Зарайский Андрей Георгиевич

Личная информация

Работает в ИБХ РАН с 1983 г. В настоящее время является руководителем лаборатории молекулярных основ эмбриогенеза ИБХ РАН.

Образование

Период обученияСтрана, городУчебное заведениеДополнительная информация
1978–1983 Россия, Москва Московский государственный университет имени М.В. Ломоносова (МГУ), биологический факультет Диплом биолога
1990 Россия, Москва Московский государственный университет имени М.В. Ломоносова (МГУ), биологический факультет Присуждена учёная степень кандидата биологических наук
2000 Россия, Москва Московский государственный университет имени М.В. Ломоносова (МГУ), биологический факультет Присуждена учёная степень доктора биологических наук
2011 Россия Диплом профессора по специальности "молекулярная биология"

Научные интересы

Основные работы А.Г. Зарайского посвящены структурно-функциональному изучению генов и белков, регулирующих эмбриональное развитие мозга.

Премии и заслуги

Лауреат премий А. А. Баева (2001 г.) и А. О. Ковалевского РАН (2006 г.).

Основные научные результаты

В лаборатории молекулярных основ эмбриогенеза под руководством А. Г. Зарайского были открыты и изучены гомеобоксные гены нового класса Anf. Данные гены кодируют специфические транскрипционные факторы, играющие ключевую роль в развитии переднего мозга в раннем эмбриогенезе позвоночных и, в том числе, человека. Были получены данные о том, что появление Anf генов в эволюции у предков позвоночных послужило предпосылкой для возникновения переднего мозга — эмбрионального отдела мозга, дающего начало таким важным структурам, как большие полушария мозга. Дальнейшие работы по изучению генной сети, связанной с функционированием генов Anf, привели к обнаружению нового, 8-ого, семейства малых ГТФаз, получивших название Ras-dva, которые специфично экспрессируются в зачатке головного мозга и регулируют его раннее развитие. Важным достижением, лаборатории молекулярных основ эмбриогенеза является также открытие двух новых семейства секретируемых белков Noggin регулируемых факторами Anf и способных связывать, и тем самым ингибировать, ряд ростовых факторов из суперсемейства TGF-β.

Членство в научных обществах

А. Г. Зарайский является членом Ученого и Диссертационного советов ИБХ РАН, редколлегий журналов «Молекулярная биология» и «Онтогенез».

Избранные публикации

  1. Eroshkin F.M., Zaraisky A.G. (2017). Mechano-sensitive regulation of gene expression during the embryonic development. Genesis , [+]

    Cell movements during embryogenesis produce mechanical tensions that shape the embryo and can also regulate gene expression, thereby affecting cell differentiation. Increasing evidence indicates that mechano-sensitive regulation of gene expression plays important roles during embryogenesis by coupling the processes of morphogenesis and differentiation. However, the molecular mechanisms of this phenomenon remain poorly understood. This review focuses on the molecular mechanisms that "translate" mechanical stimuli into gene expression. This article is protected by copyright. All rights reserved.

    ID:1707
  2. Nesterenko A.M., Kuznetsov M.B., Korotkova D.D., Zaraisky A.G. (2017). Morphogene adsorption as a Turing instability regulator: Theoretical analysis and possible applications in multicellular embryonic systems. PLoS ONE 12 (2), e0171212 [+]

    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.

    ID:1699
  3. Bayramov A.V., Ermakova G.V., Eroshkin F.M., Kucheryavyy A.V., Martynova N.Y., Zaraisky A.G. (2016). The presence of Anf/Hesx1 homeobox gene in lampreys suggests that it could play an important role in emergence of telencephalon. Sci Rep 6, 39849 [+]

    Accumulated evidence indicates that the core genetic mechanisms regulating early patterning of the brain rudiment in vertebrates are very similar to those operating during development of the anterior region of invertebrate embryos. However, the mechanisms underlying the morphological differences between the elaborate vertebrate brain and its simpler invertebrate counterpart remain poorly understood. Recently, we hypothesized that the emergence of the most anterior unit of the vertebrate brain, the telencephalon, could be related to the appearance in vertebrates' ancestors of a unique homeobox gene, Anf/Hesx1(further Anf), which is absent from all invertebrates and regulates the earliest steps of telencephalon development in vertebrates. However, the failure of Anf to be detected in one of the most basal extant vertebrate species, the lamprey, seriously compromises this hypothesis. Here, we report the cloning of Anf in three lamprey species and demonstrate that this gene is indeed expressed in embryos in the same pattern as in other vertebrates and executes the same functions by inhibiting the expression of the anterior general regulator Otx2 in favour of the telencephalic regulator FoxG1. These results are consistent with the hypothesis that the Anf homeobox gene may have been important in the evolution of the telencephalon.

    ID:1698
  4. Eroshkin F.M., Nesterenko A.M., Borodulin A.V., Martynova N.Y., Ermakova G.V., Gyoeva F.K., Orlov E.E., Belogurov A.A. Jr, Lukyanov K.A., Bayramov A.V., Zaraisky A.G. (2016). Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis. Sci Rep 6, 23049 [+]

    Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/β -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development.

    ID:1419
  5. Nesterenko A.M., Orlov E.E., Ermakova G.V., Ivanov I.A., Semenyuk P.I., Orlov V.N., Martynova N.Y., Zaraisky A.G. (2015). Affinity of the heparin binding motif of Noggin1 to heparan sulfate and its visualization in the embryonic tissues. Biochem. Biophys. Res. Commun. 468 (1-2), 331–6 [+]

    Heparin binding motifs were found in many secreted proteins and it was suggested that they are responsible for retardation of the protein diffusion within the intercellular space due to the binding to heparan sulfate proteoglycanes (HSPG). Here we used synthetic FITC labeled heparin binding motif (HBM peptide) of the Xenopus laevis secreted BMP inhibitor Noggin1 to study its diffusion along the surface of the heparin beads by FRAP method. As a result, we have found out that diffusivity of HBM-labeled FITC was indeed much lesser than those predicted by theoretical calculations even for whole protein of the Noggin size. We also compared by isothermal titration calorimetry the binding affinity of HBM and the control oligolysine peptide to several natural polyanions including heparan sulfate (HS), heparin, the bacterial dextran sulfate and salmon sperm DNA, and demonstrated that HBM significantly exceeds oligolysine peptide in the affinity to HS, heparin and DNA. By contrast, oligolysine peptide bound with higher affinity to dextran sulfate. We speculate that such a difference may ensure specificity of the morphogen binding to HSPG and could be explained by steric constrains imposed by different distribution of the negative charges along a given polymeric molecule. Finally, by using EGFP-HBM recombinant protein we have visualized the natural pattern of the Noggin1 binding sites within the X. laevis gastrula and demonstrated that these sites forms a dorsal-ventral concentration gradient, with a maximum in the dorsal blastopore lip. In sum, our data provide a quantitative basis for modeling the process of Noggin1 diffusion in embryonic tissues, considering its interaction with HSPG.

    ID:1364
  6. Matlashov M.E., Bogdanova Y.A., Ermakova G.V., Mishina N.M., Ermakova Y.G., Nikitin E.S., Balaban P.M., Okabe S., Lukyanov S., Enikolopov G., Zaraisky A.G., Belousov V.V. (2015). Fluorescent ratiometric pH indicator SypHer2: applications in neuroscience and regenerative biology. Biochim. Biophys. Acta 1850 (11), 2318–2328 [+]

     

    SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, a relatively low brightness of the indicator limits its use.

    METHODS:

     

    Here we designed a new version of pH-sensor - SypHer-2, that has up to three times brighter fluorescence signal in cultured mammalian cells compared to the SypHer.

    RESULTS:

     

    Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent temporal neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop which occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate.

    CONCLUSIONS:

     

    SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo.

    GENERAL SIGNIFICANCE:

     

    The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.

     

    ID:1309
  7. Ivanova A.S., Shandarin I.N., Ermakova G.V., Minin A.A., Tereshina M.B., Zaraisky A.G. (2015). The secreted factor Ag1 missing in higher vertebrates regulates fins regeneration in Danio rerio. Sci Rep 5, 8123 [+]

    Agr family includes three groups of genes, Ag1, Agr2 and Agr3, which encode the thioredoxin domain-containing secreted proteins and have been shown recently to participate in regeneration of the amputated body appendages in amphibians. By contrast, higher vertebrates have only Agr2 and Agr3, but lack Ag1, and have low ability to regenerate the body appendages. Thus, one may hypothesize that loss of Ag1 in evolution could be an important event that led to a decline of the regenerative capacity in higher vertebrates. To test this, we have studied now the expression and role of Ag1 in the regeneration of fins of a representative of another large group of lower vertebrates, the fish Danio rerio. As a result, we have demonstrated that amputation of the Danio fins, like amputation of the body appendages in amphibians, elicits an increase of Ag1 expression in cells of the stump. Furthermore, down-regulation of DAg1 by injections of Vivo-morpholino antisense oligonucleotides resulted in a retardation of the fin regeneration. These data are in a good agreement with the assumption that the loss of Ag1 in higher vertebrates ancestors could lead to the reduction of the regenerative capacity in their modern descendants.

    ID:1248
  8. Pereverzev A.P., Gurskaya N.G., Ermakova G.V., Kudryavtseva E.I., Markina N.M., Kotlobay A.A., Lukyanov S.A., Zaraisky A.G., Lukyanov K.A. (2015). Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep 5, 7729 [+]

    Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.

    ID:1247
  9. Tereshina M.B., Ermakova G.V., Ivanova A.S., Zaraisky A.G. (2014). 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. Biol Open , [+]

    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.

    ID:1007
  10. Martynova N.Y., Ermolina L.V., Ermakova G.V., Eroshkin F.M., Gyoeva F.K., Baturina N.S., Zaraisky A.G. (2013). The cytoskeletal protein Zyxin inhibits Shh signaling during the CNS patterning in Xenopus laevis through interaction with the transcription factor Gli1. Dev. Biol. 380 (1), 37–48 [+]

    Zyxin is a cytoskeletal protein that controls cell movements by regulating actin filaments assembly, but it can also modulate gene expression owing to its interactions with the proteins involved in signaling cascades. Therefore, identification of proteins that interact with Zyxin in embryonic cells is a promising way to unravel mechanisms responsible for coupling of two major components of embryogenesis: morphogenetic movements and cell differentiation. Now we show that in Xenopus laevis embryos Zyxin can bind to and suppress activity of the primary effector of Sonic hedgehog (Shh) signaling cascade, the transcription factor Gli1. By using loss- and gain-of-function approaches, we demonstrate that Zyxin is essential for reduction of Shh signaling within the dorsal part of the neural tube of X. laevis embryo. Thus, our finding discloses a novel function of Zyxin in fine tuning of the central neural system patterning which is based on the ventral-to-dorsal gradient of Shh signaling.

    ID:857
  11. Ivanova A.S., Tereshina M.B., Ermakova G.V., Belousov V.V., Zaraisky A.G. (2013). Agr genes, missing in amniotes, are involved in the body appendages regeneration in frog tadpoles. Sci Rep 3, 1279 [+]

    Previous studies have shown that Agr genes, which encode thioredoxin domain-containing secreted proteins, play a critical role in limb regeneration in salamanders. To determine the evolutionary conservation of Agr function, it is important to examine whether Agrs play a similar role in species with a different type of regeneration. Here, we refined the phylogeny of Agrs, revealing three subfamilies: Ag1, Agr2 and Agr3. Importantly, we established that Ag1 was lost in higher vertebrates, which correlates with their decreased regeneration ability. In Xenopus laevis tadpoles (anamniotes), which have all three Agr subfamilies and a high regenerating capacity, Agrs were activated in the stumps of tails and hindlimb buds that were amputated at stage 52. However, Agrs were not up-regulated when the hindlimb buds were amputated at stage 57, the stage at which their regeneration capacity is lost. Our findings indicate the general importance of Agrs for body appendages regeneration in amphibians.

    ID:833
  12. Borodulin A.V., Eroshkin F.M., Bayramov A.V., Zaraisky A.G. (2012). Noggin4 expression during chick embryonic development. Int. J. Dev. Biol. 56 (5), 403–6 [+]

    We describe here the expression pattern of Noggin4 during the early development of the chick embryo (Gallus gallus). The expression of this gene starts with the onset of gastrulation (stage HH4), in two bilateral bands along the primitive streak, with a local maximum around Hensens node. By the end of gastrulation, Noggin4 transcripts are distributed diffusely throughout the epiblast, with the highest concentration in the head ectoderm. Interestingly, the expression of Noggin4 during the first half of gastrulation demonstrates a clear left-right asymmetry in Hensens node, being much more intensive in its right anterior portion. During neurulation, Noggin4 is expressed mainly in the neuroectoderm, with the most intensive expression in the head and lateral neural folds. In mesoderm derivatives, expression is seen in somites but not in the notochord. In general, primarily ectodermal and diffusive expression of Noggin4 in chick embryo, with a maximum in the anterior neurectoderm, resembles that of its ortholog in Xenopus, which indicates a conservative function of this gene in evolution.

    ID:728
  13. Shemiakina I.I., Ermakova G.V., Cranfill P.J., Baird M.A., Evans R.A., Souslova E.A., Staroverov D.B., Gorokhovatsky A.Y., Putintseva E.V., Gorodnicheva T.V., Chepurnykh T.V., Strukova L., Lukyanov S., Zaraisky A.G., Davidson M.W., Chudakov D.M., Shcherbo D. (2012). A monomeric red fluorescent protein with low cytotoxicity. Nat Commun 3, 1204 [+]

    Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues.

    ID:832
  14. Bayramov A.V., Eroshkin F.M., Martynova N.Y., Ermakova G.V., Solovieva E.A., Zaraisky A.G. (2011). Novel functions of Noggin proteins: inhibition of Activin/Nodal and Wnt signaling. Development 138 (24), 5345–56 [+]

    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.

    ID:551
  15. Serebrovskaya E.O., Gorodnicheva T.V., Ermakova G.V., Solovieva E.A., Sharonov G.V., Zagaynova E.V., Chudakov D.M., Lukyanov S., Zaraisky A.G., Lukyanov K.A. (2011). Light-induced blockage of cell division with a chromatin-targeted phototoxic fluorescent protein. Biochem. J. 435 (1), 65–71 [+]

    Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)-tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B-tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B-tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B-tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B-tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B-tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo.

    ID:550
  16. Tereshina M.B., Bayramov A.V., Zaraisky A.G. (2011). Expression patterns of genes encoding small GTPases Ras-dva-1 and Ras-dva-2 in the Xenopus laevis tadpoles. Gene Expr. Patterns 11 (1-2), 156–61 [+]

    Small GTPases of the recently discovered Ras-dva family are specific to the Vertebrate phylum. In Xenopus laevis, Ras-dva-1 is expressed during gastrulation and neurulation in the anterior ectoderm where it regulates the early development of the forebrain and cranial placodes (Tereshina et al., 2006). In the present work, we studied the expression of Ras-dva-1 at later developmental stages. As a result, the Ras-dva-1 expression was revealed in the eye retina, epiphysis (pineal gland), hypophysis (pituitary), branchial arches, pharynx, oesophagus, stomach and gall bladder of swimming tadpoles. Additionally, we investigated for the first time the expression pattern of Ras-dva-2. This gene encodes a protein belonging to a novel sub-group of Ras-dva GTPases that we identified by phylogenetic analysis within Ras-dva family. In contrast to Ras-dva-1, Ras-dva-2 is not expressed before the swimming tadpole stage. At the swimming tadpole stage, however, Ras-dva-2 transcripts can be detected in the eye retina and brain. Later in development, the expression of Ras-dva-2 can also be revealed in the mesonephros and stomach.

    ID:549
  17. Shcherbo D., Shemiakina I.I., Ryabova A.V., Luker K.E., Schmidt B.T., Souslova E.A., Gorodnicheva T.V., Strukova L., Shidlovskiy K.M., Britanova O.V., Zaraisky A.G., Lukyanov K.A., Loschenov V.B., Luker G.D., Chudakov D.M. (2010). Near-infrared fluorescent proteins. Nat. Methods 7 (10), 827–9 [+]

    Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

    ID:369
  18. Shcherbo D., Murphy C.S., Ermakova G.V., Solovieva E.A., Chepurnykh T.V., Shcheglov A.S., Verkhusha V.V., Pletnev V.Z., Hazelwood K.L., Roche P.M., Lukyanov S., Zaraisky A.G., Davidson M.W., Chudakov D.M. (2009). Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418 (3), 567–74 [+]

    Разработан яркий, мономерный, фотостабильный, pH-стабильный, дальне-красный флуоресцентый белок mKate2. Белок mKate2 хорошо показал себя в качестве метки во фьюзах с рядом белков, как в культуре клеток, так и в трансгенных лягушках Xenopus laevis (совместно с лабораторией Молекулярных основ эмбриогенеза ИБХ РАН).

    ID:31
  19. Belintsev B.N., Belousov L.V., Zaraĭskiĭ A.G. (2009). [Model of epithelial morphogenesis based on elastic forces and cell contact polarization]. Ontogenez 16 (1), 5–14 [+]

    It is well known that in embryonic tissues at the key stages of morphogenesis there arise stable, stage--specific tension fields. These fields occur due to particular pattern of morphologically polarized cells. Some basic properties have been understood previously. 1. Morphologically polarized and isotropic shapes correspond to the alternative stable states of embryonic cells. 2. Polarization can be transmitted between the adjacent cells via intercellular contacts. 3. The tension fields at particular stage of development determine the pattern of morphogenetic movements. In this paper the physical model is suggested which interprets the selforganization of tension fields in embryonic tissues. The polarization in some region of tissue is assumed to generate the elastic tension in the surrounding cells thus restricting the propagation of cell polarization. It is shown that the properties underlined are sufficient to provide spontaneous subdivision of the cellular layer into the domains of polarized and unpolarized stretched cells. The proportion of polarized and unpolarized areas is determined and size--invariant.

    ID:567
  20. Zaraĭskiĭ A.G. (2009). [Self-organization in the determination of the size of the axial structures in the embryogenesis of the clawed toad]. Ontogenez 22 (4), 365–74 [+]

    Experiments were performed using X. laevis embryos during gastrulation and neurulation (stages 10, 11 1/2, 12 1/2, 13 1/2, 15 and 18). Part of presumptive epidermis and lateral plate mesoderm was removed, and embryos raised until stage 25. The size of axial structures (notochord, somite mesoderm, central nervous system) was determined using serial histological sections and compared with that of control embryos. In experimental embryos, the size of axial structures was decreased. Until a specific stage of development, close correlation was found between the volume of embryonic compartment corresponding to a particular, structure and the volume of presumptive epidermis and lateral plate mesoderm. This stage is individual for each axial organ: middle gastrula (stage 11 1/2) for notochord, late gastrula (stage 12 1/2) for somite mesoderm, and late neurula (stage 18) for central nervous system. This data suggest that differentiation pattern of ecto-mesodermal rudiment is subject to regulation during gastrulation-neurulation, and subdivision of ectoderm and mesoderm into axial and non-axial tissues is a self-organizing process.

    ID:568
  21. Belousov L.V., Luchinskaia N.N., Zaraĭskiĭ A.G. (2009). [Tensotaxis--a collective movement of embryonic cells up along the gradients of mechanical tensions]. Ontogenez 30 (3), 220–8 [+]

    We have examined the active collective movement of ectodermal cells from early gastrula of Xenopus laevis towards the point source of stretching, using techniques of videomicroscopy and scanning electron microscopy. We define this mode of cell movement as tensotaxis. This movement begins near the source of tension 5-10 min after the beginning of stretching and is spread in a relay fashion to more distant cells. As a result, a considerable fraction of observed cells more towards the source of stretching over a considerable territory at a rate of 0.6-3 mu/min. Subsequently, these movements are replaced by cell intercalation roughly oriented in the direction transverse to that of tissue stretching. It is proposed that tensotaxis is initiated by asymmetric deformation of the embryonic tissue due to the concentration (focusing) of a stretching force and contains both passive and active components. Data are presented supporting the view that, during normal development, tensotaxis may determine the movement of embryonic cells towards the blastopore and can also participate in other morphogenetic processes.

    ID:569
  22. Ivanova A.S., Shandarin I.N., Minin A.A., Tereshina M.B., Zaraisky A.G. (2009). [ag1 Is Required for the Fin Regeneration in Danio rerio]. Bioorg. Khim. 41 (4), 427–31 [+]

    In the current research, we have demonstrated that Ag1 protein is necessary for the fin regeneration in the fish Danio rerio. Robust activation of gene ag1 expression in cells of the wound epithelium is observed after caudal fin amputation. Besides, inhibition of translation of ag1 mRNA leads to retardation of the caudal tail fin regeneration. Results of our research are important because only lower vertebrates (fish and amphibians) with good regenerative capacity have ag1, whereas this gene is missing in higher vertebrates, which are not capable to effectively regenerate limbs. Our data confirm that reduction of the regenerative abilities in higher vertebrates, including human, could be explained by extinction of some genes essential for the regeneration, in particular, of ag1.

    ID:1422
  23. Martynova N.Y., Eroshkin F.M., Ermolina L.V., Ermakova G.V., Korotaeva A.L., Smurova K.M., Gyoeva F.K., Zaraisky A.G. (2008). The LIM-domain protein Zyxin binds the homeodomain factor Xanf1/Hesx1 and modulates its activity in the anterior neural plate of Xenopus laevis embryo. Dev. Dyn. 237 (3), 736–49 [+]

    The question of how subdivision of embryo into cell territories acquiring different fates is coordinated with morphogenetic movements shaping the embryonic body still remains poorly resolved. In the present report, we demonstrate that a key regulator of anterior neural plate patterning, the homeodomain transcriptional repressor Xanf1/Hesx1, can bind to the LIM-domain protein Zyxin, which is known to regulate cell morphogenetic movements via influence on actin cytoskeleton dynamics. Using a set of deletion mutants, we found that the Engrailed-type repressor domain of Xanf1 and LIM2-domain of Zyxin are primarily responsible for interaction of these proteins. We also demonstrate that Zyxin overexpression in Xenopus embryos elicits effects similar to those observed in embryos with downregulated Xanf1. In contrast, when the repressor-fused variant of Zyxin is expressed, the forebrain enlargements typical for embryos overexpressing Xanf1 develop. These results are consistent with a possible role of Zyxin as a negative modulator of Xanf1 transcriptional repressing activity.

    ID:78
  24. Shcherbo D., Merzlyak E.M., Chepurnykh T.V., Fradkov A.F., Ermakova G.V., Solovieva E.A., Lukyanov K.A., Bogdanova E.A., Zaraisky A.G., Lukyanov S., Chudakov D.M. (2007). Bright far-red fluorescent protein for whole-body imaging. Nat. Methods 4 (9), 741–6 [+]

    Разработан новый флуоресцентный белок Katushka, обладающий флуоресценцией в дальне-красной области спектра, которая является предпочтительной для анализа сигнала внутри тканей животных. Katushka в десять раз ярче, чем созданные ранее дальне-красные флуоресцентные белки и характеризуется высокой скоростью созревания, высокой рН-стабильностью и фотостабильностью. Это делает новый белок идеальным инструментом для прижизненного мечения клеток внутри целых организмов. Создан мономерный вариант белка Katushka, названный mKate, для исследования внутриклеточной локализации белков.

    ID:76
  25. Ermakova G.V., Solovieva E.A., Martynova N.Y., Zaraisky A.G. (2007). The homeodomain factor Xanf represses expression of genes in the presumptive rostral forebrain that specify more caudal brain regions. Dev. Biol. 307 (2), 483–97 [+]

    Early development of the rostral forebrain (RF) in vertebrates is accompanied by the inhibition of two homeobox regulators, Otx2 and Pax6 in the rostral sector of the anterior neural plate, further giving rise to the RF. However, the precise molecular mechanism and meaning of this inhibition is still obscure. We now demonstrate that the activity of the Anf homeodomain protein is necessary and sufficient for the anterior inhibition of Otx2 and Pax6. Specifically, we show that knockdown of the Xenopus laevis Anf, Xanf, by antisense morpholino oligonucleotides results in the anterior expansion of Otx2 and Pax6 expression into the presumptive RF territory. Furthermore, by overexpressing hormone-inducible activator- and repressor-fused variants of Xanf in the absence of protein synthesis, we present evidence that Xanf can directly downregulate Otx2 and Pax6 but not the more rostrally expressed Bf1, Bf2, Fgf8 and Nkx2.4. These results explain how the inhibitory activity of Xanf can discriminate RF regulators in favor of posterior forebrain ones. Assuming that the Anf type of homeobox is specific for vertebrates, our data suggest that the emergence of Anf in evolution could be a critical event for RF development in vertebrates through the elimination of homologues of modern posterior forebrain regulators from the rostral sector of the anterior neural plate.

    ID:77
  26. Evdokimov A.G., Pokross M.E., Egorov N.S., Zaraisky A.G., Yampolsky I.V., Merzlyak E.M., Shkoporov A.N., Sander I., Lukyanov K.A., Chudakov D.M. (2006). Structural basis for the fast maturation of Arthropoda green fluorescent protein. EMBO Rep. 7 (10), 1006–12 [+]

    Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

    ID:280
  27. Tereshina M.B., Zaraisky A.G., Novoselov V.V. (2006). Ras-dva, a member of novel family of small GTPases, is required for the anterior ectoderm patterning in the Xenopus laevis embryo. Development 133 (3), 485–94 [+]

    Ras-like small GTPases are involved in the regulation of many processes essential for the specification of the vertebrate body plan. Recently, we identified the gene of novel small GTPase Ras-dva, which is specifically expressed at the anterior margin of the neural plate of the Xenopus laevis embryo. Now, we demonstrate that Ras-dva and its homologs in other species constitute a novel protein family, distinct from the previously known families of small GTPases. We show that the expression of Ras-dva begins during gastrulation throughout the anterior ectoderm and is activated by the homeodomain transcription factor Otx2; however, later on, Ras-dva expression is inhibited in the anterior neural plate by another homeodomain factor Xanf1. Downregulation of Ras-dva functioning by the dominant-negative mutant or by the antisense morpholino oligonucleotides results in severe malformations of the forebrain and derivatives of the cranial placodes. Importantly, although the observed abnormalities can be rescued by co-injection of the Ras-dva mRNA, they cannot be rescued by the mRNA of the closest Ras-dva homolog from another family of small GTPases, Ras. This fact indicates functional specificity of the Ras-dva signaling pathway. At the molecular level, downregulation of Ras-dva inhibits the expression of several regulators of the anterior neural plate and folds patterning, such as Otx2, BF-1 (also known as Foxg1), Xag2, Pax6, Slug and Sox9, and interferes with FGF8 signaling within the anterior ectoderm. By contrast, expression of the epidermal regulator BMP4 and its target genes, Vent1, Vent2b and Msx1, is upregulated. Together, the data obtained indicate that Ras-dva is an essential component of the signaling network that patterns the early anterior neural plate and the adjacent ectoderm in the Xenopus laevis embryos.

    ID:75
  28. Efimov V.A., Birikh K.R., Staroverov D.B., Lukyanov S.A., Tereshina M.B., Zaraisky A.G., Chakhmakhcheva O.G. (2006). Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo. Nucleic Acids Res. 34 (8), 2247–57 [+]

    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.

    ID:552
  29. Eroshkin F.M., Ermakova G.V., Bayramov A.V., Zaraisky A.G. (2006). Multiple noggins in vertebrate genome: cloning and expression of noggin2 and noggin4 in Xenopus laevis. Gene Expr. Patterns 6 (2), 180–6 [+]

    Noggin is a neural inducer secreted by cells of the Spemann organizer. A single noggin gene was identified until very recently in all tested vertebrates. The only exception was zebrafish, in which two close homologs of noggin, named noggin1 and noggin3, and one gene more diverged from them, noggin2, were cloned. Nevertheless, finding of three zebrafish noggins was attributed exclusively to specific genomic duplications in the fish evolutionary branch. However, very recently it was shown that Xenopus tropicalis have additional noggin homolog, called noggin2 [Fletcher, R.B., Watson, A.L., Harland, R.M. (2004). Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod. Gene Expr. Patterns 5, 225-230], which indicates at least two independent noggin genes in vertebrate phylum. Now we report identification of two novel noggin homologs in each of so evolutionary distant species as Xenopus laevis, chicken and fugu. One of these noggins is ortholog of the X. tropicalis and zebrafish noggin2, whereas another, named noggin4, was not known previously. In the X. laevis embryos, the expression of noggin2 very resembles that of its counterpart in X. tropicalis: it begins with neurulation at the anterior margin of the neural plate and, afterward, continues mainly in the forebrain and dorsal hindbrain. At the same time, noggin4 is expressed starting from the beginning of gastrulation, throughout the ectoderm, with a local expression maximum in the prospective anterior neurectoderm. Later, it is widely expressed on the dorsal side of embryo, including neural tube, eyes, otic vesicles, cranial placodes, branchial arches, and somites. The data presented here demonstrate that the vertebrate phylum contains at least three distinct noggin genes.

    ID:553
  30. Bayramov A.V., Martynova N.Y., Eroshkin F.M., Ermakova G.V., Zaraisky A.G. (2004). The homeodomain-containing transcription factor X-nkx-5.1 inhibits expression of the homeobox gene Xanf-1 during the Xenopus laevis forebrain development. Mech. Dev. 121 (12), 1425–41 [+]

    Expression of the homeobox gene Xanf-1 starts within the presumptive forebrain primordium of the Xenopus embryo at the midgastrula stage and is inhibited by the late neurula. Such stage-specific inhibition is essential for the normal development as the experimental prolongation of the Xanf-1 expression elicits severe brain abnormalities. To identify transcriptional regulators that are responsible for the Xanf-1 inhibition, we have used the yeast one-hybrid system and identified a novel Xenopus homeobox gene X-nkx-5.1 that belongs to a family of Nkx-5.1 transcription factors. In terms of gene expression, X-nkx-5.1 shares many common features with its orthologs in other species, including expression in the embryonic brain and in the ciliated cells of the otic and lateral line placodes. However, we have also observed several features specific for X-nkx-5.1, such as expression in precursors of the epidermal ciliated cells that may indicate a possible common evolutionary origin of all ciliated cells derived from the embryonic ectoderm. Another specific feature is that the X-nkx-5.1 expression in the anterior neural plate starts early, within the area overlapping the Xanf-1 expression territory at the midneurula stage, and it correlates with the beginning of the Xanf-1 inhibition. Using various loss and gain-of-function techniques, including microinjections of antisense morpholino oligonucleotides and mRNA encoding for the X-nkx-5.1 and its dominant repressor and activator versions, we have shown that X-nkx-5.1 can indeed play a role of stage-specific inhibitor of Xanf-1 in the anterior neural plate during the Xenopus development.

    ID:554
  31. Martynova N., Eroshkin F., Ermakova G., Bayramov A., Gray J., Grainger R., Zaraisky A. (2004). Patterning the forebrain: FoxA4a/Pintallavis and Xvent2 determine the posterior limit of Xanf1 expression in the neural plate. Development 131 (10), 2329–38 [+]

    During early development of the nervous system in vertebrates, expression of the homeobox gene Anf/Hesx1/Rpx is restricted to the anterior neural plate subdomain corresponding to the presumptive forebrain. This expression is essential for normal forebrain development and ectopic expression of Xenopus Anf, Xanf1 (also known as Xanf-1), results in severe forebrain abnormalities. By use of transgenic embryos and a novel bi-colour reporter technique, we have identified a cis-regulatory element responsible for transcriptional repression of Xanf1 that defines its posterior expression limit within the neural plate. Using this element as the target in a yeast one-hybrid system, we identified two transcription factors, FoxA4a/Pintallavis and Xvent2 (also known as Xvent-2), which are normally expressed posterior to Xanf1. Overexpression of normal and dominant-negative versions of these factors, as well as inhibition of their mRNA translation by antisense morpholinos, show that they actually function as transcriptional repressors of Xanf1 just behind its posterior expression limit. The extremely high similarity of the identified Anf cis-regulatory sequences in Xenopus, chick and human, indicates that the mechanism restricting posterior expression of Anf in Xenopus is shared among vertebrates. Our findings support Nieuwkoop's activation-transformation model for neural patterning, according to which the entire neurectoderm is initially specified towards an anterior fate, which is later suppressed posteriorly as part of the trunk formation process.

    ID:74
  32. Verkhusha V.V., Kuznetsova I.M., Stepanenko O.V., Zaraisky A.G., Shavlovsky M.M., Turoverov K.K., Uversky V.N. (2003). High stability of Discosoma DsRed as compared to Aequorea EGFP. Biochemistry 42 (26), 7879–84 [+]

    Comparative analysis of conformational stabilities was performed for two widely used genetic reporters, EGFP and DsRed, proteins exhibiting similar beta-can folds, but possessing different oligomeric organization and chromophore structures. Two factors affecting protein stability in vitro, such as elevated temperatures and a chaotropic agent guanidine hydrochloride, were studied. In vivo tolerance of the fluorescence proteins to proteasomal-based degradation was studied in insect and mammalian cells, and in Xenopus embryos. The apparent rate constants of thermal and GdmCl-induced denaturation were several orders of magnitude lower for DsRed than for EGFP. DsRed lifetimes severalfold longer than those of EGFP were observed in cultured cells and in embryos. The remarkable fluorescence stability of DsRed under the all conditions that have been studied is attributed to a significant extent to its tetrameric organization. Therefore, DsRed can be used as a genetic reporter and advanced population marker with a significantly extended intracellular lifespan.

    ID:555
  33. Novoselov V.V., Alexandrova E.M., Ermakova G.V., Zaraisky A.G. (2003). Expression zones of three novel genes abut the developing anterior neural plate of Xenopus embryo. Gene Expr. Patterns 3 (2), 225–30 [+]

    We identified three novel genes that were expressed within the anterior non-neural ectoderm of Xenopus early neurula embryos. The expression of these genes was observed in the different areas complementary to the expression zone of a homeodomain gene Xanf-1 in the anterior neural plate. One of these genes, a Ras-like GTP-ase Ras-dva, marked the anterior placodal ectoderm area; a second, an Agr family homologous gene, XAgr2, was expressed in the anterior-most ectoderm in the cement gland primordium, and a third, novel gene Nlo was expressed in the lateral neural folds. The genes were transiently expressed in the developing cement and hatching gland primordia, and repressed in the mature cement and hatching glands. XAgr2 and Nlo were also expressed in the otic vesicles, and Ras-dva was expressed in the dorso-lateral column of the neural tube.

    ID:556
  34. Chudakov D.M., Belousov V.V., Zaraisky A.G., Novoselov V.V., Staroverov D.B., Zorov D.B., Lukyanov S., Lukyanov K.A. (2003). Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21 (2), 191–4 [+]

    Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.

    ID:73
  35. Terskikh A.V., Fradkov A.F., Zaraisky A.G., Kajava A.V., Angres B. (2002). Analysis of DsRed Mutants. Space around the fluorophore accelerates fluorescence development. J. Biol. Chem. 277 (10), 7633–6 [+]

    Earlier mutagenesis of the red fluorescent protein drFP583, also called DsRed, resulted in a mutant named Fluorescent Timer (Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X., Lukyanov, S., Matz, M., Kim, S., Weissman, I., and Siebert, P. (2000) Science 290, 1585--1588). Further mutagenesis generated variants with novel and improved fluorescent properties. The mutant called AG4 exhibits only green fluorescence. The mutant, called E5up (V105A), shows complete fluorophore maturation, eventually eliminating residual green fluorescence present in DsRed. Finally, the mutant, called E57 (V105A, I161T, S197A), matures faster than DsRed as demonstrated in vitro with purified protein and in vivo with recombinant protein expressed in Escherichia coli and Xenopus leavis. Comparative analysis of the mutants in the context of the crystal structure of DsRed suggests that mutants with free space around the fluorophore mature faster and more completely.

    ID:558
  36. Eroshkin F., Kazanskaya O., Martynova N., Zaraisky A. (2002). Characterization of cis-regulatory elements of the homeobox gene Xanf-1. Gene 285 (1-2), 279–86 [+]

    Investigation of molecular mechanisms underlying early patterning of the nervous system is an important task of modern developmental biology. Previously, we identified a novel homeobox gene, Anf, that is expressed in the most anterior zone at the beginning of neuroectoderm specification. The expression pattern of Anf corresponds to primordia of the telencephalon and the rostral part of the diencephalon. In the present work, we investigated cis-regulation of expression of the Xenopus laevis Anf, Xanf-1. Two elements, highly conserved in Xenopus, chick and human, were identified within the Xanf-1 promoter region. The first element, located near position -500, is necessary for overall enhancement of the Xanf-1 expression. The second element, near position -200, is crucial for maintenance of the Xanf-1 expression at moderate levels and also for specific localization of the expression in the anterior neuroectoderm. Thus, the distal part of this element is responsible for suppression of Xanf-1 posterior to the normal expression domain of this gene. The data obtained corroborate with the Nieuwkoop two-signal model of neural induction. This model states that at the first step of induction, all neuroectoderm acquires potencies to develop toward forebrain structures, but later these potencies are suppressed in posterior regions.

    ID:557
  37. Brockmann B., Smith M.W., Zaraisky A.G., Harrison K., Okada K., Kamiya Y. (2001). Subcellular localization and targeting of glucocorticoid receptor protein fusions expressed in transgenic Arabidopsis thaliana. Plant Cell Physiol. 42 (9), 942–51 [+]

    An animal system of inducible activation of protein fusions with the binding domain of glucocorticoid receptor (BDGR) was tested in Arabidopsis thaliana by monitoring dexamethasone (DEX)-induced nuclear targeting of reporter constructs. Two constructs containing green fluorescent protein (GFP), human homeobox protein Hanf-1 and Xenopus laevis BDGR were used, GFP/Hanf-1/BDGR and GFP/BDGR. The control construct contained GFP alone. In the absence of DEX both fusion proteins were uniformly distributed in the cytoplasm of root cells, but showed strong association with plastids in plant aerial parts. DEX treatment of roots prompted a strong and reversible nuclear accumulation of GFP/Hanf-1/BDGR, but not GFP/BDGR. Thus, in roots, the specific nuclear translocation of GFP/Hanf-1/BDGR was driven by Hanf-1 and tightly regulated by BDGR. However, in plant aerial parts treated with DEX, nuclear translocation of GFP/Hanf-1/BDGR was observed only in a few cases, and most part of the fusion protein was incorrectly and irreversibly targeted to plastids. Protease X digestion of isolated chloroplasts showed that BDGR fusion proteins were translocated into the chloroplast envelope and bound to envelope membranes, probably due to association with the chloroplast import apparatus. Thus, for efficient use of the glucocorticoid-inducible system in plants, it will be necessary to modify BDGR structure to prevent incorrect targeting of fusion proteins.

    ID:559
  38. Terskikh A., Fradkov A., Ermakova G., Zaraisky A., Tan P., Kajava A.V., Zhao X., Lukyanov S., Matz M., Kim S., Weissman I., Siebert P. (2000). "Fluorescent timer": protein that changes color with time. Science 290 (5496), 1585–8 [+]

    We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

    ID:72
  39. Lukyanov K.A., Fradkov A.F., Gurskaya N.G., Matz M.V., Labas Y.A., Savitsky A.P., Markelov M.L., Zaraisky A.G., Zhao X., Fang Y., Tan W., Lukyanov S.A. (2000). Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275 (34), 25879–82 [+]

    It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.

    ID:560
  40. Matz M.V., Fradkov A.F., Labas Y.A., Savitsky A.P., Zaraisky A.G., Markelov M.L., Lukyanov S.A. (1999). Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17 (10), 969–73 [+]

    Открыты новые флуоресцентные белки из коралловых полипов класса Anthozoa с разными цветами флуоресценции от сине-зеленого до красного. Выявление флуоресцентных и окрашенных GFP-подобных белков у неспособных к биолюминесценции коралловых полипов опровергло распространенное мнение, что такие белки функционируют только в составе биолюминисцентных систем и прояснило природу разнообразной флуоресцентной окраски коралловых рифов — явления, которое на протяжении многих лет не находило правильного объяснения.

    ID:70
  41. Ermakova G.V., Alexandrova E.M., Kazanskaya O.V., Vasiliev O.L., Smith M.W., Zaraisky A.G. (1999). The homeobox gene, Xanf-1, can control both neural differentiation and patterning in the presumptive anterior neurectoderm of the Xenopus laevis embryo. Development 126 (20), 4513–23 [+]

    From the onset of neurectoderm differentiation, homeobox genes of the Anf class are expressed within a region corresponding to the presumptive telencephalic and rostral diencephalic primordia. Here we investigate functions of the Xenopus member of Anf, Xanf-1, in the differentiation of the anterior neurectoderm. We demonstrate that ectopic Xanf-1 can expand the neural plate at expense of adjacent non-neural ectoderm. In tadpoles, the expanded regions of the plate developed into abnormal brain outgrowths. At the same time, Xanf-1 can inhibit terminal differentiation of primary neurones. We also show that, during gastrula/neurula stages, the exogenous Xanf-1 can downregulate four transcription regulators, XBF-1, Otx-2, Pax-6 and the endogenous Xanf-1, that are expressed in the anterior neurectoderm. However, during further development, when the exogenous Xanf-1 was presumably degraded, re-activation of XBF-1, Otx-2 and Pax-6 was observed in the abnormal outgrowths developed from blastomeres microinjected with Xanf-1 mRNA. Other effects of the ectopic Xanf-1 include cyclopic phenotype and inhibition of the cement gland, both by Otx-2-dependent and -independent mechanisms. Using fusions of Xanf-1 with the repressor domain of Drosophila engrailed or activator domain of herpes virus VP16 protein, we showed that most of the observed effects of Xanf-1 were probably elicited by its functioning as a transcription repressor. Altogether, our data indicate that the repressor function of Xanf-1 may be necessary for regulation of both neural differentiation and patterning in the presumptive anterior neurectoderm.

    ID:71
  42. Bogdanova E., Matz M., Tarabykin V., Usman N., Shagin D., Zaraisky A., Lukyanov S. (1998). Inductive interactions regulating body patterning in planarian, revealed by analysis of expression of novel gene scarf. Dev. Biol. 194 (2), 172–81 [+]

    Subtractive hybridization was used to search for the early difference in gene expression between anteriorly and posteriorly regenerating tissues of the same region of the planarian body. A sequence (named scarf) specific for posteriorly regenerating tissue was isolated, encoding a soluble C-type lectin consisting of two slightly different carbohydrate-recognition domains. Such an unusual bivalent structure allows attribution of the protein to a novel subfamily of C-type lectins. scarf expression in intact planarian is rather abundant and follows a characteristic pattern not linked to any known morphological structure. We performed a series of experiments using scarf as a molecular marker. Its expression was monitored during different types of regeneration by whole-mount in situ hybridization and reverse-transcription polymerase chain reaction. The obtained data suggest that scarf expression is positively regulated by anterior tissues closely adjacent to the scarf-expressing region, so that their surgical removal results in rapid scarf switch-off. In turn, tissues expressing scarf seem to inhibit its activation anteriorly. This indicates that at least some of the body patterning events in planarians are based on a system of reciprocal inductive interactions rather than on a global morphogen gradient.

    ID:561
  43. Vasiliev O.L., Lukyanov S.A., Belyavsky A.V., Kazanskaya O.V., Zaraisky A.G. (1997). A novel marker of early epidermal differentiation: cDNA subtractive cloning starting on a single explant of Xenopus laevis gastrula epidermis. Int. J. Dev. Biol. 41 (6), 877–82 [+]

    To understand the molecular mechanism underlying in the earliest steps of the embryonic ectoderm subdivision into epidermis and neuroectoderm, it would be important to isolate differentially expressed genes in presumptive neuroectoderm and epidermis at the gastrula stage, the period of the divergence of the two adjacent ectodermal compartments. Meanwhile, the most direct approach for such a task, i.e. subtractive enrichment of cDNA from neuroectodermal and epidermal explants with differentially expressed gene sequences, was difficult to realize because of the high number of explants needed for this technique. In the present paper we report a novel effective and quite simple method of cDNA subtractive enrichment, based on amplification of cDNA in vitro by polymerase chain reaction (PCR) and allowing to use a very small amount of initial cDNA samples. With this method we have cloned cDNA of a novel gene of Xenopus laevis, which was named XEP-1 for its specific expression in the presumptive epidermis starting from the midgastrula stage.

    ID:562
  44. Kazanskaya O.V., Severtzova E.A., Barth K.A., Ermakova G.V., Lukyanov S.A., Benyumov A.O., Pannese M., Boncinelli E., Wilson S.W., Zaraisky A.G. (1997). Anf: a novel class of vertebrate homeobox genes expressed at the anterior end of the main embryonic axis. Gene 200 (1-2), 25–34 [+]

    Five novel genes homologous to the homeobox-containing genes Xanf-1 and Xanf-2 of Xenopus and Hesx-1/Rpx of mouse have been identified as a result of a PCR survey of cDNA in sturgeon, zebrafish, newt, chicken and human. Comparative analysis of the homeodomain primary structure of these genes revealed that they belong to a novel class of homeobox genes, which we name Anf. All genes of this class investigated so far have similar patterns of expression during early embryogenesis, characterized by maximal transcript levels being present at the anterior extremity of the main embryonic body axis. The data obtained also suggest that, despite considerable high structural divergence between their homeodomains, all known Anf genes may be orthologues, and thus represent one of the most quickly evolving classes of vertebrate homeobox genes.

    ID:69
  45. Ecochard V., Cayrol C., Foulquier F., Zaraisky A., Duprat A.M. (1995). A novel TGF-beta-like gene, fugacin, specifically expressed in the Spemann organizer of Xenopus. Dev. Biol. 172 (2), 699–703 [+]

    Using a differential screening strategy, we have cloned a novel Xenopus gene, fugacin, related to the transforming growth factor beta superfamily. Transcripts were detected primarily in the dorsal marginal zone of late blastula. Thereafter, they became highly localized to the blastopore lip of early gastrula and were not observed at later stages. This gene, which is most homologous to the mouse gene nodal, displays a new pattern of cysteine residues. These findings highlight the potential role of these growth factors during early vertebrate development.

    ID:563
  46. Zaraisky A.G., Ecochard V., Kazanskaya O.V., Lukyanov S.A., Fesenko I.V., Duprat A.M. (1995). The homeobox-containing gene XANF-1 may control development of the Spemann organizer. Development 121 (11), 3839–47 [+]

    At the beginning of gastrulation the homeobox-containing gene, XANF-1, is expressed at a low level throughout the animal hemisphere of Xenopus laevis embryos, with a local maximum of expression in the region of the dorsal blastopore lip. By the end of gastrulation expression ceases everywhere except in the most anterior part of the neurectoderm. We have investigated the functions of this gene by microinjecting XANF-1 mRNA in the blastomeres of the 32-cell stage embryo and have observed the following effects. First, microinjections of the mRNA in the animal blastomeres and the blastomeres of the marginal zone elicited massive migration of cells to the interior of the embryo at the early gastrula stage. Second, overexpression of XANF-1 in the ventral marginal zone (VMZ) resulted in the appearance of an additional centre of gastrulation movements and the formation of a secondary axis. In addition we showed that synthetic XANF-1 mRNA was able to cause dorsal-type differentiation in VMZ explants extirpated from the microinjected embryos at the beginning of gastrulation. These results suggest that XANF-1 may control the main functions of cells of the Spemann organizer.

    ID:68
  47. Lukyanov K.A., Launer G.A., Tarabykin V.S., Zaraisky A.G., Lukyanov S.A. (1995). Inverted terminal repeats permit the average length of amplified DNA fragments to be regulated during preparation of cDNA libraries by polymerase chain reaction. Anal. Biochem. 229 (2), 198–202 [+]

    A simple polymerase chain reaction (PCR)-based technique for construction of cDNA libraries starting with very small amounts of cells or tissues is described. The technique is based on the insertion of inverted terminal repeats into amplified cDNAs which permit short molecules to generate "pan"-type structures at each cycle of PCR amplification and thus to escape annealing with primers. This allows one to avoid amplification of primer dimers and makes it possible to perform oligonucleotide tailing of the first cDNA strands followed by PCR amplification in the same tube. Moreover, the average size of amplified cDNAs can be controlled by varying the primer concentration.

    ID:564
  48. Zaraisky A.G., Lukyanov S.A., Vasiliev O.L., Smirnov Y.V., Belyavsky A.V., Kazanskaya O.V. (1992). A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo. Dev. Biol. 152 (2), 373–82 [+]

    To obtain gene sequences controlling the early steps of amphibian neurogenesis, we have performed differential screening of a subtractive cDNA library prepared by a novel PCR-based method from a single presumptive neural plate of a Xenopus laevis late-gastrula embryo. As a result we have isolated a fragment of a novel homeobox gene (named XANF-1, for Xenopus anterior neural folds). This gene is expressed predominantly in the anterior part of the developing nervous system. Such preferential localization of XANF-1 mRNA is established from its initially homogenous distribution in ectoderm of early gastrula. This change in the expression pattern is conditioned by a differential influence of various mesoderm regions on ectoderm: anterior mesoderm activates XANF-1 expression in the overlying ectoderm, whereas posterior axial and ventral mesoderm areas inhibit it. The data obtained demonstrate for the first time that selection of genes for specific expression in the CNS of the early vertebrate embryo is affected not only by chordamesoderm (a neural inductor) but also by ventral mesoderm.

    ID:67
  49. Belintsev B.N., Beloussov L.V., Zaraisky A.G. (1987). Model of pattern formation in epithelial morphogenesis. J. Theor. Biol. 129 (4), 369–94 [+]

    One of the most universal events in morphogenesis is the formation of domains of morphologically polarized cells in the initially homogeneous epithelial sheets. We investigate the possibility of considering this process as a phenomenon of self-organization which is based upon the following experimentally proven mechanochemical cell properties: (1) a capacity of individual cells for morphological polarization considered as a bistable "all-or-none" transition of a cell from a non-polarized to a polarized state; (2) transmission of this capacity from one cell to another on their contacts; (3) feedback relations between co-operative cell polarization and tangential elastic tensions in a cell sheet: cell polarization increases tangential tensions whereas the latter inhibit further cell polarization. We have constructed a phenomenological model which formally expresses the above properties. Its mathematical description includes but few macroscopic parameters available to experimental investigation and controlled changes. The analysis of the collective dynamic regimes of cell polarization demonstrates that variations of some non-specific parameters leads to spontaneous transition in the morphology of cell layers accompanied by symmetry breaking (Turing's instability). Under these conditions either long-range ordered patterns of cell polarization (including hexagonal cell nets) or non-regular spotted structures can emerge. In the particular case of a sheet having fixed complete dimensions and lacking any external elastic bonds a stable macrostate is created; it corresponds to the sheet's binary subdivision into polarized and non-polarized cell domains of size-invariant proportions. The model conclusions are compared with the morphogenetical processes in sea-urchin development, the morphogenesis of skin derivates and artificially induced budding in hydrozoa.

    ID:565
  50. Savic D., Belintzev B.N., Beloussov L.V., Zaraisky A.G. (1986). Morphogenetic activity prepattern in embryonic epithelia. Prog. Clin. Biol. Res. 217A, 101–4 ID:566