非洲爪蟾早期胚胎发育中的Wnt信号

非洲爪蟾是脊椎动物胚胎发育研究中的几种重要模式生物之一,为揭示早期胚胎发育中的分子调控机制做出了显著的贡献.其中一个重要的发现就是细胞信号通路在胚胎发育中起到非常关键的调控作用.本文简单介绍Wnt信号在爪蟾早期胚胎发育不同时期的几种调控作用.     

In vitro induction and transplantation of eye during early Xenopus development

A vertebrate eye was induced via a series of coordinated inductive interactions. Here, we describe a novel in vitro system to induce eye formation at high frequency using Xenopus early gastrulae. The eye formed in vitro is morphologically similar to the normal eye. When the in vitro eye was transplanted into a stage-33 tadpole, the optic nerve was seen extending from the grafted eye to the tectum of the host brain and the in vitro eye graft was retained after metamorphosis. In addition, we transplanted the eye formed in vitro into a tadpole with both eyes removed. The resultant juvenile frogs could perceive brightness using the grafted eye and thereby control their skin color, suggesting that the eye formed in vitro could function normally.     

Rap2 is required for Wnt/beta-catenin signaling pathway in Xenopus early development

The Wnt/beta-catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Here, we present evidence that Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/beta-catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer-specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits beta-catenin stabilization and the induction of ectopic dorsal axis and Wnt-responsive genes caused by XWnt8, Dsh or beta-catenin, but has no effect on the signaling activities of a stabilized beta-catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh-mediated beta-catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, we suggest that XRap2 is involved in Wnt/beta-catenin signaling as a modulator of the subcellular localization of Dsh.     

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.     

Roles of Wnt proteins in neural development and maintenance

Many constituents of Wnt signaling pathways are expressed in the developing and mature nervous systems. Recent work has shown that Wnt signaling controls initial formation of the neural plate and many subsequent patterning decisions in the embryonic nervous system, including formation of the neural crest. Wnt signaling continues to be important at later stages of development. Wnts have been shown to regulate the anatomy of the neuronal cytoskeleton and the differentiation of synapses in the cerebellum. Wnt signaling has been demonstrated to regulate apoptosis and may participate in degenerative processes leading to cell death in the aging brain.     

Expression of Wnt signaling components during Xenopus pronephros development

Background

The formation of the vertebrate kidney is tightly regulated and relies on multiple evolutionarily conserved inductive events. These are present in the complex metanephric kidney of higher vertebrates, but also in the more primitive pronephric kidney functional in the larval stages of amphibians and fish. Wnts have long been viewed as central in this process. Canonical β-Catenin-dependent Wnt signaling establishes kidney progenitors and non-canonical β-Catenin-independent Wnt signaling participate in the morphogenetic processes that form the highly sophisticated nephron structure. While some individual Wnt signaling components have been studied extensively in the kidney, the overall pathway has not yet been analyzed in depth.

Methodology/Principal Findings

Here we report a detailed expression analysis of all Wnt ligands, receptors and several downstream Wnt effectors during pronephros development in Xenopus laevis using in situ hybridization. Out of 19 Wnt ligands, only three, Wnt4, Wnt9a and Wnt11, are specifically expressed in the pronephros. Others such as Wnt8a are present, but in a broader domain comprising adjacent tissues in addition to the kidney. The same paradigm is observed for the Wnt receptors and its downstream signaling components. Fzd1, Fzd4, Fzd6, Fzd7, Fzd8 as well as Celsr1 and Prickle1 show distinct expression domains in the pronephric kidney, whereas the non-traditional Wnt receptors, Ror2 and Ryk, as well as the majority of the effector molecules are rather ubiquitous. In addition to this spatial regulation, the timing of expression is also tightly regulated. In particular, non-canonical Wnt signaling seems to be restricted to later stages of pronephros development.

Conclusion/Significance

Together these data suggest a complex cross talk between canonical and non-canonical Wnt signaling is required to establish a functional pronephric kidney.     

BMP signalling in early Xenopus development

Bone morphogenetic proteins (BMPs) are typically members of the transforming growth factor β (TGF-β) family with diverse roles in embryonic development. At least five genes with homology to BMPs are expressed during Xenopus development, along with their receptors and intracellular signalling pathways. The evidence suggests that BMPs have roles to play in both mesoderm induction and dorsoventral patterning. Studies in Xenopus have also identified a number of inhibitory binding proteins for the classical BMPs, encoded by genes such as chordin and noggin. These proteins appear to be responsible for establishing a morphogen gradient of BMP4 activity, which specifies different dorsoventral fates in early gastrulae. An emerging theme is that inhibition of BMP signalling is an important mechanism regulating cell fate decisions in early development. BioEssays 21:751–760, 1999. © 1999 John Wiley & Sons, Inc.     

Xenopus ADAM 13 is a metalloprotease required for cranial neural crest-cell migration.

BACKGROUND: Cranial neural-crest (CNC) cells originate from the lateral edge of the anterior neuroepithelium and migrate to form parts of the peripheral nervous system, muscles, cartilage, and bones of the face. Neural crest-cell migration involves the loss of adhesion from the surrounding neuroepithelium and a corresponding increase in cell adhesion to the extracellular matrix (ECM) present in migratory pathways. While proteolytic activity is likely to contribute to the regulation of neural crest-cell adhesion and migration, the role of a neural crest-specific protease in these processes has yet to be demonstrated. We previously showed that CNC cells express ADAM 13, a cell surface metalloprotease/disintegrin. Proteins of this family are known to act in cell-cell adhesion and as sheddases. ADAMs have also been proposed to degrade the ECM, but this has not yet been shown in a physiological context. RESULTS: Using a tissue transplantation technique, we show that Xenopus CNC cells overexpressing wild-type ADAM 13 migrate along the same hyoid, branchial, and mandibular pathways used by normal CNC cells. In contrast, CNC cell grafts that express protease-defective ADAM 13 fail to migrate along the hyoid and branchial pathways. In addition, ectopic expression of wild-type ADAM 13 results in a gain-of-function phenotype in embryos, namely the abnormal positioning of trunk neural-crest cells. We further show that explanted embryonic tissues expressing wild-type, but not protease-defective, ADAM 13 display decreased cell-matrix adhesion. Purified ADAM 13 can cleave fibronectin, and tissue culture cells that express wild-type, but not protease-defective, ADAM 13 can remodel a fibronectin substrate. CONCLUSIONS: Our findings support the hypothesis that the protease activity of ADAM 13 plays a critical role in neural crest-cell migration along defined pathways. We propose that the ADAM 13-dependent modification of ECM and/or other guidance molecules is a key step in the directed migration of the CNC.     

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.     

TGF-beta signalling pathways in early Xenopus development

Many different ligands of the TGF-beta superfamily signal in the early Xenopus embryo and are required for the specification and patterning of the three germ layers as well as for gastrulation. Recent advances in the field are helping us understand how ligand activity is regulated both spatially and temporally, the mechanism by which the signals are transduced to the nucleus and how essentially the same signalling pathway can activate completely different sets of genes in different regions of the embryo.     

Xenopus hoxc8 during early development

Vertebrate hoxc8 homologous genes have been shown to be involved in the formation of lower thoracic/lumbar vertebrae during early embryonic development. We report the isolation of a Xenopus hoxc8 (Xhoxc8), which shows 94% amino acid sequence identity to the mouse counterpart. Xhoxc8 is initially expressed in a broad region of blastopore lip at gastrular stage; however, at later stages, the region of expression is progressively restricted to the dorsal region caudal to the third somite and to the central trunk region of abdomen. Retinoic acid treatment that caused a severe malformation in antero-posterior axis did not induce any significant change in the spatio-temporal expression pattern of Xhoxc8 mRNA. Antisense RNA injection into 2- or 4-cell stage embryos resulted in a severe malformation in the abdominal structure leading to embryonic death. The results strongly indicate that Xhoxc8 expression is critical for the formation of abdominal structure.     

Localization and induction in early development of Xenopus

Our experimental results, as well as those of others, lead us to suggest the following steps in the dorsalization and axialization of the Xenopus egg and embryo: the sperm aster determines the direction of rotation of the cortex relative to the deeper cytoplasm (endoplasm); the rotation of the cortex activates latent dorsalizing-axializing agents in the vegetal hemisphere. The extent of rotation determines the amount of activation. The direction of rotation determines the location of the activated agents. The activated agents determine the level of mesoderm-inducing activity of the vegetal cells cleaved from that cytoplasmic region. The level of inducing activity determines at least the time at which marginal zone cells will begin gastrulation movements. The time of its initiation of gastrulation may determine how anterior and dorsal a particular marginal zone cell can become.     

Emerin expression in early development of Xenopus laevis

Emerin is an integral protein of the inner nuclear membrane in the majority of differentiated vertebrate cells. In humans, deficiency of emerin causes a progressive muscular dystrophy of the Emery-Dreifuss type. The physiological role of emerin is poorly understood. By screening and sequencing of EST clones we have identified two emerin homologues in Xenopus laevis, Xemerin1 and Xemerin2. Xemerins share with mammalian emerins the N-terminal LEM domain and a single transmembrane domain at the C-terminus. As shown by immunoblot analysis with Xemerin-specific antibodies, both proteins have an apparent molecular mass of 24 kDa but differ in their isoelectric points. Xemerin1 and Xemerin2 proteins are not detectable in oocytes nor during early embryogenesis. Protein expression is first found at stage 43 and persists in somatic cells. However, RT-PCR and Northern blot analysis show Xemerin mRNAs of approximately 4.0 kb to be present in oocytes and throughout embryogenesis. During embryogenesis the level of Xemerin mRNAs increases at stage 22 and is particularly abundant in mesodermal and neuro-ectodermal regions of the embryo. These data provide the necessary background to further investigate the role of emerin in nuclear envelope assembly, gene expression and organ development of X. laevis as a model organism.     

Muscarinic drugs affect cholinesterase activity and development of eye structures during early chick development

During neurogenesis, markers of the cholinergic system are present in the eye and visual cortex of vertebrates. In adult vertebrates, a role for these molecules, including muscarinic acetylcholine receptors (mAChRs), in eye growth non-accommodative regulation is also known. In order to understand the biological mechanisms triggered by the cholinergic system in these events, we analysed the effects of a cholinergic agonist (10(-4) M carbachol) and an antagonist (10(-4) M atropine) of the muscarinic receptors, on early chick development. To establish if the cholinergic system also plays a role in the regulation of early neurogenetic signals, the drug treatments were made at stage 5-6 HH, during the formation of the cephalic process. Specific effects on forehead, and in particular on eye development were found; carbachol treated embryos presented huge and well pigmented eyes, significantly different from controls. The eyes of atropine-exposed embryos presented anomalies with different phenotypes ranging from strongly affected features to normal-like appearance. Generally, the eyes were smaller as compared to the controls, with a number of anomalies, also in the normal-like phenotype, including retina and lens defects. In these structures, distribution of cholinesterase activities was checked by histochemical methods, and the amount of cells undergoing nuclear disgregation was revealed by DAPI staining. We propose that the drugs affected the known nervous and pre-nervous functions of the cholinergic markers, such as cell signalling during primary induction, and regulation of cell death by ACh receptors.     

Roles of Xenopus chemokine ligand CXCLh (XCXCLh) in early embryogenesis

Several chemokine molecules control cell movements during early morphogenesis. However, it is unclear whether chemokine molecules affect cell fate. Here, we identified and characterized the CXC‐type chemokine ligand in Xenopus laevis, Xenopus CXCLh (XCXCLh), during early embryogenesis. XCXCLh is expressed in the dorsal vegetal region at the gastrula stage. Both overexpression and knockdown of XCXCLh in the dorsal region inhibited gastrulation. XCXCLh contributed to the attraction of mesendodermal cells and accelerated the reassembly of scratched culture cells. Also, XCXCLh contributed to early endodermal induction. Overexpression of VegTmRNA or high concentrations of calcium ions induced XCXCLh expression. XCXCLh may play roles in both cell movements and differentiation during early Xenopus embryogenesis.