Yu. A. Smirnova

Expression of Regulatory GenesOct-4, Pax-6, Prox-1, and Ptx-2 at the Initial Stages of Differentiation of Embryonic Stem Cells 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-4involved 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.

Expression of Transforming Growth Factor-β2 in Vitreous Body and Adjacent Tissues during Prenatal Development of Human Eye

Expression of transforming growth factor-β2 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-β2 and the development of intraocular hyaloid vascular network, its regression, formation of the vitreous body, and development of definite retinal vessels.

Analysis of the Expression Pattern of Regulatory Genes Pax6, Prox1, and Six3 during Regeneration of Eye Structures in the Newt

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 Biological Approaches

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 α-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.

Identification of the OCT4-pg1 retrogene and NANOG gene expression in the human fetal eye

This study is part of the project aimed at identification and structural-functional analysis of the regulatory genes specific for multipotent embryonic stem cells (ESCs). For the first time, primers constructed on the basis of OCT4 and NANOG mRNAs were used for PCR analysis of cDNA derived from the eyes of a 9.5-week human fetus. PCR-amplified DNA fragments were sequenced, and sequence alignment confirmed their 100% homology with the OCT4-pg1 retrogene and NANOG gene. The expression of these genes was reliably detected in the cornea, lens, retina, and eye coats of a 10.5-week fetus. Localization of the NANOG and OCT4-pg1 gene products in the cell nuclei indicates that these proteins probably belong to the class of transcription factors. The role of the OCT4-pg1 retrogene and NANOG gene in self-renewal and differentiation of multipotent cells in the developing eye is discussed.

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.

Constructive Synergism of Regulatory Genes Expressed in the Course of Eye and Muscle Development and Regeneration

The expression patterns of regulatory genes involved in the formation of the eye in Drosophilaand 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/Eyaand dac/Dacwere also revealed. Transcription factors produced by expressing gene cascades—ey/eya/dac/soin invertebrates and Pax/Eya/Dac/Sixin vertebrates—form 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.

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.

Community of aquatic insects in forest-steppe lakes of Baraba (South of West Siberia)

This paper gives unique data on the ecology of aquatic insects inhabiting lakes in the Baraba forest-steppe region of West Siberia, where the lakeside zone is represented by thick reeds. It is shown that reeds, along with other lake biotopes, are an optimal habitat for many hydrobionts and especially for larvae of the orders Odonata and Diptera.

Temporal and spatial distribution of PAX6 gene expression in the developing human eye

264 The study of the molecular-genetic mechanisms of regulation of organogenesis and ontogenesis is one of the key directions in modern biology. The eye is a unique model to studying these mechanisms. It is known that eye morphogenesis is under the control of a cascade of evolutionarily conserved homeobox-containing genes which are differentially expressed in the course of ontogenesis. Transcription factors of Pax, Prox, Six, Pitx, and some other families, which are activated at the earliest stages of embryogenesis, play the key role in the regulation of eye development in vertebrates. Mutations at the genes encoding these factors affect the function of respective tissue-specific genes and cause various eye pathologies.