R. D. Zinovieva

Expression of regulatory genes Px6, 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 normal 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 (βII-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 β II-tubulin (βII-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, βII-tubulin, and Rho genes, provide indirect evidence for the interaction of regulatory and specific genes during retina regeneration.

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.

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.

Intracellular Localization of Transcription Factor PROX1 in the Human Retina in Ontogeny

The spatiotemporal intracellular localization of the transcription factor PROXl 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 PROXl 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 PROXl 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 PROXl 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 Proxl/PROXl in the vertebrate retina.

Molecular-genetic mechanisms of cornea morphogenesis

In this paper, we analyzed our own results and published data on the expression of regulatory genes encoding transcription factors Pax6/PAX6, Pitx2/PITX2, Fox1/FOXC1, Prox1/PROX1, Oct4/OCT4, Nanog/NANOG, and TGFβ2 signaling protein during morphogenesis of the cornea in vertebrates. We considered the results obtained for the cornea of model animals, primarily mice, and human fetal cornea. The main possibility of establishing common mechanisms of eye development in vertebrates in health and disease is comparative studies of eye morphogenesis of humans and animal models.

Study of β-III tubulin expression in human eye tissues during prenatal development

The expression of β-III tubulin, a marker protein of early neuronal cells, was studied by molecular genetic and immunochemical techniques. The study was performed with the eyes of human fetuses on weeks 8.5 to 27–28 of intrauterine development. Expression of β-III tubulin was detected immunochemically in the retina and lens fibers of fetuses at stages of 8.5 to 22–23 weeks. By means of PCR, a strong expression of the β-III tubulin gene was revealed in the retina of 9.5-week fetuses. Its level remained high until week 18, became slightly lower by week 24, and decreased to zero by week 27–28. The expression of this gene was also revealed in the lens of 9.5-week fetuses. Its level at stages of 15 to 24 weeks was very low, and no expression could be detected in 27-to 28-week fetuses. The results of PCR analysis were consistent with immunochemical data.

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.