Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo

To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms

Protocol for separation of the nuclear and the cytoplasmic fractions of Xenopus laevis embryonic cells for studying protein shuttling

Summary: This protocol for the separation of nuclear and cytoplasmic fractions of cells of Xenopus laevis embryos was developed to study changes in the intracellular localization of the Zyxin and Ybx1 proteins, which are capable of changing localization in response to certain stimuli. Western blot analysis allows the quantification of changes in the distribution of these proteins between the cytoplasm and nucleus, whereas the posttranslational modifications specific to each compartment can be identified by changes in electrophoretic mobility.For complete details on the use and execution of this protocol, please refer to Parshina et al. (2020)

The Molecular Mechanism of Body Axis Induction in Lampreys May Differ from That in Amphibians

Lamprey homologues of the classic embryonic inducer Noggin are similar in expression pattern and functional properties to Noggin homologues of jawed vertebrates. All noggin genes of vertebrates apparently originated from a single ancestral gene as a result of genome duplications. nogginA, nogginB and nogginC of lampreys, like noggin1 and noggin2 of gnathostomes, demonstrate the ability to induce complete secondary axes with forebrain and eye structures when overexpressed in Xenopus laevis embryos. According to current views, this finding indicates the ability of lamprey Noggin proteins to suppress the activity of the BMP, Nodal/Activin and Wnt/beta-catenin signaling pathways, as shown for Noggin proteins of gnathostomes. In this work, by analogy with experiments in Xenopus embryos, we attempted to induce secondary axes in the European river lamprey Lampetra fluviatilis by injecting noggin mRNAs into lamprey eggs in vivo. Surprisingly, unlike what occurs in amphibians, secondary axis induction in the lampreys either by noggin mRNAs or by chordin and cerberus mRNAs, the inductive properties of which have been described, was not observed. Only wnt8a mRNA demonstrated the ability to induce secondary axes in the lampreys. Such results may indicate that the mechanism of axial specification in lampreys, which represent jawless vertebrates, may differ in detail from that in the jawed clade

Using RNA-binding proteins for immunoprecipitation of mRNAs from Xenopus laevis embryos

Summary: 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)

Ras-dva1 small GTPase regulates telencephalon development in Xenopus laevis embryos by controlling Fgf8 and Agr signaling at the anterior border of the neural plate

We previously found that the small GTPase Ras-dva1 is essential for the telencephalic development in Xenopus laevis because Ras-dva1 controls the Fgf8-mediated induction of FoxG1 expression, a key telencephalic regulator. In this report, we show, however, that Ras-dva1 and FoxG1 are expressed in different groups of cells; whereas Ras-dva1 is expressed in the outer layer of the anterior neural fold, FoxG1 and Fgf8 are activated in the inner layer from which the telencephalon is derived. We resolve this paradox by demonstrating that Ras-dva1 is involved in the transduction of Fgf8 signal received by cells in the outer layer, which in turn send a feedback signal that stimulates FoxG1 expression in the inner layer. We show that this feedback signal is transmitted by secreted Agr proteins, the expression of which is activated in the outer layer by mediation of Ras-dva1 and the homeodomain transcription factor Otx2. In turn, Agrs are essential for maintaining Fgf8 and FoxG1 expression in cells at the anterior neural plate border. Our finding reveals a novel feedback loop mechanism based on the exchange of Fgf8 and Agr signaling between neural and non-neural compartments at the anterior margin of the neural plate and demonstrates a key role of Ras-dva1 in this mechanism

The Cytoskeletal Protein Zyxin Inhibits Retinoic Acid Signaling by Destabilizing the Maternal mRNA of the RXRγ Nuclear Receptor

Zyxin is an LIM-domain-containing protein that regulates the assembly of F-actin filaments in cell contacts. Additionally, as a result of mechanical stress, Zyxin can enter nuclei and regulate gene expression. Previously, we found that Zyxin could affect mRNA stability of the maternally derived stemness factors of Pou5f3 family in Xenopus laevis embryos through binding to Y-box factor1. In the present work, we demonstrate that Zyxin can also affect mRNA stability of the maternally derived retinoid receptor Rxrγ through the same mechanism. Moreover, we confirmed the functional link between Zyxin and Rxrγ-dependent gene expression. As a result, Zyxin appears to play an essential role in the regulation of the retinoic acid signal pathway during early embryonic development. Besides, our research indicates that the mechanism based on the mRNA destabilization by Zyxin may take part in the control of the expression of a fairly wide range of maternal genes

Morphogene adsorption as a Turing instability regulator: Theoretical analysis and possible applications in multicellular embryonic systems

<div><p>The Turing instability in the reaction-diffusion system is a widely recognized mechanism of the morphogen gradient self-organization during the embryonic development. One of the essential conditions for such self-organization is sharp difference in the diffusion rates of the reacting substances (morphogens). In classical models this condition is satisfied only for significantly different values of diffusion coefficients which cannot hold for morphogens of similar molecular size. One of the most realistic explanations of the difference in diffusion rate is the difference between adsorption of morphogens to the extracellular matrix (ECM). Basing on this assumption we develop a novel mathematical model and demonstrate its effectiveness in describing several well-known examples of biological patterning. Our model consisting of three reaction-diffusion equations has the Turing-type instability and includes two elements with equal diffusivity and immobile binding sites as the third reaction substance. The model is an extension of the classical Gierer-Meinhardt two-components model and can be reduced to it under certain conditions. Incorporation of ECM in the model system allows us to validate the model for available experimental parameters. According to our model introduction of binding sites gradient, which is frequently observed in embryonic tissues, allows one to generate more types of different spatial patterns than can be obtained with two-components models. Thus, besides providing an essential condition for the Turing instability for the system of morphogen with close values of the diffusion coefficients, the morphogen adsorption on ECM may be important as a factor that increases the variability of self-organizing structures.</p></div

Simulation of follicle formation by using the extended GM model.

<p>WNT activation pattern in wild type (a) and DKK+ transgenic mice (b) redrawn from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171212#pone.0171212.ref030" target="_blank">30</a>]. Turing structures formed from homogenious noise in 5 minutes (internal time) with different levels of base DKK expression: 0.1, 0.15 and 0.2 <i>μ</i>M/s (c-d). Other parameters used are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171212#pone.0171212.t001" target="_blank">Table 1</a>.</p

Parameters of the extended GM model and their values fitted for reproducing Wnt/DKK hair follicle patterning.

<p>Parameters of the extended GM model and their values fitted for reproducing Wnt/DKK hair follicle patterning.</p