Избранные публикации

1. 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).

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.