Preparation and analysis of biological activity of the secreted proteins of the Noggin family

Methods for preparation of recombinant physiologically active proteins of the Noggin family (Noggin1 and Noggin2) of the clawed frog Xenopus laevis that are capable of interaction with BMP factors of the TGF-beta superfamily have been developed. Structures were designed for the expression of Noggin1 and Noggin2 proteins allowing us to produce them from the directly microinjected synthetic mRNA in the cells of developing embryos of the clawed frog, as well as in the prokaryotic expression system. Target proteins contained three Myc-epitopes at the N-terminal. Introduction of these’ tags’ allowed comparison of the expression level of the Noggin1 and Noggin2 proteins, isolation on the affinity immunosorbent, and demonstration of biological activity of the isolated Noggin-proteins through the analysis of their ability to bind the BMP4 factor of the TGF-beta superfamily with coimmunoprecipitation method.     

The interaction of secreted proteins Noggin4 and Wnt8 from <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos

We demonstrated that the secreted protein Noggin4 from Xenopus laevis was capable of the in vitro binding to the secreted factor Wnt8, one of the ligands of the Wnt/betaCatenin signaling pathway. It was also shown that posttranslational modifications occurring during secretion of these proteins from the embryonic cells were necessary for their effective interaction. Also, we proposed a method for the preparation of physiologically active secreted morphogenic proteins from the intercellular space of the Xenopus laevis embryos.     

Vizualizing the morphogen adsorption gradient in the <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryo using fluorescently labeled heparin-binding motif of BMP4 morphogen

Spatial distribution of heparan sulfates in the embryonic extracellular matrix at midgastrula stage has been demonstrated in the Xenopus laevis embryo model. Towards this end, fluorescently labeled fusion protein EGFP-hbmBMP4 made up by green fluorescent protein (EGFP) and heparin-binding motif of Bone Morphogenetic Protein 4 (BMP4) was produced in the E. coli expression system. Xenopus laevis embryos at midgastrula stage (stage 11) were fixed and cut along the anteroposterior axis and then incubated with EGFP-hbmBMP4. The fluorescently labeled samples were analyzed in fluorescence microscope. The spatial distribution of fluorescence intensity reflecting BMP4 adsorption on the embryonic extracellular matrix proved to be similar to the corresponding distribution pattern for the Noggin1 heparin-binding motif obtained previously. The highest intensity zone was detected around the dorsal blastopore lip; another high intensity zone, although slightly less prominent, was observed in the ventral blastopore lip region. Since on one hand, heparin-binding sites significantly differ in their organization in BMP4 and Noggin1 proteins and, on the other hand, spatial adsorption distribution patterns for these proteins coincide in the embryo, it appears that all secreted morphogens containing a heparin-binding site share a single adsorption gradient in the embryonic extracellular space.     

The secreted protein Noggin4 is an activator of the Wnt/PCP-signaling pathway

The protein Noggin4 of the African clawed frog Xenopus laevis has been shown to act as a modulator of the “noncanonical” Wnt/PCP-signaling pathway that plays an important role in the regulation of cell motility. Induction of disturbances in the expression of Noggin4 led to the activation of Wnt/PCP-pathway and the related anomalies of early embryonic development. The Noggin4 protein can bind the Wnt11 protein that normally contributes to the activation of the Wnt/PCP-pathway and of enhancing the activator effect of this protein in luciferase assays. Thus, Noggin4 can be used as a tool for specific experimental regulation of the activity of the Wnt/PCP pathway.     

Interaction of secreted factor Agr2 with its potential receptors from the family of three-finger proteins

Interactions of the secreted protein XAgr2 of the Agr (anterior gradient proteins) family with six submembrane proteins, Tfp1–6, from the family of three-finger proteins Ly6 were studied in Xenopus laevis embryos. Earlier, other authors have shown that the newt homologue of XAgr2 is able to bind the Prod1 three-finger protein, which participates in the establishment of the proximal-to-distal pattern of cell differentiation in the regenerating blastema of a newt’s limb bud. Here, we identified six homologues of Prod1 in Xenopus laevis genome named Tfp1–6. By co-immunoprecipitation, we demonstrated that among these homologues, Tfp4 is the most probable receptor of XAgr2. Further study of the revealed interactions between XAgr2 and Tfp4 is of significant interest because XAgr2 is involved in the signaling pathways that regulate neural system development and the body appendages regeneration.     

Protein synthesis in interspecies hybrid embryos of the amphibian Xenopus

The newly synthesized abundant proteins of early Xenopus laevis and Xenopus borealis embryos have been examined by two-dimensional electrophoresis after labelling with [³⁵S]methionine. Six prominent polypeptides specific to Xenopus laevis embryos and a further six specific to Xenopus borealis have been identified. Overall, embryos of the two species are estimated to differ by approx. 15% in their protein synthetic patterns from blastula to tailbud stage. Interspecific hybrid embryos (Xenopus laevis (♀)/Xenopus borealis (♂)) synthesise only the maternally specified set of proteins until gastrulation after which they produce the full complement of both Xenopus laevis and Xenopus borealis specific proteins. The possible use of such molecular markers of parental genome activity in facilitating further embryological study is discussed.     

Permeability of Xenopus laevis embryos: Specific incorporation of precursors into eukaryotic proteins and nucleic acids

All amino acids and several nucleic acid precursors are taken up by Xenopus laevis embryos. The embryos are completely intact and not modified in any way. These precursors are directly incorporated into the macromolecules of Xenopus embryos and not prokaryotic contaminants as has been previously claimed. Radioactive leucine is incorporated into Xenopus laevis ribosomal proteins as characterized by sucrose gradient centrifugation. The uptake of the amino acids is cycloheximide sensitive and unaffected by chloramphenicol. Radioactive adenosine and orotic acid are taken up and incorporated into tRNA and rRNA at high levels as characterized by sucrose gradients and electrophoresis. These characterizations of labeled macromolecules unequivocally show that normal Xenopus laevis embryos will take up and incorporate labeled precursors to levels which are sufficient to study cellular biochemical events at such early stages of development.     

Gene expression analysis of the Tao kinase family of Ste20p-like map kinase kinase kinases during early embryonic development in Xenopus laevis

Development of the vertebrate embryo requires strict coordination of a highly complex series of signaling cascades, that drive cell proliferation, differentiation, migration, and the general morphogenetic program. Members of the Map kinase signaling pathway are repeatedly required throughout development to activate the downstream effectors, ERK, p38, and JNK. Regulation of these pathways occurs at many levels in the signaling cascade, with the Map3Ks playing an essential role in target selection. The thousand and one amino acid kinases (Taoks) are Map3Ks that have been shown to activate both p38 and JNK and are linked to neurodevelopment in both invertebrate and vertebrate organisms. In vertebrates, there are three Taok paralogs (Taok1, Taok2, and Taok3) which have not yet been ascribed a role in early development. Here we describe the spatiotemporal expression of Taok1, Taok2, and Taok3 in the model organism Xenopus laevis. The X. laevis Tao kinases share roughly 80% identity to each other, with the bulk of the conservation in the kinase domain. Taok1 and Taok3 are highly expressed in pre-gastrula and gastrula stage embryos, with initial expression localized to the animal pole and later expression in the ectoderm and mesoderm. All three Taoks are expressed in the neural and tailbud stages, with overlapping expression in the neural tube, notochord, and many anterior structures (including branchial arches, brain, otic vesicles, and eye). The expression patterns described here provide evidence that the Tao kinases may play a central role in early development, in addition to their function during neural development, and establish a framework to better understand the developmental roles of Tao kinase signaling.     

Novel functions of Noggin proteins: inhibition of Activin/Nodal and Wnt signaling

The secreted protein Noggin1 is an embryonic inducer that can sequester TGFβ cytokines of the BMP family with extremely high affinity. Owing to this function, ectopic Noggin1 can induce formation of the headless secondary body axis in Xenopus embryos. Here, we show that Noggin1 and its homolog Noggin2 can also bind, albeit less effectively, to ActivinB, Nodal/Xnrs and XWnt8, inactivation of which, together with BMP, is essential for the head induction. In support of this, we show that both Noggin proteins, if ectopically produced in sufficient concentrations in Xenopus embryo, can induce a secondary head, including the forebrain. During normal development, however, Noggin1 mRNA is translated in the presumptive forebrain with low efficiency, which provides the sufficient protein concentration for only its BMP-antagonizing function. By contrast, Noggin2, which is produced in cells of the anterior margin of the neural plate at a higher concentration, also protects the developing forebrain from inhibition by ActivinB and XWnt8 signaling. Thus, besides revealing of novel functions of Noggin proteins, our findings demonstrate that specification of the forebrain requires isolation of its cells from BMP, Activin/Nodal and Wnt signaling not only during gastrulation but also at post-gastrulation stages.     

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin–fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular adhesion. These data indicate that Wnt/Glypican4/Frizzled signaling regulates ECM assembly through effects on cadherin-mediated cell cohesion. Together, our results demonstrate that zebrafish Vangl2/Prickle1a and non-canonical Wnt/Frizzled signaling have opposing effects on ECM organization underlying PCP and gastrulation cell movements.     

Noggin Expression in the Adult Retina Suggests a Conserved Role during Vertebrate Evolution

Vertebrates share common mechanisms in the control of development and in the maintenance of neural and retinal function. The secreted factor Noggin, a BMP inhibitor, plays a crucial role in neural induction during embryonic development. Moreover, we have shown its involvement in retinal differentiation of pluripotent cells. Here we show Noggin expression in the adult retina in three vertebrate species. Four Noggin genes are present in zebrafish (Danio rerio; ZbNog1, 2, 3, 5), three in frog (Xenopus laevis; XenNog1, 2 and 4), and one in mouse (Mus musculus; mNog). Quantitative RT-PCR experiments show the presence of ZbNog3 and ZbNog5 mRNAs, but not ZbNog1 and ZbNog2, in the adult zebrafish retina. All three genes are expressed in the frog retina, and mNog in the mouse. Immunohistochemistry data show that Noggin proteins are predominantly localized in the Golgi apparatus of photoreceptors and in the fibers of the outer plexiform layer. Lower expression levels are also found in inner plexiform layer fibers, in ganglion cells, in the ciliary marginal zone, and in retinal pigmented epithelium. Our results show that Noggin has a specific cellular and sub-cellular expression in the adult vertebrate retina, which is conserved during evolution. In addition to its established role during embryonic development, we postulate that Noggin also exerts a functional role in the adult retina.     

An amphibian with ambition: a new role for Xenopus in the 21st century

Much of our knowledge about the mechanisms of vertebrate early development comes from studies using Xenopus laevis. The recent development of a remarkably efficient method for generating transgenic embryos is now allowing study of late development and organogenesis in Xenopus embryos. Possibilities are also emerging for genomic studies using the closely related diploid frog Xenopus tropicalis.     

The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5′-untranslated region (5′-UTR) of the mouse mammary tumor virus (MMTV). The 5′-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5′-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5′-UTR was resistant to the addition of m⁷GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5′-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.     

Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos

This study was conducted to investigate quantitatively the luciferase activity of gene constructs with viral and hybrid enhancers and promoters in bovine preimplantation embryos by using firefly luciferase reporter genes. In Experiment I, to examine the stability of the luciferase, bioluminescence intensity of bovine embryos injected with the luciferase gene driven by the SV40 early promoter and enhancer (SVEluc) was measured with a luminometer at 2 days after microinjection. The results indicated that the bioluminescence could be analysed at any time within 30 min because the luciferase activity was constant during the measurement period from 5 to 30 min. In Experiment II, the luciferase expression of fertilized oocytes injected with four gene constructs (TKEluc, TK6WEluc, SVEluc, and Miwluc) was analysed by using a photon imaging system at 2 or 6 days following microinjection. The results from Experiment II indicated that the reporter gene governed by the Miw promoter (RSV LTR and chicken β-actin promoter) was expressed more intensively in bovine morulae and blastocysts than three other gene constructs. In Experiment III, the effect of SV40 enhancer was investigated when fused downstream to the luciferase cDNA of the Miwluc vector. The results showed that SV40 enhancer further activated the luciferase activity of the Miw promoter in bovine preimplantation embryos. It was concluded, therefore, that the Miw promoter together with the SV40 enhancer would confer the strongest expression of the firefly luciferase reporter gene among the gene constructs tested in preimplantation bovine embryos. Mol. Reprod. Dev. 49:368–373, 1998. © 1998 Wiley-Liss, Inc.     

Studies on the appearance and nature of a maturation-inducing factor in the cytoplasm of amphibian oocytes exposed to progesterone

Full-grown oocytes of amphibians respond in vitro to exogenous progesterone by undergoing physiological maturation (breakdown of the germinal vesicle (GVBD), meiosis, and acquisition of the capacity for activation). Both cytoplasm and “cytosol” from maturing oocytes have been shown to produce similar events when injected into unstimulated oocytes. This activity appeared within 4 hr after hormone treatment in Rana pipiens and Xenopus laevis and represents the earliest detectable, specific response of the oocyte yet observed, i.e., 6–8 hr before GVBD in Rana. Maturing oocytes retained activity as long as 100 hr after exposure to progesterone, and activity was also obtained from ovulated eggs and cleaving embryos. In addition, cytoplasm from Rana pipiens, Xenopus laevis, or Ambystoma mexicanum was effective in inducing maturation in oocytes of each other, indicating a lack of specificity.Recipient oocytes of Xenopus laevis consistently began to mature within 1.5–3 hr after injection of maturing cytoplasm, well before progesterone-treated controls. The timing of the response was closely related to the quantity of cytoplasm transferred, suggesting the presence of both a minimum and threshold level of cytoplasmic factor. Serial cytoplasmic transfer in Xenopus oocytes showed no significant loss of activity through 10 injections.