found to be dependent on endogenous Wnt signaling. We conclude that neural induction occurs initially in a Wnt-independent manner, but that generation of complete anteroposterior neural pattern requires the cooperative actions of Wnt and FGF pathways.

Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development.

When overexpressed in Xenopus embryos, Xwnt-1, -3A, -8 and -8b define a functional class of Wnts (the Wnt-1 class) that promotes duplication of the embryonic axis, whereas Xwnt-5A, -4, and -11 define a distinct class (the Wnt-5A class) that alters morphogenetic movements (Du, S., S. Purcell, J. Christian, L. McGrew, and R. Moon. 1995. Mol. Cell. Biol. 15:2625-2634). Since come embryonic cells may be exposed to signals from both functional classes of Wnt during vertebrate development, this raises the question of how the signaling pathways of these classes of Wnts might interact. To address this issue, we coexpressed various Xwnts and components of the Wnt-1 class signaling pathway in developing Xenopus embryos. Members of the Xwnt-5A class antagonized the ability of ectopic Wnt-1 class to induce goosecoid expression and a secondary axis. Interestingly, the Wnt-5A class did not block goosecoid expression or axis induction in response to overexpression of cytoplasmic components of the Wnt-1 signaling pathway, beta-catenin or a kinase-dead gsk-3, or to the unrelated secreted factor, BVg1. The ability of the Wnt-5A class to block responses to the Wnt-1 class may involve decreases in cell adhesion, since ectopic expression of Xwnt-5A leads to decreased Ca2+-dependent cell adhesion and the activity of Xwnt-5A to block Wnt-1 class signals is mimicked by a dominant negative N-cadherin. These data underscore the importance of cell adhesion in modulating the responses of embryonic cells to signaling molecules and suggest that the Wnt-5A functional class of signaling factors can interact with the Wnt-1 class in an antagonistic manner.

Specification of the anteroposterior neural axis through synergistic interaction of the Wnt signaling cascade with noggin and follistatin.

Embryological data and the activities of the neural-inducing factors noggin and follistatin are consistent with the hypothesis that the nervous system is initially induced with an anterior character, with subsequent signals imparting posterior pattern. We report that Xwnt3a is a candidate for involvement in anteroposterior neural patterning, as it synergizes with the neural-inducing factors noggin and follistatin to increase the expression of posterior neural genes. Furthermore we show that beta-catenin, an intracellular protein implicated in the Wnt signal transduction cascade, mimics the activity of Xwnt3a. These data suggest that the generation of pattern within the vertebrate nervous system may rely on synergism between a Wnt signaling pathway and multiple neural-inducing factors.

Distinct expression and shared activities of members of the hedgehog gene family of Xenopus laevis.

The hedgehog family of signaling proteins is associated with a variety of spatial patterning activities in insects and vertebrates. Here we show that new members of this family isolated from Xenopus laevis are expressed embryonically in patterns suggestive of roles in patterning in the ectoderm, nervous system and somites. Banded hedgehog is expressed throughout the neural plate and subsequently in both the nervous system and in the dermatome of somites. Cephalic hedgehog is expressed in anterior ectoderm and endodermal structures, and sonic hedgehog is expressed in patterns which parallel those in other species. Injection of RNAs encoding Xenopus hedgehogs induces ectopic cement gland formation in embryos. Similar to reported activities of noggin and follistatin, Xenopus hedgehogs share a common ability to induce cement glands in animal cap explants. However, hedgehog activities in naive ectoderm appear capable of acting independently of noggin and follistatin since, although all three are induced by activin in animal cap explants, X-hh expression does not induce noggin or follistatin.

Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos.

Wnts are secreted signaling factors which influence cell fate and cell behavior in developing embryos. Overexpression in Xenopus laevis embryos of a Xenopus Wnt, Xwnt-8, leads to a duplication of the embryonic axis. In embryos ventralized by UV irradiation, Xwnt-8 restores expression of the putative transcription factor goosecoid, and rescues normal axis formation. In contrast, overexpression of Xwnt-5A in normal embryos generates defects in dorsoanterior structures, without inducing goosecoid or a secondary axis. To determine whether Xwnt-4 and Xwnt-11 fall into one of these two previously described classes of activity, synthetic mRNAs were introduced into animal caps, normal embryos, and UV-treated embryos. The results indicate that Xwnt-4, Xwnt-5A, and Xwnt-11 are members of a single functional class with activities that are indistinguishable in these assays. To investigate whether distinct regions of Xwnt-8 and Xwnt-5A were sufficient for eliciting the observed effects of overexpression, we generated a series of chimeric Xwnts. RNAs encoding the chimeras were injected into normal and UV-irradiated Xenopus embryos. Analysis of the embryonic phenotypes and goosecoid levels reveals that chimeras composed of carboxy-terminal regions of Xwnt-8 and amino-terminal regions of Xwnt-5A are indistinguishable from the activities of native Xwnt-8 and that are the reciprocal chimeras elicit effects indistinguishable from overexpression of native Xwnt-5A. We conclude that the carboxy-terminal halves of these Xwnts are candidate domains for specifying responses to Xwnt signals.

Xwnt-5A: a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis.

To contribute to an understanding of the roles and mechanisms of action of Wnts in early vertebrate development, we have characterized the normal expression of Xenopus laevis Wnt-5A, and investigated the consequences of misexpression of this putative signalling factor. Xwnt-5A transcripts are expressed throughout development, and are enriched in both the anterior and posterior regions of embryos at late stages of development, where they are found primarily in ectoderm, with lower levels of expression in mesoderm. Overexpression of Xwnt-5A in Xenopus embryos leads to complex malformations distinct from those achieved by ectopic expression of Xwnts -1, -3A, or -8. This phenotype is unlikely to result from Xwnt-5A acting as an inducing agent, as overexpression of Xwnt-5A does not rescue dorsal structures in UV-irradiated embryos, does not induce mesoderm in blastula caps, and Xwnt-5A does not alter the endogenous patterns of expression of goosecoid, Xbra, or Xwnt-8. To pursue whether Xwnt-5A has the capacity to affect morphogenetic movements, we investigated whether overexpression of Xwnt-5A alters the normal elongation of blastula cap explants induced by activin. Intriguingly, Xwnt-5A blocks the elongation of blastula caps in response to activin, without blocking the differentiation of either dorsal or ventral mesoderm within these explants. The data are consistent with Xwnt-5A having the potential activity of modifying the morphogenetic movements of tissues.

Dissecting Wnt signalling pathways and Wnt-sensitive developmental processes through transient misexpression analyses in embryos of Xenopus laevis.

We review evidence that Xenopus Wnts (Xwnts) have activities consistent with their hypothesized roles as secreted signalling factors involved in multiple developmental processes. Transient misexpression of different Xwnts has distinct effects upon early development, and upon the formation of tissues in UV-irradiated embryos. Misexpression of Xwnts also has distinct effects on the in vitro differentiation of blastula cap explants. Cellular responses to Xwnt signals include changes in gap junctional permeability, altered responsiveness to growth factors, and possibly changes in cell adhesion. Current data suggest that a maternal Xwnt- or noggin-like activity is involved in the Nieuwkoop center activity during mesoderm induction, that Xwnt-8 participates in a pathway of differentiation as ventral mesoderm, and that Xwnt-5A is a potential modulator of morphogenetic movements.

Expression and potential functions of G-protein alpha subunits in embryos of Xenopus laevis.

During early embryonic development, many inductive interactions between tissues depend on signal transduction processes. We began to test the possibility that G-proteins participate in the signal transduction pathways that mediate neural induction. The expression during Xenopus development of three G alpha subunits, G alpha 0, G alpha i-1 and G alpha s-1, was characterized. The three maternally expressed genes showed different expression patterns during early development. Whole-mount in situ hybridization revealed that all three genes were expressed almost exclusively in the gastrula ectoderm and predominantly in the neuroectoderm in the neurula embryo. In order to investigate the involvement of these proteins in neural induction, we overexpressed the G-protein alpha subunits by injecting the G alpha mRNAs into fertilized eggs. Overexpression of G alpha s-1 increased the ability of gastrula ectoderm to become induced to neural tissue approximately four-fold. Overexpression of G alpha 0 and G alpha i-1 had less pronounced effects on neural competence, and inhibition of the G alpha 0 and G alpha i-1 proteins by pertussis toxin did not change the neural competence of the exposed gastrula ectoderm. Overexpression of the G alpha 0 and G alpha i-1 genes did, however, inhibit the normal disappearance of the blastocoel during gastrulation, suggesting a role for these G-proteins in regulating this process. The data also suggest a specific role for the G alpha s subunit in mediating the initial phases of neural induction.

Analysis of Xwnt-4 in embryos of Xenopus laevis: a Wnt family member expressed in the brain and floor plate.

This study characterizes the temporal and spatial expression during early Xenopus development of Xwnt-4, a member of the Wnt gene family. The Xwnt-4 protein contains all of the sequence motifs that are hallmarks of the Wnt gene family and is 84% identical to the mouse homolog, Wnt-4. The highest level of Xwnt-4 expression occurs during the early neurula stage of development although its expression persists throughout embryogenesis and can be found in the adult testis, brain and epithelium. Consistent with its localization to head and dorsal regions of microdissected embryos, the expression of Xwnt-4 is enhanced in anterodorsalized embryos resulting from treatment with LiCl, and the expression of Xwnt-4 is suppressed in UV-ventralized embryos that lack anterior neural tissue. These results suggested that expression of Xwnt-4 is dependent on the induction of neural tissue. This idea was tested using induction experiments with dorsal or ventral ectoderm from a stage 10 embryo, recombined with dorsal marginal zone mesoderm from the same embryo. Recombinant tissue and ectoderm alone were cultured until stage 14, when Xwnt-4 expression was assayed using Northern analysis. In the recombinant assay, Xwnt-4 expression does not occur in the uninduced ectoderm but is expressed in both the dorsal and ventral recombinants. Xwnt-4 expression in neural ectoderm was confirmed in isolated, induced neural ectoderm, dissected away from the dorsal mesoderm, in a stage 12.5 embryo. Whole-mount in situ hybridization confirmed the dissection studies and demonstrated that Xwnt-4 transcripts are expressed in the dorsal midline of the midbrain, hindbrain and the floor plate of the neural tube. Collectively, the data indicate that Xwnt-4 is a unique member of the Wnt family whose expression is dependent on neural induction. The specific pattern of expression following neural induction suggests that Xwnt-4 plays a role in the early patterning events responsible in the formation of the nervous system in Xenopus.

Translational control by cytoplasmic polyadenylation during Xenopus oocyte maturation: characterization of cis and trans elements and regulation by cyclin/MPF.

The expression of certain maternal mRNAs during oocyte maturation is regulated by cytoplasmic polyadenylation. To understand this process, we have focused on a maternal mRNA from Xenopus termed G10. This mRNA is stored in the cytoplasm of stage 6 oocytes until maturation when the process of poly(A) elongation stimulates its translation. Deletion analysis of the 3' untranslated region of G10 RNA has revealed that two sequence elements, UUUUUUAU and AAUAAA were both necessary and sufficient for polyadenylation and polysomal recruitment. In this communication, we have defined the U-rich region that is optimal for polyadenylation as UUUUUUAUAAAG, henceforth referred to as the cytoplasmic polyadenylation element (CPE). We have also identified unique sequence requirements in the 3' terminus of the RNA that can modulate polyadenylation even in the presence of wild-type cis elements. A time course of cytoplasmic polyadenylation in vivo shows that it is an early event of maturation and that it requires protein synthesis within the first 15 min of exposure to progesterone. MPF and cyclin can both induce polyadenylation but, at least with respect to MPF, cannot obviate the requirement for protein synthesis. To identify factors that may be responsible for maturation-specific polyadenylation, we employed extracts from oocytes and unfertilized eggs, the latter of which correctly polyadenylates exogenously added RNA. UV crosslinking demonstrated that an 82 kd protein binds to the U-rich CPE in egg, but not oocyte, extracts. The data suggest that progesterone, either in addition to or through MPF/cyclin, induces the synthesis of a factor during very early maturation that stimulates polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal mRNA expression in early development: regulation at the 3' end.

Early development in many animals is programmed by maternal mRNAs inherited by the fertilized egg. Many of these RNAs are translationally dormant in immature oocytes, but are recruited onto polysomes during meiotic maturation or fertilization. Polyadenylation plays a major role in controlling the translation of maternal mRNA during these times of development. Polyadenylation, in turn, is dependent upon two cis elements that reside in the 3'-terminal region of responsive mRNAs. In two cases, the factors that interact with these regions have been examined. The half-life of maternal mRNA also is regulated by polyadenylation, which again is controlled by 3'-terminal cis elements. The recent literature covering these topics is reviewed.

Xenopus oocyte poly(A) RNAs that hybridize to a cloned interspersed repeat sequence are not translatable.

A cDNA clone encoding an interspersed repeat RNA from Xenopus oocytes was isolated. Each strand of the cDNA clone hybridized to several different oocyte transcripts of diverse size. Many of these transcripts were present in poly(A) RNA at least up to the neurula stage. DNA sequence analysis and hybrid selection and in vitro translation show that molecules of this repeat family are not translatable.

Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element.

Xenopus oocytes contain several mRNAs that are mobilized into polysomes only at the completion of meiosis (maturation) or at specific times following fertilization. To investigate the mechanisms that control translation during early development, we have focused on an mRNA, termed G10, that is recruited for translation during oocyte maturation. Coincident with its translation, the poly(A) tail of this message is elongated from approximately 90 to 200 adenylate residues. To identify the cis sequence that is required for this cytoplasmic adenylation and recruitment, we have synthesized wild-type and deletion mutant G10 mRNAs with SP6 polymerase. When injected into oocytes that subsequently were induced to mature with progesterone, wild-type G10 mRNA, but not mutant transcripts lacking a 50-base sequence in the 3'-untranslated region, was polyadenylated and recruited for translation. The 50-base sequence was sufficient to confer polyadenylation and translation when fused to globin mRNA, which does not normally undergo these processes during oocyte maturation. Further mutational analysis of this region revealed that a U-rich sequence 5' to the AAUAAA hexanucleotide nuclear polyadenylation signal, as well as the hexanucleotide itself, were both required for polyadenylation and translation. The 50-base cis element directs polyadenylation, but not translation per se, as a transcript that terminates with 3'-deoxyadenosine (cordycepin) is not recruited for translation. The available data suggest that the dynamic process of polyadenylation, and not the length of the poly(A) tail, is required for translational recruitment during oocyte maturation.