WNT/β-Catenin Signalling and Epithelial Patterning in the Homoscleromorph Sponge Oscarella

Sponges branch basally in the metazoan phylogenetic tree and are thus well positioned to provide insights into the evolution of mechanisms controlling animal development, likely to remain active in adult sponges. Of the four sponge clades, the Homoscleromorpha are of particular interest as they alone show the “true” epithelial organization seen in other metazoan phyla (the Eumetazoa). We have examined the deployment in sponges of Wnt signalling pathway components, since this pathway is an important regulator of many developmental patterning processes. We identified a reduced repertoire of three divergent Wnt ligand genes in the recently-sequenced Amphimedon queenslandica (demosponge) genome and two Wnts from our EST collection from the homoscleromorph Oscarella lobularis, along with well-conserved genes for intracellular pathway components (β-catenin, GSK3β). Remarkably, the two O. lobularis Wnt genes showed complementary expression patterns in relation to the evenly spaced ostia (canal openings) of the exopinacoderm (ectoderm), highly reminiscent of Wnt expression during skin appendage formation in vertebrates. Furthermore, experimental activation of the Wnt/β-catenin pathway using GSK3β inhibitors provoked formation of ectopic ostia, as has been shown for epithelial appendages in Eumetazoa. We thus suggest that deployment of Wnt signalling is a common and perhaps ancient feature of metazoan epithelial patterning and morphogenesis.     

Wnt/��-catenin signaling in cerebral cortical development

The cerebral cortex is the multilayered sheet of neurons that underlies our highest cognitive abilities. Canonical Wnt/β-catenin signaling has well-known activities in tissue patterning in regulating rostral-caudal and medial-lateral patterning in the developing cortex. In addition, recent studies suggest that Wnt/β-catenin signaling also plays important roles in establishing the radial inside to outside organization of the cerebral cortex. Different Wnts, Wnt receptors and inhibitors are expressed in overlapping radial compartments of the cerebral cortex, and in vivo functional studies have provided evidence for Wnt/β-catenin regulation of neural precursor self-renewal, laminar fate determination and establishing or stabilizing the patterns of neuronal communication of cortical neurons. Wnt/β-catenin alterations have been observed in human brain tumors, and understanding its many diverse functions during normal neural development may provide greater insight into the mechanisms underlying the development and progression of neural tumors.Key words: cerebral cortex, neural stem cell, neural precursor, ventricular zone, laminar fate, regional specification, radial patterning     

Wnt signal transduction pathways

The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans hinting to a daunting complexity of signaling regulation, function and biological output. To date major signaling branches downstream of the Fz receptor have been identified including a canonical or Wnt/β-catenin dependent pathway and the non-canonical or β-catenin-independent pathway which can be further divided into the Planar Cell Polarity and the Wnt/Ca²⁺ pathways, and these branches are being actively dissected at the molecular and biochemical levels. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of these pathways in regulating key events during embryonic patterning and morphogenesis.Key words: Wnt, frizzled, dishevelled, canonical, non-canonical, β-catenin, Planar Cell Polarity     

Wnt signaling in limb organogenesis

Secreted signaling molecules of the Wnt family have been found to play a central role in controlling embryonic development of a wide range of taxa from Hydra to humans. The most extensively studied Wnt signaling pathway is the canonical Wnt pathway, which controls gene expression by stabilizing β-catenin, and regulates a multitude of developmental processes. More recently, noncanonical Wnt pathways, which are β-catenin-independent, have been found to be important developmental regulators. Understanding the mechanisms of Wnt signaling is essential for the development of novel preventive and therapeutic approaches of human diseases. Limb development is a paradigm to study the principles of Wnt signaling in various developmental contexts. In the developing vertebrate limb, Wnt signaling has been shown to have important functions during limb bud initiation, limb outgrowth, early limb patterning, and later limb morphogenesis events. This review provides a brief overview on the diversity of Wnt-dependent signaling events during embryonic development of the vertebrate limb.Key words: Wnts, limb initiation, outgrowth, patterning, morphogenesis     

<Emphasis Type="Italic">Wnt</Emphasis> gene loss in flatworms

Wnt genes encode secreted glycoproteins that act in cell–cell signalling to regulate a wide array of developmental processes, ranging from cellular differentiation to axial patterning. Discovery that canonical Wnt/β-catenin signalling is responsible for regulating head/tail specification in planarian regeneration has recently highlighted their importance in flatworm (phylum Platyhelminthes) development, but examination of their roles in the complex development of the diverse parasitic groups has yet to be conducted. Here, we characterise Wnt genes in the model tapeworm Hymenolepis microstoma and mine genomic resources of free-living and parasitic species for the presence of Wnts and downstream signalling components. We identify orthologs through a combination of BLAST and phylogenetic analyses, showing that flatworms have a highly reduced and dispersed complement that includes orthologs of only five subfamilies (Wnt1, Wnt2, Wnt4, Wnt5 and Wnt11) and fewer paralogs in parasitic flatworms (5–6) than in planarians (9). All major signalling components are identified, including antagonists and receptors, and key binding domains are intact, indicating that the canonical (Wnt/β-catenin) and non-canonical (planar cell polarity and Wnt/Ca²⁺) pathways are functional. RNA-Seq data show expression of all Hymenolepis Wnts and most downstream components in adults and larvae with the notable exceptions of wnt1, expressed only in adults, and wnt2 expressed only in larvae. The distribution of Wnt subfamilies in animals corroborates the idea that the last common ancestor of the Cnidaria and Bilateria possessed all contemporary Wnts and highlights the extent of gene loss in flatworms.     

Multiple roles of Activin/Nodal,bone morphogenetic protein,fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification.     

LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling

R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/β-catenin and Wnt/PCP signalling. R-spondin-triggered β-catenin signalling requires Clathrin, while Wnt3a-mediated β-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling.     

Wnt5a and Wnt11 interact in a maternal Dkk1-regulated fashion to activate both canonical and non-canonical signaling in Xenopus axis formation

Wnt signaling in development and adult tissue homeostasis requires tight regulation to prevent patterning abnormalities and tumor formation. Here, we show that the maternal Wnt antagonist Dkk1 downregulates both the canonical and non-canonical signaling that are required for the correct establishment of the axes of the Xenopus embryo. We find that the target Wnts of Dkk activity are maternal Wnt5a and Wnt11, and that both Wnts are essential for canonical and non-canonical signaling. We determine that Wnt5a and Wnt11 form a previously unrecognized complex. This work suggests a new aspect of Wnt signaling: two Wnts acting in a complex together to regulate embryonic patterning.     

Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/β-catenin signalling

Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise β-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3], [4], [5] and [6], the effect of other Dkks on the Wnt/β-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/β-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/β-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.     

Remote Activation of the Wnt/β-Catenin Signalling Pathway Using Functionalised Magnetic Particles

Wnt signalling pathways play crucial roles in developmental biology, stem cell fate and tissue patterning and have become an attractive therapeutic target in the fields of tissue engineering and regenerative medicine. Wnt signalling has also been shown to play a role in human Mesenchymal Stem Cell (hMSC) fate, which have shown potential as a cell therapy in bone and cartilage tissue engineering. Previous work has shown that biocompatible magnetic nanoparticles (MNP) can be used to stimulate specific mechanosensitive membrane receptors and ion channels in vitro and in vivo. Using this strategy, we determined the effects of mechano-stimulation of the Wnt Frizzled receptor on Wnt pathway activation in hMSC. Frizzled receptors were tagged using anti-Frizzled functionalised MNP (Fz-MNP). A commercially available oscillating magnetic bioreactor (MICA Biosystems) was used to mechanically stimulate Frizzled receptors remotely. Our results demonstrate that Fz-MNP can activate Wnt/β-catenin signalling at key checkpoints in the signalling pathway. Immunocytochemistry indicated nuclear localisation of the Wnt intracellular messenger β-catenin after treatment with Fz-MNP. A Wnt signalling TCF/LEF responsive luciferase reporter transfected into hMSC was used to assess terminal signal activation at the nucleus. We observed an increase in reporter activity after treatment with Fz-MNP and this effect was enhanced after mechano-stimulation using the magnetic array. Western blot analysis was used to probe the mechanism of signalling activation and indicated that Fz-MNP signal through an LRP independent mechanism. Finally, the gene expression profiles of stress response genes were found to be similar when cells were treated with recombinant Wnt-3A or Fz-MNP. This study provides proof of principle that Wnt signalling and Frizzled receptors are mechanosensitive and can be remotely activated in vitro. Using magnetic nanoparticle technology it may be possible to modulate Wnt signalling pathways and thus control stem cell fate for therapeutic purposes.     

The dual regulator Sufu integrates Hedgehog and Wnt signals in the early Xenopus embryo

Hedgehog (Hh) and Wnt proteins are important signals implicated in several aspects of embryonic development, including the early development of the central nervous system. We found that Xenopus Suppressor-of-fused (XSufu) affects neural induction and patterning by regulating the Hh/Gli and Wnt/β-catenin pathways. Microinjection of XSufu mRNA induced expansion of the epidermis at the expense of neural plate tissue and caused enlargement of the eyes. An antisense morpholino oligonucleotide against XSufu had the opposite effect. Interestingly, both gain- and loss-of-function experiments resulted in a posterior shift of brain markers, suggesting a biphasic effect of XSufu on anteroposterior patterning. XSufu blocked early Wnt/β-catenin signaling, as indicated by the suppression of XWnt8-induced secondary axis formation in mRNA-injected embryos, and activation of Wnt target genes in XSufu-MO-injected ectodermal explants. We show that XSufu binds to XGli1 and Xβ-catenin. In Xenopus embryos and mouse embryonic fibroblasts, Gli1 inhibits Wnt signaling under overexpression of β-catenin, whereas β-catenin stimulates Hh signaling under overexpression of Gli1. Notably, endogenous Sufu is critically involved in this crosstalk. The results suggest that XSufu may act as a common regulator of Hh and Wnt signaling and contribute to intertwining the two pathways.     

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Background

Wnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.

Methodology/Principal Findings

We tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4.

Conclusions/Significance

Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.