An anterior signalling centre in Xenopus revealed by the homeobox gene XHex.

BACKGROUND: Signals from anterior endodermal cells that express the homeobox gene Hex initiate development of the most rostral tissues of the mouse embryo. The dorsal/anterior endoderm of the Xenopus gastrula, which expresses Hex and the putative head-inducing gene cerberus, is proposed to be equivalent to the mouse anterior endoderm. Here, we report the origin and signalling properties of this population of cells in the early Xenopus embryo. RESULTS: Xenopus anterior endoderm was found to derive in part from cells at the centre of the blastocoel floor that express XHex, the Xenopus cognate of Hex. Like their counterparts in the mouse embryo, these Hex-expressing blastomeres moved to the dorsal side of the Xenopus embryo as gastrulation commenced, and populated deep endodermal adjacent to Spemann's organiser. Experiments involving the induction of secondary axes confirmed that XHex expression was associated with anterior development. Ventral misexpression of XHex induced ectopic cerberus expression and conferred anterior signalling properties to the endoderm. Unlike the effect of misexpressing cerberus, these signals could not neuralise overlying ectoderm. CONCLUSIONS: XHex expression reveals the unexpected origin of an anterior signalling centre in Xenopus, which arises in part from the centre of the blastula and localises to the deep endoderm adjacent to Spemann's organiser. Signals originating from these endodermal cells impart an anterior identity to the overlying ectoderm, but are insufficient for neural induction. The anterior movement of Hex-expressing cells in both Xenopus and mouse embryos suggests that this process is a conserved feature of vertebrate development.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

Bottle cell formation in relation to mesodermal patterning in the Xenopus embryo

The appearance of bottle cells at the dorsal vegetal/marginal boundary of Xenopus embryos marks the onset of blastopore formation. The conditions leading to this epithelial activity were investigated by inducing bottle cells ectopically in the animal region with VegT or different members of the transforming growth factor (TGF)-beta family. Morphological studies on the ectopic bottle cells indicate their close similarity to the endogenous bottle cells at the dorsal blastopore lip. The subepithelial cells of the induced animal region express mesodermal genes in a pattern reminiscent to that observed on the dorsal lip. Relating this expression pattern to the position of the ectopic bottle cells leads to the conclusion that bottle cells form in regions of high TGF-beta signalling. The specific inhibitory effects of cerberus on ectopically induced bottle cells revealed that nodal related growth factors are the intrinsic signals that elicit bottle cell formation in the normal embryo. In addition, fibroblast growth factor signalling is an essential precondition for this epithelial response as it is for mesoderm formation. We conclude that bottle cell formation in the epithelial layer of the gastrula is closely linked to mesodermal patterning in the subepithelial tissues.     

Spatially distinct head and heart inducers within the Xenopus organizer region.

BACKGROUND: The mouse anterior visceral endoderm, an extraembryonic tissue, expresses several genes essential for normal development of structures rostral to the anterior limit of the notochord and has been termed the head organizer. This tissue also has heart-inducing activity and expresses mCer1 which, like its Xenopus homolog cerberus, can induce markers of cardiac specification and anterior neural tissue when ectopically expressed. We investigated the relationship between head and heart induction in Xenopus embryos, which lack extraembryonic tissues. RESULTS: We found three regions of gene expression in the Xenopus organizer: deep endoderm, which expressed cerberus; prechordal mesoderm, which showed overlapping but non-identical expression of genes characteristic of the murine head organizer, such as XHex and XANF-1; and leading-edge dorsoanterior endoderm, which expressed both cerberus and a subset of the genes expressed by the prechordal mesoderm. Microsurgical ablation of the cerberus-expressing endoderm decreased the incidence of heart, but not head, formation. Removal of prechordal mesoderm, in contrast, caused deficits of anterior head structures. Finally, although misexpression of cerberus induced ectopic heads, it was unable to induce genes thought to participate in head induction. CONCLUSIONS: In Xenopus, the cerberus-expressing endoderm is required for heart, but not head, inducing activity. Therefore, this tissue is not the topological equivalent of the murine anterior visceral endoderm. We propose that, in Xenopus, cerberus is redundant to other bone morphogenetic protein (BMP) and Wnt antagonists located in prechordal mesoderm for head induction, but may be necessary for heart induction.     

Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm

Formation of mesoderm from the pluripotent epiblast depends upon canonical Wnt/beta-catenin signaling, although a precise molecular basis for this requirement has not been established. To develop a robust model of this developmental transition, we examined the role of Wnt signaling during the analogous stage of embryonic stem cell differentiation. We show that the kinetics of Wnt ligand expression and pathway activity in vitro mirror those found in vivo. Furthermore, inhibition of this endogenous Wnt signaling abrogates the functional competence of differentiating ES cells, reflected by their failure to generate Flk1(+) mesodermal precursors and subsequent mature mesodermal lineages. Microarray analysis at various times during early differentiation reveal that mesoderm- and endoderm-associated genes fail to be induced in the absence of Wnt signaling, indicating a lack of germ layer induction that normally occurs during gastrulation in vivo. The earliest genes displaying Wnt-dependent expression, however, were those expressed in vivo in the primitive streak. Using an inducible form of stabilized beta-catenin, we find that Wnt activity, although required, does not autonomously promote primitive streak-associated gene expression in vitro. Our results suggest that Wnt signaling functions in this model system to regulate the thresholds or stability of responses to other effector pathways and demonstrate that differentiating ES cells represent a useful model system for defining complex regulatory interactions underlying primary germ layer induction.     

Reiterated Wnt signaling during zebrafish neural crest development

While Wnt/beta-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 we employed a zebrafish line that was transgenic for an inducible inhibitor of Wnt/beta-catenin signaling, and inhibited endogenous Wnt/beta-catenin signaling at discrete times in development. Using this approach, we defined a critical period for Wnt signaling in the initial induction of neural crest, which is distinct from the later period of development when pigment cells are specified from neural crest. Blocking Wnt signaling during this early period interfered 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, we established that wnt8 is 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.     

Wnt signals are targets and mediators of Gli function

There is growing evidence that Gli proteins participate in the mediation of Hedgehog and FGF signaling in neural and mesodermal development. However, little is known about which genes act downstream of Gli proteins. Here we show the regulation of members of the Wnt family by Gli proteins in different contexts. Our findings indicate that Gli2 regulates Wnt8 expression in the ventral marginal zone of the early frog embryo: activating Gli2 constructs induce ectopic Wnt8 expression in animal cap explants, whereas repressor forms inhibit its endogenous expression in the marginal zone. Using truncated Frizzled and dominant-negative Wnt constructs, we then show the requirement of at least two Wnt proteins, Wnt8 and Wnt11, for Gli2/3-induced posterior mesodermal development. Blocking Wnt signals, however, inhibits Gli2/3-induced morphogenesis, but not mesodermal specification. Gli2/3 may therefore normally coordinate the action of these two Wnt proteins, which regulate distinct downstream pathways. In addition, the finding that Gli1 consistently induces a distinct set of Wnt genes in animal cap explants and in skin tumors suggests that Wnt regulation by Gli proteins is general. Such a mechanism may link signals that induce Gli activity, such as FGFs and Hedgehogs, with Wnt function.     

Wnt6 induces the specification and epithelialization of F9 embryonal carcinoma cells to primitive endoderm

Epithelial-to-mesenchymal transitions (EMTs) play key roles in the normal development of an organism as well as its demise following the metastasis of a malignant tumour. An EMT during early mouse development results in the differentiation of primitive endoderm into the parietal endoderm that forms part of the parietal yolk sac. In the embryo, primitive endoderm develops from cells in the inner cell mass, but the signals that instruct these cells to become specified and adopt an epithelial fate are poorly understood. The mouse F9 teratocarcinoma cell line, a model that can recapitulate the in vivo primitive to parietal endoderm EMT, has been used extensively to elucidate the signalling cascades involved in extraembryonic endoderm differentiation. Here, we identified Wnt6 as a gene up-regulated in F9 cells in response to RA and show that Wnt6 expressing cells or cells exposed to Wnt6 conditioned media form primitive endoderm. Wnt6 induction of primitive endoderm is accompanied by beta-catenin and Snail1 translocation to the nucleus and the appearance of cytokeratin intermediate filaments. Attenuating glycogen synthase kinase 3 activity using LiCl gave similar results, but the fact that cells de-differentiate when LiCl is removed reveals that other signalling pathways are required to maintain cells as primitive endoderm. Finally, Wnt6-induced primitive endodermal cells were tested to determine their competency to complete the EMT and differentiate into parietal endoderm. Towards that end, results show that up-regulating protein kinase A activity is sufficient to induce markers of parietal endoderm. Together, these findings indicate that undifferentiated F9 cells are responsive to canonical Wnt signalling, which negatively regulates glycogen synthase kinase 3 activity leading to the epithelialization and specification of primitive endoderm competent to receive additional signals required for EMT. Considering the ability of F9 cells to mimic an in vivo EMT, the identification of this Wnt6-beta-catenin-Snail signalling cascade has broad implications for understanding EMT mechanisms in embryogenesis and metastasis.     

A role for maternal beta-catenin in early mesoderm induction in Xenopus

Mesoderm formation results from an inducing process that requires maternal and zygotic FGF/MAPK and TGFbeta activities, while maternal activation of the Wnt/beta-catenin pathway determines the anterior-dorsal axis. Here, we show a new role of Wnt/beta-catenin signaling in mesoderm induction. We find that maternal beta-catenin signaling is not only active dorsally but also all around the equatorial region, coinciding with the prospective mesoderm. Maternal beta-catenin function is required both for expression of dorsal genes and for activation of MAPK and the mesodermal markers Xbra and eomesodermin. beta-catenin acts in a non- cell-autonomous manner upstream of zygotic FGF and nodal signals. The Wnt/beta-catenin activity in the equatorial region of the early embryo is the first example of a maternally provided mesoderm inducer restricted to the prospective mesoderm.     

Wnt/beta-catenin signaling: a promising new target for fibrosis diseases

Wnt/beta-catenin signaling is involved in virtually every aspect of embryonic development and also controls homeostatic self-renewal in a number of adult tissues. Recently, emerging evidence from researches of organ fibrosis suggest that sustained Wnt/beta-catenin pathway reactivation is linked to the pathogenesis of fibrotic disorders. Here we focus on Wnt/beta-catenin-related pathogenic effects in different organs, such as lung fibrosis, liver fibrosis, skin fibrosis and renal fibrosis. Additionally, Wnt/beta-catenin signaling works in a combinatorial manner with TGF-beta signaling in the process of fibrosis, and TGF-beta signaling can induce expression of Wnt/beta-catenin superfamily members and vice versa. Moreover, network analysis, based on pathway databases, revealed that key factors in the Wnt pathway were targeted by some differentially expressed microRNAs detected in fibrosis diseases. These findings demonstrated the crosstalks between Wnt/beta-catenin pathway and TGF-beta signalings, and microRNAs, highlighting the role of Wnts in organ fibrogenesis. Most importantly, nowadays there is a variety of Wnt pathway inhibitors which give us the potential therapeutic feasibility, modulation of the Wnt pathway may, therefore, present as a suitable and promising therapeutic strategy in the future.     

WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo

A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Here, we show that three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm and the lateral plate mesoderm in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, we also show that the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo.     

Transforming growth factor-beta and Wnt signals regulate chondrocyte differentiation through Twist1 in a stage-specific manner

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.