[Intracellular localization of transcription factor PROX1 in the human retina in ontogeny].

The spatiotemporal intracellular localization of the transcription factor PROX1 in the human retina during prenatal development (fetal weeks 9.5 to 31) and in the adult human retina was studied for the first time. The PROX1 protein was identified in the cell nuclei of the neuroblast retinal layers at the stage of active cell proliferation (fetal week 9.5) as well as in the nuclei of differentiating neurons of the inner nuclear retinal layer (horizontal, amacrine, and bipolar cells) from weeks 13 to 31 of prenatal development. The PROX1 protein localization in the adult retina was the same as at the late stage of prenatal development. Our results indicate the involvement of the transcription factor PROX1 in the regulation of proliferation of progenitor cells and differentiation of the inner nuclear layer cells of the human retina. These results confirm the conservative functions of Prox1/PROX1 in the vertebrate retina.

Comparative analysis of the expression of neural stem cell-associated genes during neocortex and retina development in human.

We compared the expression of Sox2, Oct4, Nanog, Pax6, Prox1 genes associated with plasticity of neural stem and progenitor cells during human neocortex and retina development and in cell cultures. At the analyzed stages of neurogenesis, Pax6 gene is expressed in the neocortex and retina at constant levels, the expression is by one order of magnitude higher in the retina. The dynamics of Sox2 and Pax6 expression in the neocortex was similar. The expression of Oct4 and Nanog genes during neurogenesis in the neocortex and human fetal retina reflects the existence of a high-plasticity cell pool. The dynamics of ßIII-tubulin expression indicates that the retina develops more rapidly than the neocortex. Our experiments showed that genetically determined cell potencies typical of native cells are realized in primary cultures without specific stimulation.

[Analysis of TGFbeta2 expression in human eye tissues in prenatal development].

In this work we focus on the temporal and spatial characteristics of TGFbeta2 expression in various human eye tissues during prenatal development from fetal weeks 8 to 22. We used the complex approach: the analysis of TGFbeta2 gene expression by PCR and the localization of TGFbeta2 protein by immunohistochemistry. TGFbeta2 was detected in all eye tissues. Our results suggest that differential expression pattern of TGFbeta2 in the lens and retina is correlated with the cell type and differentiation state. The data obtained give evidence to the TGFbeta2 contribution to forming of eye tissues of various embryonic origins.

Expression of transforming growth factor-ß2in vitreous body and adjacent tissues during prenatal development of human eye.

Expression of transforming growth factor-ß2 was detected by PCR in the vitreous body, lens, retina, and ciliary-iris complex of human eye at early stages of fetal development. Immunochemical assay of the corresponding protein in eye tissues revealed a correlation between the localization of transforming growth factor-ß2 and the development of intraocular hyaloid vascular network, its regression, formation of the vitreous body, and development of definite retinal vessels.

[Identification of the OCT4-pg1 retrogene and NANOG gene expression in the human embryonic eye].

The goal of this study was the search for and structure-function analysis of the regulatory genes specific for pluripotent embryonic stem cells (ESCs). This was the first study in which PCR was used to obtain DNA fragments with primers constructed on the basis of OCT4 and NANOG mRNA. cDNA synthesized on mRNA isolated from human embryonic eye in the 9.5th week of development was used as a template in PCR analysis. PCR fragment DNA was sequenced. A comparative analysis of the nucleotide sequences demonstrated their 100% homology with the OCT4-pg1 retrogene and NANOG gene. Expression of the genes of interest was reliably detected in the cornea, crystalline lens, retina, and eye tunics in the 10.5th week of development. The nuclear localization of the products of the NANOG gene and OCT4-pg1 retrogene indicates that these proteins are classified with transcription factors. The role of the OCT4-pg1 retrogene and NANOG gene in self-renewal and differentiation of pluripotent cells in a developing eye is discussed.

[Study of expression of beta-III tubulin in human eye tissues during prenatal development].

Expression of beta-III tubulin, a marker protein of early neuronal cells, was studied by molecular genetic and immunochemical techniques. The study was performed with human eyes in the 8.5th to 27-28th weeks of prenatal development. Expression of beta-III tubulin was detected immunochemically in the retina and lens fibers in the 8.5 to 22-23 weeks of development. PCR revealed a high level of expression of the gene for beta-III tubulin in the retina of 9.5-week embryos. The level of expression of this gene remained high until the 18th week of prenatal development, slightly decreased to the 24th week, and became negligible in 27- to 28-week embryos. In the 15th to 24th weeks of prenatal development, the level of expression of this gene in the lens was very low and became undetectable in 27- to 28-week embryos. The results of PCR analysis are consistent with immunochemical data.

[Localization of the PITX2 gene expression in human eye cells in the course of prenatal development].

The pattern of the PITX2 gene expression was studied in the cornea, lens, retina, iridocorneal complex (ICC), and eye coats of human fetuses at weeks 9.5-22 of intrauterine development. Using the PCR method, PITX2 expression in all these tissues was revealed already at the earliest stage studied (9.5 weeks), being especially strong in the anterior eye complex (the cornea and lens) and weaker in the retina and sclera. The level of PITX2 expression in all eye tissues slightly decreased by week 15, increased to a high level in the ICC on week 18, and further decreased in all tissues by week 22. Using cDNA derived from the whole eyes of 8-, 9-, 10.5-, and 11-week fetuses, the expression of two PITX2 isoforms specific for eye tissues (A and B) was revealed. By means of in situ hybridization, the PITX2 mRNA was localized in the eye tissues of ectodermal and neuroectodermal origin.

[Molecular genetic aspects of human eye development].

Multicomponent signal pathways and the main cascade of regulatory genes play a key role in eye morphogenesis. There are about 15 conservative transcription factors and several signal proteins involved in the regulation of eye development. The review is focused on the regulatory factors and their interaction that determines the development of the eye at different stages of embryogenesis.

[Expression of regulatory genes Pax6, Otx2, Six3, and FGF2 during newt retina regeneration].

Molecular-genetic mechanisms of regeneration of adult newt (Pleurodeles waltl) retina were studied. For the first time, a comparative analysis of the expression of regulatory genes Pax6, Otx2, and Six3 and Fgf2 genes encoding signal molecules was performed in the native retinal pigment epithelium (RPE) and retina and at successive stages of retina regeneration. Cell differentiation types were determined using genetic markers of cell differentiation in the RPE (RPE65) and the retina (betaII-tubulin and Rho). Activation of the expression of neurospecific genes Pax6 and Six3 and the growth factor gene Fgf2 and suppression of activation of the regulatory gene Otx2 and the RPE65 were observed at the stage of multipotent neuroblast formation in the regenerating retina. The expression of genes Pax6, Six3, and Fgf2 was retained at a later stage of retina regeneration at which the expression of retinal differentiation markers, the genes encoding betaII-tubulin (betaII-tubulin) and rhodopsin (Rho), was also detected. We assume that the above regulatory genes are multifunctional and control not only transdifferentiation of RPE cells (the key stage of retina regeneration) but also differentiation of regenerating retina cells. The results of this study, demonstrating coexpression of Pax6, Six3, Fgf2, betaII-tubulin, and Rho genes, provide indirect evidence for the interaction of regulatory and specific genes during retina regeneration.

[Analysis of expression of regulatory genes Pax6, Prox1, and Pitx2 in differentiating eye cells in human fetus].

Expression of transcription factors PAX6, PROX1, and PITX2 was evaluated in eye tissues after 9.5 and 22.0 weeks of human fetus development using polymerase chain reaction. Pax6, Prox1 and Pitx2 expression has been revealed in the cornea, lens, retina, and eye membranes (total preparation of the pigment epithelium, choroid, and sclera) after 9.5 weeks of prenatal development with the maximum expression of Pax6 gene in all studied tissues. After 22.0 weeks of development, Pax6 expression increased in the retina and lens but decreased in the cornea. Insignificant levels of Pax6 transcription have been detected in the eye membranes. Prox1 expression was apparent in the cornea, lens, retina, and eye membranes after 9.5 weeks. After 22.0 weeks, Prox1 expression increased in the lens and retina, decreased in the cornea, and was undetectable in the eye membranes. High level of Pitx2 expression has been revealed in all studied eye tissues after 9.5 weeks with the lowest transcription level observed in the retina. After 22.0 weeks, Pitx2 expression notably decreased in the lens and cornea and became undetectable in the retina and eye membranes. The differential pattern of Pax6, Prox1 and Pitx2 expression in developing human eye tissues after 9.5 and 22.0 weeks of development agrees with our histological data.

In vivo and in vitro proliferative and differentiation activity of human embryonic retinal cells.

Differentiation of human embryonic retinal cells (20-22 weeks gestation) was studied using morphological, immunohistochemical, and biomolecular approaches. The retina included several regions differing by the degree of cell differentiation. Mitoses were rarely found in the marginal zone. This zone contained low differentiated cells. The central retinal area consisted of typical layers with differentiated cells. Culturing was accompanied by the formation of aggregates and neurospheres, where mitoses and progenitor or differentiated cells expressing markers of photoreceptors, neurons, and glia were found.

[Analysis of the expression pattern of regulatory genes Pax6, Prox1, and Six3 during regeneration of eye structures in the newt]. / Issledovanie patterna ékspressii reguliatornykh genov Pax6, Prox1 i Six3 v khode regeneratsii struktur glaza tritona.

We studied tissue-specific expression of homeobox genes Pax6, Prox1, and Six3 during regeneration of the retina and lens. In the native retina, mRNA of Pax6, Prox1, and Six3 was predominantly localized in ganglion cells and in the inner nuclear layer of the retina. Active Pax6, Prox1, and Six3 expression was detected at early stages of regeneration in all proliferating neuroblasts forming the retinal primordium. Low levels of Pax6, Prox1, and Six3 mRNA were revealed in depigmented cells of the pigment epithelium as compared to the proliferating neuroblasts. At the intermediate stage of retinal regeneration, the distribution of Pax6, Prox1, and Six3 mRNA was diffuse and even all over the primordium. During differentiation of the cellular layers in the course of retinal regeneration, Pax6, Prox1, and Six3 mRNA was predominantly localized in ganglion cells and in the inner part of the inner nuclear layer, which was similar to the native retina. An increased expression was revealed in the peripheral regenerated retina where multipotent cells were localized. The dual role of regulatory genes Pax6, Prox1, and Six3 during regeneration of eye structures has been revealed; these genes controlled cell proliferation and subsequent differentiation of ganglion, amacrine, and horizontal cells. High hybridization signal of all studied genes was revealed in actively proliferating epithelial cells of the native and regenerating lens, while the corneal epithelium demonstrated a lower signal. Pax6 and Prox1 expression was also revealed in single choroid cells of the regenerating eye.

[Pattern of skeletal muscle differentiation in fish: molecular and biological approaches]. / Osobennosti differentsirovki skeletnoi muskulatury u ryb: molekuliarno-biologicheskie podkhody.

The initial stages of myogenesis going in myoblasts include the stages of induction, determination, and differentiation. The induction and determination of cells in the myotomes are controlled by morphogenetic signals from neighboring tissues of the notochord and neural tube manifested as expression of genes of Shh and Wnt families, respectively. In fish (at the example of danio), this signal is passed to somite cells neighboring the notochord; later the cells migrate to the embryo surface and differentiate into slow muscle fibers. Synthesis of the main contractile proteins, primarily the components of myosin molecule--heavy chain (MHC) and individual isoforms of light chains (MLC1, MLC2, and MLC3)--are encoded by different genes during different ontogenetic stages. The peptide maps obtained after alpha-chymotrypsin digestion of MHCs from larvae, fast and slow skeletal muscle of loach are different, which points to differences in their primary structure. In addition, considerable differences were revealed in the structure of MLC isoforms at different ontogenetic stages. The definitive fast muscle contained three light chain types, MLC1, MLC2, and MLC3; slow muscle, MLC1 and MLC3; while the larval muscle fibers included a specific larval MLCL in addition to MLC3.

[Expression of regulatory genes Oct-4, Pax-6, Prox-1, Ptx-2 at the initial stages of differentiation of embryonic stem cells in vitro]. / Ekspressiia reguliatornykh genov Oct-4, Pax-6, Prox-1, Ptx-2 na nachal'nykh stadiiakh differentsirovki émbrional'nykh stvolovykh kletok in vitro.

The expression of regulatory genes of the POU, Pax, Prox, and Ptx gene families was studied at the initial stages of differentiation of murine embryonic stem cells of R1 line. mRNAs were isolated from undifferentiated embryonic stem cells and embryoid bodies formed at the early stages of in vitro differentiation and cDNA sequences were synthesized for comparative PCR analysis of the expression of studied genes. The levels of expression of the gene Oct-4 involved in maintenance of the pluripotent status of embryonic stem cells proved to be practically indistinguishable in undifferentiated cells and embryoid bodies, while the expression of Pax-6 markedly increased in the latter. The levels and patterns of expression of the homeobox transcription factors Prox-1 and Ptx-2 were compared on this cell model for the first time. A probable role of these genes in differentiation of the murine embryonic stem cells is discussed.

[Effect of thermal acclimation on the expression of gene coding for lactate dehydrogenase A4 in loach skeletal muscle]. / Vliianie temperaturnoi akklimatsii na ékspressiiu gena, kodiruiushchego laktatdegidrogenazy-A4 iz skeletnykh myshts v'iuna.

Acclimation of Misgurnus fossilis to 5 and 18 degrees C induced considerable changes in LDH-A gene expression in white skeletal muscle. Qualities of total and messenger RNA isolated from weighted portions of muscle are considerably higher after acclimation to 18 degrees C as compared to 5 degrees C. However, a PCR assay of cDNA synthesized from these mRNA and equalized by optical density demonstrated that the level of LDH-A gene expression was indistinguishable for high and low acclimation temperatures, while expression of other genes (glyceraldehyde-3-phosphate dehydrogenase and alpha-actin) considerably increased at 18 degrees C as compared to 5 degrees C. The specific enzymatic activity of LDH from white skeletal muscle of the fish acclimated to low temperature is by 20% higher than that for high-temperature acclimation. Structural analysis of the PCR products synthesized on cDNA-5 degrees C and cDNA-18 degrees C has revealed no differences. However, there are indirect indications of the differences in the C-thermal region of the LDH-A molecule. Northern hybridization reveals the differences at the RNA level: one (1400 bp) or two (about 1600 and 1400 bp) hybridization signals have been found in mRNA-5 degrees C and mRNA-18 degrees C, respectively. The presence of two fractions in the mRNA-18 degrees C indicates alternative splicing.

[Expression of regulatory homeobox genes during retina regeneration in adult newts]. / Ekspressiia reguliatornykh gomeobokssoderzhashchikh genov v khode regeneratsii setchatki u vzroslykh tritonov.

We studied molecular-genetic mechanisms of retina regeneration in amphibians and, specifically, expression of the homeobox genes Pax6, Prox1, and Six3 in normal development and during retina regeneration in the newt. Based on the structural analysis of genes in closely related amphibian species, primers were constructed that flank certain regions of these genes. PCR fragments of calculated length were obtained. The affiliation of PCR products to the above genes was confirmed by sequencing. A comparative PCR analysis of expression of Pax6, Prox1, and Six3 was carried out in the native and regenerating newt retina, which allowed estimation of the level of expression. cDNA libraries obtained from the native and regenerating retina were used as templates. The libraries were preliminary standardized according to glyceraldehydes-3-phosphate dehydrogenase, an enzyme of general cell metabolism. The genes we studied were expressed in both native and regenerating retina. The level of Pax6 and Prox1 expression increased during regeneration, while that of Six3 decreased. The decrease in the level of Six3 expression could be due to antagonistic interrelations of Prox1 and Six3. The changed level of Prox1 and Six3 expression is a new fact and requires further studies. The interactions between these and other regulatory genes and localization of their expression in the cells of native and regenerating retina will be studied using in situ hybridization and immunohistochemistry.

[Constructive synergism of regulatory genes expressed in the course of the eye and muscle development and regeneration]. / Konstruktivnyi sinergizm ékspressiruiushchikhsia reguliatornykh genov v protsesse razvitiia i regeneratsii glaza i myshts.

The expression patterns of regulatory genes involved in the formation of the eye in Drosophila and vertebrates during early development were analyzed comparatively. The results demonstrated that, although the compound eyes of invertebrates and the camera eyes of vertebrates markedly differ in their structure and development, they exhibit a striking similarity at the molecular level. This similarity manifests itself in the fact that the homologous regulatory genes ey/Pax, eya/Eya, dac/Dac, and so/Six, which control the early stages of eye development, are expressed in both groups. Not only was synergism shown in the expression of early regulatory genes, but direct interactions of ey/Pax- and so/Six-encoded transcription factors with DNA and protein-protein interactions between nuclear transcription factors encoded by eya/Eya and dac/Dac were also revealed. Transcription factors produced by expressing gene cascades--ey/eya/dac/so in invertebrates and Pax/Eya/Dac/Six in vertebrates--from the transcription complexes that control eye morphogenesis. Paradoxically, the development of muscles in vertebrates proved to involve the expression of genes homologous to the same regulatory genes that control eye morphogenesis in invertebrates and vertebrates. In the developing muscles, regulatory genes also produce transcription factors that form transcription complexes with the mechanism of action based on protein-DNA and protein-protein interactions. The processes of regeneration in the eye and skeletal muscles are controlled by the homologues of the same regulatory genes. Thus, the Pax/Eya/Dac/Six regulatory network is a general system involved in regeneration as well as in development.