Лаборатория молекулярных основ эмбриогенеза

Научно-исследовательские подразделения

Руководитель: Зарайский Андрей Георгиевич, д. б. н., профессор
azaraisky@yahoo.com+7(495)336-36-22

Развитие мозга, регуляция экспрессии генов, трансгенные организмы, гомеобоксные гены, регуляторы TGF-beta сигнализация, малые ГТФазы, Zyxin

Лаборатория изучает молекулярно-генетические механизмы раннего развития и эволюции нервной системы, а также регенерации больших придатков тела на моделях низших позвоночных.

Один из проектов посвящен  исследованию у позвоночных животных моногенного семейства гомеобоксных генов Anf (Dev Biol, 1992, 152, 373-382; Development, 1995, 121, 3839-3847; Gene, 1997, 200, 25-34). Впервые установлено, что ген присутствует в геномах исключительно у позвоночных животных, включая человека, и отсутствует у беспозвоночных. Он контролирует развитие уникального отдела мозга позвоночных – конечного мозга – и в клетках зачатка конечного мозга играет роль репрессора транскрипции, подавляя экспрессию генов, которые усиливают формирование задних отделов мозга (Development, 1999, 126, 4513- 4523; Gene, 2002, 285, 279-286; Development, 2004, 131, 2329-2338; Mech Dev, 2004, 121, 1425-1441; Dev Biol, 2007; 307, 483-497). Известные мутации в этом гене у мыши и человека имеют рецессивный характер, но в гомозиготном состоянии приводят к серьезным аномалиям развития мозга, в диапазоне от недоразвития гипофиза и дисплазии оптического нерва и перегородки больших полушарий до слияния желудочков конечного мозга и отсутствия его структур. Поэтому исследователи предположили, что возникновение гена Anf у предков позвоночных могло послужить одной из ключевых предпосылок к возникновению конечного мозга в эволюции (Dev Biol, 2007; 307, 483-497).  Поскольку Anf пока не был обнаружен у представителей самой древней группы современных позвоночных – у бесчелюстных (миноги и миксины), –  одной из задач, решаемых в настоящее время в Лаборатории, является поиск этого гена у миноги.

А. Гомеобоксный ген Anf и конечный мозг присутствуют только у позвоночных животных, в том числе у человека, и отсутствуют у всех остальных животных. Б. Искусственное изменение уровня экспрессии гомеобоксного гена Anf вызывает изменения размеров конечного мозга у головастика шпорцевой лягушки.

Лаборатория также изучает роль генов, пропавших в ходе эволюции позвоночных, – в регуляции регенерации. Исследователи установили, что некоторые из найденных генов-мишеней гомеобоксного гена Anf  (гены секретируемого фактора Ag1 и гены малых ГТФаз Ras-dva) присутствуют только в геномах низших позвоночных, рыб и амфибий, но отсутствуют у высших, у рептилий, птиц и млекопитающих (Gene Expr Patterns, 2003, 325-30; Development, 2006, 133, 485-494; Nucleic Acids Res, 2006, 34, 2247-2257; Gene Expr Patterns, 2011, 11, 156–161). При этом у низших позвоночных эти гены, кодирующие разные типы белков, регулируют два важнейших процесса – развитие мозга и регенерацию конечностей (Sci Rep, 2013, 3, 1279; Biol Open, 2014, 3, 192-203; Sci Rep, 2015, 5:8123). Это дало основание выдвинуть гипотезу о том, что исчезновение в эволюции найденных генов могло быть своеобразной платой, сделанной предками высших позвоночных за возможность прогрессивного развития мозга. В рамках этого проекта сотрудники с коллегами разработали алгоритм и программу, которые позволяют на основе сравнения геномов идентифицировать гены, возникшие или исчезнувшие на заданном этапе эволюции. Функции некоторых из генов, найденных с помощью этой программы, сейчас изучаются в Лаборатории.

Инъекции анти-смыслового морфолинового олигонуклеотида блокируют трансляцию мРНК гена Ag1, что приводит к ингибированию регенерации

Изучение генной сети, связанной с функционированием гена Anf в клетках раннего зачатка переднего мозга, привело к открытию ряда неизвестных ранее генов, играющих важную роль в эмбриогенезе. Так, были открыты и изучены регуляторы раннего развития головного мозга – секретируемые белки Noggin2 и Noggin4 (Gene Expr Patterns, 2006 6:180-6; Development, 2011, 138, 5345-5356;  Int J Dev Biol, 2012;56: 403-6; Sci Rep, 1996, 14, 6: 23049). Эти белки в отличие от их широко известного гомолога, ингибитора BMP – белка Noggin1, имеют способность связывать и ингибировать функцию  регулятора задних отделов центральной нервной системы – секретируемого белка Wnt8.

Секретируемый белок Noggin2 ингибирует три сигнальных каскада - BMP, Activin/Nodal и Wnt, что является достаточным условием для индукции второй головы при искусственно вызванной экспрессии Noggin2 на брюшной стороне эмбриона шпорцевой лягушки.

Кроме того, исследователи разработали ряд методов для изучения диффузии и взаимодействий секретируемых белков-морфогенов в межклеточном пространстве эмбриональных тканей (Sci Rep, 2016, 6:23049;  Biochem Biophys Res Commun, 2015; 468:331-6). Впервые измерили коэффициенты диффузии белков семейств Noggin и Wnt in vivo и показали роль адсорбции на внеклеточном матриксе в их диффузии. Впервые с помощью математического моделирования показали, что такая адсорбция может играть роль важного фактора, необходимого для создания пространственно-упорядоченных структур в эмбриогенезе.

Секретируемый белок Noggin4, меченный EGFP (EGFP-Noggin4), диффундирует из пересаженного кусочка эктодермы (граница выделена пунктиром), клетки которого экспрессируют EGFP-Noggin4, по межклетникам эмбриона-реципиента на большие расстояния. Стрелки показывают направление диффузии.

Также в Лаборатории впервые была изучена роль в раннем эмбриональном развитии цитоскелетного белка Zyxin, который способен связываться с гомеодоменным белком Anf и с эффектором Shh сигнального каскада – транскрипционным фактором Gli1, ингибируя его активность (Dev Dyn, 2008, 237, 736-749; Dev Biol, 2013, 380, 37-48). Полученные данные важны, так как они впервые демонстрируют связь между одним из цитоскелетных белков-регуляторов морфогенетических движений клеток и важным сигнальным каскадом, управляющим дифференцировкой клеток в нейральном зачатке.

В Лаборатории проводятся работы по тестированию возможности использования генетически кодируемых флуоресцентных репортеров и сенсоров для изучения различных процессов в раннем эмбриогенезе и при регенерации (Nat Biotechnol, 1999, 17, 969-973; Science, 2000, 290:1478-1479; Nat Biotechnol, 2003, 21:191-194; Nat Methods, 2007, 4, 741-746; Nat Methods, 2010, 7, 827-829; Nat Commun, 2012 13; 3 :1204). В частности,  впервые была показана возможность использования флуоресцентных белков из коралловых полипов для прижизненного мониторинга клеток в эмбрионах шпорцевой лягушки (Nat Biotechnol, 1999, 17, 969-973).

Аквариальная комната, где содержатся лягушки и головастики некоторых трансгенных линий, а также взрослые лягушки, экспрессирующие флуоресцентные белки под контролем промоторов различных генов.

Лаборатория сотрудничает с другими лабораториями Института, а также с кафедрой биофизики МГУ, кафедрой эмбриологии МГУ, Центром «Биоинженерия» РАН, НИИ физико-химической биологии им. А.Н. Белозерского МГУ, Институтом проблем передачи информации РАН, НИЦ Курчатовский институт, Массачусетским технологическим институтом (США), Университетом Виргинии (США).

Лаборатория была образована в 2005 году на основе группы с аналогичным названием, выделившейся в 1995 году из Лаборатории структуры и функции генов человека.

  • Изучение молекулярно-генетических механизмов раннего развития и эволюции нервной системы.
  • Изучение процессов регенерации больших придатков тела на моделях низших позвоночных.
  • Проведение уникальных для нашей страны работ по созданию и поддержанию линий трансгенных лягушек Xenopus, экспрессирующих генетически кодируемые флуоресцентные репортеры и сенсоры.
  • Установлено, что некоторые из найденных генов-мишеней гомеобоксного гена Anf  (гены секретируемого фактора Ag1 и гены малых ГТФаз Ras-dva) присутствуют только в геномах низших позвоночных, рыб и амфибий, но отсутствуют у высших, у рептилий, птиц и млекопитающих.
  • Открыты и изучены регуляторы раннего развития головного мозга – секретируемые белки Noggin2 и Noggin4.
  • Разработан ряд методов для изучения диффузии и взаимодействий секретируемых белков-морфогенов в межклеточном пространстве эмбриональных тканей.
  • Впервые была изучена роль в раннем эмбриональном развитии цитоскелетного белка Zyxin, который способен связываться с гомеодоменным белком Anf и с эффектором Shh сигнального каскада – транскрипционным фактором Gli1, ингибируя его активность.
  • Разработана технология двухцветного  репортерного вектора, существенно повышающая эффективность функционального промоторного анализа, благодаря возможности сравнивать экспрессионный потенциал двух делеционных мутантов изучаемого генного промотора в одном и том же трансгенном эмбрионе (Development, 2004, 131, 2329-2338).
  • С помощью генетически кодируемого pH-индикатора SypHer2 на модели головастиков шпорцевой лягушки обнаружен ранее неизвестный эффект – быстрое закисление цитоплазмы клеток вблизи места ампутации хвоста (Biochim Biophys Acta, 2015 1850: 2318-28). Этот эффект является одной из первых реакций организма на ампутацию и может иметь важное регуляторное значение для последующей регенерации.

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. Suntsova M., Gogvadze E.V., Salozhin S., Gaifullin N., Eroshkin F., Dmitriev S.E., Martynova N., Kulikov K., Malakhova G., Tukhbatova G., Bolshakov A.P., Ghilarov D., Garazha A., Aliper A., Cantor C.R., Solokhin Y., Roumiantsev S., Balaban P., Zhavoronkov A., Buzdin A. (2013). Human-specific endogenous retroviral insert serves as an enhancer for the schizophrenia-linked gene PRODH. Proc. Natl. Acad. Sci. U.S.A. , [+]

    Using a systematic, whole-genome analysis of enhancer activity of human-specific endogenous retroviral inserts (hsERVs), we identified an element, hsERVPRODH, that acts as a tissue-specific enhancer for the PRODH gene, which is required for proper CNS functioning. PRODH is one of the candidate genes for susceptibility to schizophrenia and other neurological disorders. It codes for a proline dehydrogenase enzyme, which catalyses the first step of proline catabolism and most likely is involved in neuromediator synthesis in the CNS. We investigated the mechanisms that regulate hsERVPRODH enhancer activity. We showed that the hsERVPRODH enhancer and the internal CpG island of PRODH synergistically activate its promoter. The enhancer activity of hsERVPRODH is regulated by methylation, and in an undermethylated state it can up-regulate PRODH expression in the hippocampus. The mechanism of hsERVPRODH enhancer activity involves the binding of the transcription factor SOX2, whch is preferentially expressed in hippocampus. We propose that the interaction of hsERVPRODH and PRODH may have contributed to human CNS evolution.

    ID:920
  11. 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
  12. 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
  13. 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
  14. 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
  15. 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
  16. 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
  17. 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
  18. 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
  19. 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
  20. 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
  21. 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
  22. 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
  23. 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
  24. 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
  25. 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
  26. 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
  27. 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
  28. 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
  29. 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
  30. 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
  31. 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

Зарайский Андрей Георгиевич

  • Москва, ул. Миклухо-Маклая, 16/10 — На карте
  • ИБХ РАН, корп. 51, комн. 261
  • Тел.: +7(495)336-36-22
  • Эл. почта: azaraisky@yahoo.com

Ген Ag1, исчезнувший у высших позвоночных, регулирует регенерацию у рыбы Danio rerio. (2016-03-30)

На модели регенерации хвостового плавника рыбы Danio rerio впервые установлено, что секретируемый белок Ag1 необходим для регенерации. Показано, что ампутация хвостового плавника индуцирует быструю активацию экспрессии гена этого белка в клетках раневого эпителия. Вместе с тем, ингибирование трансляции мРНК Ag1 вызывает замедление регенерации. Результаты этой работы важны потому, что ген Ag1 имеется только у низших, хорошо регенерирующих позвоночных, включая рыб и амфибий, но отсутствует у высших позвоночных, не способных к эффективной регенерации конечностей. Полученные данные свидетельствуют о том, что снижение потенциальной

возможности регенерации у высших позвоночных, в том числе у человека, может объясняться исчезновением у них некоторых генов, важных для регенерации, в частности, гена Ag1.

Публикации

  1. 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.

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