Formation of the head organizer in hydra involves the canonical Wnt pathway

Stabilization of beta-catenin by inhibiting the activity of glycogen synthase kinase-3beta has been shown to initiate axis formation or axial patterning processes in many bilaterians. In hydra, the head organizer is located in the hypostome, the apical portion of the head. Treatment of hydra with alsterpaullone, a specific inhibitor of glycogen synthase kinase-3beta, results in the body column acquiring characteristics of the head organizer, as measured by transplantation experiments, and by the expression of genes associated with the head organizer. Hence, the role of the canonical Wnt pathway for the initiation of axis formation was established early in metazoan evolution.     

β-catenin plays a central role in setting up the head organizer in hydra

In an adult hydra the head organizer, located in the hypostome, is constantly active in maintaining the structure of the animal in the context of its steady state tissue dynamics. Several Wnt genes, TCF, and elevated levels of β-catenin are expressed in the hypostome as well as during the formation of a new organizer region in developing buds suggesting they play a role in the organizer. Transgenic hydra were generated in which a modified hydra β-catenin gene driven by an actin promoter is continuously expressed at a high level throughout the animal. These animals formed heads and secondary axes in multiple locations along the body column. Transplantation experiments indicate they have a high and stable level of head organizer activity throughout the body columns. However, none of the Wnt genes are expressed in the body columns of these transgenic animals. Further, in alsterpaullone-treated animals, which results in a transient rise in head organizer activity throughout the body column, the time of expression of the Wnt genes is much shorter than the time of the elevated level of head inducing activity. These results for the first time provide direct functional evidence that β-catenin plays a crucial role in the maintenance and activity of the head organizer and suggest that Wnt ligands may be required only for the initiation but not in maintenance of the organizer in Hydra.     

Characterization of the head organizer in hydra

A central process in the maintenance of axial patterning in the adult hydra is the head activation gradient, i.e. the potential to form a secondary axis, which is maximal in the head and is graded down the body column. Earlier evidence suggested that this gradient was based on a single parameter. Using transplantation experiments, we provide evidence that the hypostome, the apical part of the head, has the characteristics of an organizer in that it has the capacity to induce host tissue to form most of the second axis. By contrast, tissue of the body column has a self-organizing capacity, but not an inductive capacity. That the inductive capacity is confined to the hypostome is supported by experiments involving a hypostome-contact graft. The hypostome, but not the body column, transmits a signal(s) leading to the formation of a second axis. In addition, variations of the transplantation grafts and hypostome-contact grafts provide evidence for several characteristics of the organizer. The inductive capacity of the head and the self-organizing capacity of the body column are based on different pathways. Head inhibition, yya signal produced in the head and transmitted to the body column to prevent head formation, represses the effect of the inducing signal by interfering with formation of the hypostome/organizer. These results indicate that the organizer characteristics of the hypostome of an adult hydra are similar to those of the organizer region of vertebrate embryos. They also indicate that the Gierer-Meinhardt model provides a reasonable framework for the mechanisms that underlie the organizer and its activities. In addition, the results suggest that a region of an embryo or adult with the characteristics of an organizer arose early in metazoan evolution.     

PI3K and ERK 1-2 regulate early stages during head regeneration in hydra

Different signaling systems coordinate and regulate the development of a multicellular organism. In hydra, the canonical Wnt pathway and the signal transduction pathways mediated by PKC and Src regulate early stages of head formation. In this paper, we present evidence for the participation of a third pathway, the PI3K-PKB pathway, involved in this process. The data presented here are consistent with the participation of ERK 1-2 as a point of convergence for the transduction pathways mediated by PKC, Src and PI3K for the regulation of the regeneration of the head in hydra. The specific developmental point regulated by them appears to be the commitment of tissue at the apical end of the regenerate to form the head organizer.     

Wnt signaling in hydroid development: ectopic heads and giant buds induced by GSK-3beta inhibitors

In Hydractinia, a colonial marine hydroid representing the basal phylum Cnidaria, Wnt signaling plays a major role in the specification of the primary body axis in embryogenesis and in the establishment of the oral pole during metamorphosis. Here we report supplementing investigations on head regeneration and bud formation in post-metamorphic development. Head and bud formation were accompanied by the expression of Wnt, frizzled and Tcf. Activation of Wnt signaling by blocking GSK-3beta affected regeneration, the patterning of growing polyps and the asexual formation of new polyps in the colony. In the presence of lithium ions or paullones, gastric segments excised from adult polyps showed reversal of tissue polarity as they frequently regenerated heads at both ends. Phorbol myristate acetate, a known activator of protein kinase C increased this effect. Global activation of the Wnt pathway caused growing polyps to form ectopic tentacles and additional heads along their body column. Repeated treatment of colonies evoked the emergence of many and dramatically oversized bud fields along the circumference of the colony. These giant fields fell apart into smaller sub-fields, which gave rise to arrays of multi-headed polyps. We interpret the morphogenetic effects of blocking GSK-3beta as reflecting increase in positional value in terms of positional information and activation of Wnt target genes in molecular terms.     

Zygotic Wnt activity is required for Brachyury expression in the early Xenopus laevis embryo

The canonical, beta-catenin-dependent Wnt pathway is a crucial player in the early events of Xenopus development. Dorsal axis formation and mesoderm patterning are accepted effects of this pathway, but the regulation of expression of genes involved in mesoderm specification is not. This conclusion is based largely on the inability of the Wnt pathway to induce mesoderm in animal cap explants. Using injections of inhibitors of canonical Wnt signaling, we demonstrate that expression of the general mesodermal marker Brachyury (Xbra) requires a zygotic, ligand-dependent Wnt activity throughout the marginal zone. Analysis of the Xbra promoter reveals that putative TCF-binding sites mediate Wnt activation, the first sites in this well-studied promoter to which an activation role can be ascribed. However, established mesoderm inducers like eFGF and activin can bypass the Wnt requirement for Xbra expression. Another mesoderm promoting factor, VegT, activates Xbra in a Wnt-dependent manner. We also show that the activin/nodal signaling is necessary for ectopic Xbra induction by the Wnt pathway, but not by VegT. Our data significantly change the understanding of Brachyury regulation in Xenopus, implying the existence of an unknown zygotic Wnt ligand in Spemann's organizer. Since Brachyury is considered to have a major role in mesoderm formation, it is possible that Wnts might play a role in mesoderm specification, in addition to patterning.     

Wnt11/beta-catenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin

Current models of canonical Wnt signaling assume that a pathway is active if beta-catenin becomes nuclearly localized and Wnt target genes are transcribed. We show that, in Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. We provide evidence that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, we find that axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain beta-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizes beta-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality.     

Nuclear beta-catenin localization is not sufficient for canonical Wnt signaling activation in human meianoma cell lines

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called beta-catenin. Mutations promoting beta-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers, but rarely observed in melanomas. Nevertheless, beta-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why, the aim of the investigation was to elucidate the relation between beta-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular beta-catenin localization, and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of beta-catenin does not always correspond to active status canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear beta-catenin canonical Wnt signaling can't be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, the pathway-targeted potential antineoplastic therapy requires the formation of a "molecular pattern of cancer" for localization of the defect in Wnt signaling cascade in the each case.     

Axial Patterning in Hydra

Morphogen gradients play an important role in pattern formation during early stages of embryonic development in many bilaterians. In an adult hydra, axial patterning processes are constantly active because of the tissue dynamics in the adult. These processes include an organizer region in the head, which continuously produces and transmits two signals that are distributed in gradients down the body column. One signal sets up and maintains the head activation gradient, which is a morphogenetic gradient. This gradient confers the capacity of head formation on tissue of the body column, which takes place during bud formation, hydra''s mode of asexual reproduction, as well as during head regeneration following bisection of the animal anywhere along the body column. The other signal sets up the head inhibition gradient, which prevents head formation, thereby restricting bud formation to the lower part of the body column in an adult hydra. Little is known about the molecular basis of the two gradients. In contrast, the canonical Wnt pathway plays a central role in setting up and maintaining the head organizer.Morphogen gradients play a critical role in the early stages of embryogenesis in a number of metazoans in that they initiate and are involved in axial patterning processes. Such a gradient also plays a role in axial patterning in hydra, a primitive metazoan. However, unlike in most metazoans, this gradient is continuously active in an adult hydra as part of the tissue dynamics of the adult animal.The structure of a hydra is fairly simple (Fig. ​(Fig.1).1). It consists of a single axis with radial symmetry, which contains a head, body column, and foot along the axis. The head consist of two parts: the hypostome in the apex, and the tentacle zone from which the tentacles emerge in the basal part of the head. The body column has three parts: the gastric region and peduncle in the apical, and basal parts with a budding zone between the gastric region and peduncle. Buds, hydra''s mode of asexual reproduction, emerge from the budding zone between the gastric region and peduncle.Open in a separate windowFigure 1.Longitudinal cross section of an adult hydra. The multiple regions are labeled. The two protrusions from the body column are early and late stages of bud development. The arrows indicate the direction of tissue displacement. (Reprinted from Bode 2001.)Three cell lineages are involved. The axis consists of a cylindrical shell that is made up of two concentric epithelial layers, the ectoderm and endoderm, which are separated by a basement membrane. Interspersed among the epithelial cells of both layers are the cells of the third lineage, the interstitial cell lineage. It consists of interstitial cells, which are multipotent stem cells (David and Murphy 1977), located primarily in the ectoderm throughout the body column. They give rise to neurons, secretory cells, and nematocytes, which are the stinging cells that are typical of cnidarians, as well as gametes when a hydra undergoes sexual reproduction (David and Murphy 1977).In an adult hydra, the epithelial cells of both layers are constantly in the mitotic cycle (David and Campbell 1972; Campbell and David 1974). The expanding tissue in the upper part of the body column is continuously displaced apically into the head (Fig. 1). Once there, it is displaced onto and along the tentacles or into the hypostome, and eventually sloughed when reaching the extremities (Campbell 1967; Otto and Campbell 1977). Tissue in the remainder of the body column is displaced basally either onto developing buds, or further down onto the foot, where it is sloughed at the bottom of the foot. Thus, the tissues of an adult hydra are continuously in a steady state of production and loss. As a hydra has no defined lifetime (Martinez 1998), this activity goes on indefinitely.     

Canonical Wnt signaling is required for ophthalmic trigeminal placode cell fate determination and maintenance

Cranial placodes are ectodermal regions that contribute extensively to the vertebrate peripheral sensory nervous system. The development of the ophthalmic trigeminal (opV) placode, which gives rise only to sensory neurons of the ophthalmic lobe of the trigeminal ganglion, is a useful model of sensory neuron development. While key differentiation processes have been characterized at the tissue and cellular levels, the signaling pathways governing opV placode development have not. Here we tested in chick whether the canonical Wnt signaling pathway regulates opV placode development. By introducing a Wnt reporter into embryonic chick head ectoderm, we show that the canonical pathway is active in Pax3+ opV placode cells as, or shortly after, they are induced to express Pax3. Blocking the canonical Wnt pathway resulted in the failure of targeted cells to adopt or maintain an opV fate, as assayed by the expression of various markers including Pax3, FGFR4, Eya2, and the neuronal differentiation markers Islet1, neurofilament, and NeuN, although, surprisingly, it led to upregulation of Neurogenin2, both in the opV placode and elsewhere in the ectoderm. Activating the canonical Wnt signaling pathway, however, was not sufficient to induce Pax3, the earliest specific marker of the opV placode. We conclude that canonical Wnt signaling is necessary for normal opV placode development, and propose that other molecular cues are required in addition to Wnt signaling to promote cells toward an opV placode fate.     

Injury-induced activation of the MAPK/CREB pathway triggers apoptosis-induced compensatory proliferation in hydra head regeneration

After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process.     

Dkk1在器官发育及肿瘤发生中的调控作用

Wnt信号通路在脊椎动物的胚胎发育过程中发挥重要作用. Dkk1(Dickkopf1)是Dkk基因家族的成员之一,通过编码一种分泌型的糖蛋白与Wnt信号蛋白竞争细胞表面受体,来维持Wnt信号通路的稳态,从而调控胚胎器官的正常发育. 同时,在人类成体中,Dkk1基因活性的改变与肿瘤、代谢性骨病和骨关节炎等疾病的发生密切相关. 本文对Dkk1在头部、肢、眼和牙齿等器官的胚胎发育过程中的相关分子调控机制以及Dkk1与肿瘤发生的关系进行综述.     

Role of crescent in convergent extension movements by modulating Wnt signaling in early Xenopus embryogenesis

The Xenopus gene crescent encodes a member of the secreted Frizzled-related protein (sFRP) family and is expressed in the head organizer region. However, the target and function of Crescent in early development are not well understood. Here, we describe a role of Crescent in the regulation of convergent extension movements (CEMs) during gastrulation and neurulation. We show that overexpression of Crescent in whole embryos or animal caps inhibits CEMs without affecting tissue specification. Consistent with this, Crescent efficiently forms complexes with Xwnt11 and Xwnt5a, in contrast to another sFRP, Frzb1. As expected, the inhibitory effect of Crescent or Xwnt11 on CEMs is cancelled when both proteins are coexpressed in the neuroectoderm. Interestingly, when coexpressed in the dorsal mesoderm, the activity of Xwnt11 is rather enhanced by Crescent. Supporting this finding, the inhibition of CEMs by Crescent in mesodermalized but not neuralized animal caps is reversed by the dominant-negative form of Cdc42, a putative mediator of Wnt/Ca2+ pathway. Antisense morpholino oligos for Crescent impair neural plate closure and elicit microcephalic embryos with a shortened trunk without affecting early tissue specification. These data suggest a potential role for Crescent in head formation by regulating a non-canonical Wnt pathway positively in the adjacent posterior mesoderm and negatively in the overlying anterior neuroectoderm.     

Transplantation analysis of developmental mechanisms in Hydra

Since the pioneering work of Ethel Browne (1909) who demonstrated for the first time the concept of organizer activity, i.e. the potency of an apical Hydra tissue to induce a secondary axis when transplanted onto a host, Hydra flourished as a fruitful model system for developmental studies. Over the next 60 years this efficient transplantation approach identified graded biological activities along the body column of Hydra named Head Acti-vation and Head Inhibition. These properties inspired theoretical modelers including Lewis Wolpert, Alfred Gierer and Hans Meinhardt to propose models for morphogenesis, respectively the positional information (1969) and reaction-diffusion (1972) models. In 1973, Tsutomu Sugiyama and Toshitaka Fujisawa initiated in Mishima a unique project to analyze the properties of Hydra strains with distinct morphological and developmental characters. To this end, they collected in several areas of Japan multiple Hydra strains that they subsequently characterized and crossed. They also established a lateral transplantation strategy that was much more powerful than the previous ones, as it combined quantitative measurements with cellular analyses thanks to the chimera procedures developed by Campbell and colleagues. In-deed this approach provided a paradigm to quantify in any morphological phenotype the Head Activation and Head Inhibition levels along the body column. In this article, I review the various strains identified by Sugiyama and colleagues, the principles and the main results deduced from the quantitative lateral transplantation strategy. In addition, I briefly discuss the relevance of this approach in the era of molecular biology.