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Protein Tyrosine Kinase 7 (PTK7) is as a critical regulator of canonical and non-canonical Wnt-signaling during embryonic development and cancer cell formation. Disrupting PTK7 activity perturbs vertebrate nervous system development, and also promotes human cancer formation. Observations in different model systems suggest a complex cross-talk between PTK7 protein and Wnt signaling. During Xenopus laevis nervous system development, we previously showed that PTK7 protein positively regulates canonical Wnt signaling by maintaining optimal LRP6 protein levels, but PTK7 also acts in concert with LRP6 protein to repress non-canonical Wnt activity. PTK7 is a transmembrane protein, but studies in cancer cells showed that PTK7 undergoes "shedding" by metalloproteases to different proteolytic fragments. Some PTK7 proteolytic fragments are oncogenic, being localized to alternative cytoplasmic and nuclear cell compartments. In this study we examined the biological activity of two proteolytic carboxyl-terminal PTK7 proteolytic fragments, cPTK7 622-1070 and cPTK7 726-1070 during early Xenopus nervous system development. We found that these smaller PTK7 proteolytic fragments have similar activity to full-length PTK7 protein to promote canonical Wnt-signaling via regulation of LRP6 protein levels. In addition to cancer systems, this study shows in vivo proof that these smaller PTK7 proteolytic fragments can recapitulate full-length PTK7 protein activity in diverse systems, such as vertebrate nervous system development.
14-3-3 proteins are intracellular dimeric phosphoserine/threonine-binding molecules that participate in signal transduction and checkpoint control pathways. 14-3-3 proteins are required for normal eye development, brain function, and terminal patterning in Drosophila melanogaster, but the role of 14-3-3 proteins in vertebrate development is undefined. In this work an unphosphorylated peptide inhibitor of 14-3-3, R18, was used to determine the role of 14-3-3 proteins in Xenopus embryonic development. Biochemical analysis demonstrated that R18 was specific and efficient at attenuating global 14-3-3 activities in Xenopus embryos. Microinjection experiments showed a requirement for 14-3-3 function in mesodermal specification. Inhibition of 14-3-3 resulted in embryos with axial patterning defects and reduced expression of mesodermal marker genes. These phenotypic defects were caused by impaired fibroblast growth factor signaling in R18-injected embryos. These results establish the importance of 14-3-3 proteins in vertebrate embryonic development.
EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells.
RGS family members are GTPase activating proteins (GAPs) that antagonize signaling by heterotrimeric G proteins. Injection of Xenopus embryos with RNA encoding rat RGS4 (rRGS4), a GAP for G(i) and G(q), resulted in shortened trunks and decreased skeletal muscle. This phenotype is nearly identical to the effect of injection of either frzb or dominant negative Xwnt-8. Injection of human RGS2, which selectively deactivates G(q), had similar effects. rRGS4 inhibited the ability of early Xwnt-8 but not Xdsh misexpression to cause axis duplication. This effect is distinct from axin family members that contain RGS-like domains but act downstream of Xdsh. We identified two Xenopus RGS4 homologs, one of which, Xrgs4a, was expressed as a Spemann organizer component. Injection of Xenopus embryos with Xrgs4a also resulted in shortened trunks and decreased skeletal muscle. These results suggest that RGS proteins modulate Xwnt-8 signaling by attenuating the function of a G protein.
CENP-A is an essential histone H3 variant found in all eukaryotes examined to date. To begin to determine how CENP-A is assembled into chromatin, we developed a binding assay using sperm chromatin in cell-free extract derived from Xenopus eggs. Our data suggest that the catalytic activities of an unidentified deoxycytidine deaminase and UNG2, a uracil DNA glycosylase, are involved in CENP-A assembly. In support of this model, inhibiting deoxycytidine deaminase with zebularine, or uracil DNA glycosylase with Ugi, uracil or UTP results in a lack of detectable CENP-A on sperm DNA. Conversely, inducing DNA damage increases the level of CENP-A detected on sperm chromatin. Our data suggest that base excision repair may be involved in assembly of this histone H3 variant.
Members of the disintegrin metalloproteinase (ADAM) family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST) databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species.
The epithelial barrier is crucial for proper gastrointestinal function, preventing the unwanted passage of solutes and therefore representing a prerequisite for vectorial transport. Claudin-4 and claudin-18.2, two critical tight junction proteins of the gastric epithelium, seal neighboring cells in a physically and mechanically challenging environment. As the Xenopus laevis oocyte allows the functional and molecular analyses of claudin interaction, we have addressed the hypothesis that this interaction is not only dependent on mechanical force but also on pH. We expressed human claudin-4 and claudin-18 in Xenopus oocytes, and analyzed them in a two-cell model approach. Cells were clustered in pairs to form contact areas expressing CLDN18 + CLDN18, CLDN4/18 + CLDN4/18, and compared to controls, respectively. Contact areas in cells incubated in medium at pH 5.5 and 7.4 were quantified by employing transmitted light microscopy. After 24 h at pH 5.5, clustering of CLDN18 + CLDN18 and CLDN4/18 + CLDN4/18-expressing oocytes revealed a contact area reduced by 45% and 32%, compared with controls, respectively. A further approach, high-pressure impulse assay, revealed a stronger tight junction interaction at pH 5.5 in oocyte pairs expressing CLDN18 + CLDN18 or CLDN4/18 + CLDN4/18 indicating a protective role of claudin-18 for tight junction integrity during pH challenge. Thus, our current analysis of gastric tight junction proteins further establishes oocytes as an expression and two-cell screening model for tight junction integrity analysis of organ- and tissue-specific claudins by the characterization of homo- and heterophilic trans-interaction dependent on barrier effectors.
Although most RNA-binding proteins recognize a complex set of structural motifs in their RNA target, the double-stranded (ds) RNA-binding proteins are limited to interactions with double helices. Recently, it has been discovered that some dsRNA-binding proteins share regions of amino-acid similarity known as dsRNA-binding motifs.
This protocol is developed for identifying mRNAs that form complexes with mRNA-binding proteins (mRBPs) in Xenopus laevis embryos at different developmental stages. Here, we describe the use of the Ybx1 mRBP for immunoprecipitation-based mRNA isolation. This protocol features the translation of the mRBP of interest directly in living embryos following injection of synthetic mRNA templates encoding a hybrid of this protein with a specific tag. This approach allows precipitation of mRNA-protein complexes from embryonic lysates using commercially available anti-tag antibodies. For complete details on the use and execution of this protocol, please refer to Parshina et al. (2020).
Monoclonal antibodies (mABs) have been raised against oocyte nuclear proteins of Xenopus laevis and X. borealis and have been screened for species specificity. Although about 40% of all germinal vesicle polypeptides differ between the two species as judged by two-dimensional gel analysis, most mABs react with polypeptides of both species. Biochemical analysis of the antigens by immunoblotting revealed that a homologue of each antigen of one species could be detected in the other species, despite frequent differences in molecular structure. Nevertheless, five strictly species-specific mABs have been identified. All five are directed against the same acidic polypeptide B3 of X. borealis, which is a structurally altered homologue of the protein N1, previously described in X. laevis germinal vesicles. In oocyte nuclei of hybrids between X. laevis females and X. borealis males, polypeptide of both species appear to be accumulated equivalently. Exceptions to this rule are most easily explained by differences between individuals and by the loss of certain alleles resulting from the cross.
Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.
The kindlin/fermitin family includes three proteins involved in regulating integrin ligand-binding activity and adhesion. Loss-of-function mutations in kindlins1 and 3 have been implicated in Kindler Syndrome and Leukocyte Adhesion Deficiency III (LAD-III) respectively, whereas kindlin2 null mice are embryonic lethal. Post translational regulation of cell-cell and cell-ECM adhesion has long been presumed to be important for morphogenesis, however, few specific examples of activation-dependent changes in adhesion molecule function in normal development have been reported. In this study, antisense morpholinos were used to reduce expression of individual kindlins in Xenopus laevis embryos in order to investigate their roles in early development. Kindlin1 knockdown resulted in developmental delays, gross malformations of the gut and eventual lethality by tadpole stages. Kindlin2 morphant embryos displayed late stage defects in vascular maintenance and angiogenic branching consistent with kindlin2 loss of function in the mouse. Antisense morpholinos were also used to deplete maternal kindlin2 protein in oocytes and eggs. Embryos lacking maternal kindlin2 arrested at early cleavage stages due to failures in cytokinesis. Kindlin3 morphant phenotypes included defects in epidermal ciliary beating and partial paralysis at tailbud stages but these embryos recovered eventually as morpholino levels decayed. These results indicate a remarkably diverse range of kindlin functions in vertebrate development.
The metameric organization of the vertebrate body plan is established during somitogenesis as somite pairs sequentially form along the anteroposterior axis. Coordinated regulation of cell shape, motility and adhesion are crucial for directing the morphological segmentation of somites. We show that members of the Ena/VASP family of actin regulatory proteins are required for somitogenesis in Xenopus. Xenopus Ena (Xena) localizes to the cell periphery in the presomitic mesoderm (PSM), and is enriched at intersomitic junctions and at myotendinous junctions in somites and the myotome, where it co-localizes with beta1-integrin, vinculin and FAK. Inhibition of Ena/VASP function with dominant-negative mutants results in abnormal somite formation that correlates with later defects in intermyotomal junctions. Neutralization of Ena/VASP activity disrupts cell rearrangements during somite rotation and leads to defects in the fibronectin (FN) matrix surrounding somites. Furthermore, inhibition of Ena/VASP function impairs FN matrix assembly, spreading of somitic cells on FN and autophosphorylation of FAK, suggesting a role for Ena/VASP proteins in the modulation of integrin-mediated processes. We also show that inhibition of FAK results in defects in somite formation, blocks FN matrix deposition and alters Xena localization. Together, these results provide evidence that Ena/VASP proteins and FAK are required for somite formation in Xenopus and support the idea that Ena/VASP and FAK function in a common pathway to regulate integrin-dependent migration and adhesion during somitogenesis.
Piwi proteins utilize small RNAs (piRNAs) to recognize target transcripts such as transposable elements (TE). However, extensive piRNA sequence diversity also suggests that Piwi/piRNA complexes interact with many transcripts beyond TEs. To determine Piwi target RNAs, we used ribonucleoprotein-immunoprecipitation (RIP) and cross-linking and immunoprecipitation (CLIP) to identify thousands of transcripts associated with the Piwi proteins XIWI and XILI (Piwi-protein-associated transcripts, PATs) from early stage oocytes of X. laevis and X. tropicalis Most PATs associate with both XIWI and XILI and include transcripts of developmentally important proteins in oogenesis and embryogenesis. Only a minor fraction of PATs in both frog species displayed near perfect matches to piRNAs. Since predicting imperfect pairing between all piRNAs and target RNAs remains intractable, we instead determined that PAT read counts correlate well with the lengths and expression levels of transcripts, features that have also been observed for oocyte mRNAs associated with Drosophila Piwi proteins. We used an in vitro assay with exogenous RNA to confirm that XIWI associates with RNAs in a length- and concentration-dependent manner. In this assay, noncoding transcripts with many perfectly matched antisense piRNAs were unstable, whereas coding transcripts with matching piRNAs were stable, consistent with emerging evidence that Piwi proteins both promote the turnover of TEs and other RNAs, and may also regulate mRNA localization and translation. Our study suggests that Piwi proteins play multiple roles in germ cells and establishes a tractable vertebrate system to study the role of Piwi proteins in transcript regulation.
The melanocortin system consists of five G protein-coupled receptors (MC1R-MC5R), the bidirectional endogenous ligands (MSH and Agouti families), and accessory proteins (MRAP1 and MRAP2). Accumulative studies of vertebrate species find high expression level of melanocortin 1 receptor (MC1R) in the dermal melanocyte and elucidate the essential roles in the skin and fur pigmentation, morphological background adaptation, and stress response. The diploid amphibian Xenopus tropicalis (xt) has been utilized as a fantastic animal model for embryonic development and studies of physiological cryptic colouring and environmental adaptiveness. However, the interaction of xtMc1r signaling with xtMrap proteins has not been assessed yet. In this study, we carried out in silico evolutionary analysis of protein alignment and genetic phylogenetic and genomic synteny of mc1r among various vertebrates. Ubiquitous expression of mrap1 and mrap2 and the co-expression with mc1r transcripts in the skin were clearly observed. Co-immunoprecipitation (ip) and fluorescent complementary approach validated the direct functional interaction of xtMc1r with xtMrap1 or xtMrap2 proteins on the plasma membrane. Pharmacological assay showed the improvement of the constitutive activity and alpha melanocyte-stimulating hormone (α-MSH) stimulated plateau without dramatic alteration of the cell surface translocation of xtMc1r in the presence of xtMrap proteins. Overall, the pharmacological modulation of xtMc1r by dual xtMrap2 proteins elucidated the potential role of this protein complex in the regulation of proper dermal function in amphibian species.
The Receptor Transporter Protein (RTP) family is present in most, if not all jawed vertebrates. Most of our knowledge of this protein family comes from studies on mammalian RTPs, which are multi-function proteins that regulate cell-surface G-protein coupled receptor levels, influence olfactory system development, regulate immune signaling, and directly inhibit viral infection. However, mammals comprise less than one-tenth of extant vertebrate species, and our knowledge about the expression, function, and evolution of non-mammalian RTPs is limited. Here, we explore the evolutionary history of RTPs in vertebrates. We identify signatures of positive selection in many vertebrate RTP clades and characterize multiple, independent expansions of the RTP family outside of what has been described in mammals. We find a striking expansion of RTPs in the African clawed frog, Xenopus laevis, with 11 RTPs in this species as opposed to 1 to 4 in most other species. RNA sequencing revealed that most X. laevis RTPs are upregulated following immune stimulation. In functional assays, we demonstrate that at least three of these X. laevis RTPs inhibit infection by RNA viruses, suggesting that RTP homologs may serve as antiviral effectors outside of Mammalia.
The proneural basic-helix-loop-helix (bHLH) transcription factor Ascl1 is a master regulator of neurogenesis in both central and peripheral nervous systems in vivo, and is a central driver of neuronal reprogramming in vitro. Over the last three decades, assaying primary neuron formation in Xenopus embryos in response to transcription factor overexpression has contributed to our understanding of the roles and regulation of proneural proteins like Ascl1, with homologues from different species usually exhibiting similar functional effects. Here we demonstrate that the mouse Ascl1 protein is twice as active as the Xenopus protein in inducing neural-β-tubulin expression in Xenopus embryos, despite there being little difference in protein accumulation or ability to undergo phosphorylation, two properties known to influence Ascl1 function. This superior activity of the mouse compared to the Xenopus protein is dependent on the presence of the non-conserved N terminal region of the protein, and indicates species-specific regulation that may necessitate care when interpreting results in cross-species experiments.
Microtubules are a component of the cytoskeleton and are important for maintaining cell structure and providing platforms for intracellular transport in diverse cellular processes. Microtubule plus-end tracking proteins (+TIPs), a structurally and functionally diverse group of proteins, are specifically accumulated in the microtubule plus end and regulate dynamic microtubule behavior. We characterized the +TIPs, Clip1, p150(glued), Clasp1, Lis1 and Stim1, in Xenopus laevis and report their expression patterns during embryogenesis in this paper. All the five +TIP genes are maternally expressed and have similar expression patterns during Xenopus embryo development. The expression of +TIPs is localized in the animal hemisphere and ectoderm region at early stages of embryonic development. As development progresses to later stages, the ectodermal expression of +TIPs persists in head and neural tube structures. Clasp1, p150(glued) and Lis1 in particular are specifically expressed in the cranial nerves. Importantly, +TIPs are also expressed in the involuting mesoderm during gastrulation. This is the first study of developmental expression patterns of +TIPs, and our analysis provides insight that could serve as the basis for future research of microtubules in vertebrate development, cell movements during gastrulation and neurogenesis.
In eukaryotes, chromosomal DNA is licensed for a single round of replication in each cell cycle. Xenopus MCM3 protein has been implicated in the licensing of replication in egg extract. We have cloned cDNAs encoding five immunologically distinct proteins associated with Xenopus MCM3 as members of the MCM/P1 family. Six Xenopus MCM proteins formed a physical complex in the egg extract, bound to unreplicated chromatin before the formation of nuclei, and apparently displaced from replicated chromatin. The requirement of six XMCM proteins for the replication activity of the egg extract before nuclear formation suggests that their re-association with replicated chromatin at the end of the mitotic cell cycle is a key step for the licensing of replication.
The transmembrane domains (TMDs) of dengue virus type-1 M protein (DENV-1M) were reported to form cation-selective channels in artificial lipid bilayers. We further explored this observation using the two-electrode voltage clamp (TEVC) method on the Xenopus laevis oocytes expressing DENV PrM and M proteins. Using myc epitope tagged M proteins, M was first shown to adopt its predicted native topology in mammalian cells when expressed on its own. The recombinant proteins were then successfully expressed on the surface of Xenopus oocytes. Using influenza A M2 (Inf A/M2) protein as a control, we measured the conductance of oocytes expressing DENV proteins under hyperpolarized or low-pH conditions. Inf A/M2 showed pH-dependent, amantadine-sensitive channel activity that was consistent with previously published reports. However, no activity was detected for DENV proteins. We conclude that DENV PrM and M proteins do not show pH-activated ion channel activity.
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