Yu. V. Markitantova

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.

Study of regeneration in amphibians in age of molecular-genetic approaches and methods

The results of molecular-genetic mechanisms of regeneration in amphibians are reviewed. Based on the examples of traditional and well-studied models of the restoration of the retinas and lenses of eyes, as well as limbs and tails in amphibians, we analyze the current state of regeneration problems and questions linked to cell reprogramming, growth, and morphogenesis. The development of the Kol’tsov school of thought in the age of molecular-genetic approaches and methods are monitored. The contemporary interpretation of organ regeneration in terms of molecular-genetic regulation and a new look at the definition of regeneration as repeated development is proposed. We also emphasize the current problems that exist in that field of developmental biology and are caused by the many difficulties of genome sequencing and the introduction of transgenesis in Urodela, the animal species with the highest regeneration abilities.

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.

Transcriptional factor Pitx2: Localization during triton retina regeneration

For the first time immune-chemical analysis of transcriptional factor Pitx2 localization during triton retina regeneration after its removal and also in tissues of a nonoperated eye of an adult triton has been carried out. Protein Pitx2 has been found in the nucleus of the earliest neuroblasts that form the germ of the retina. At a later stage of retina regeneration, Pitx2 was found in the nucleus of differentiating cells of ganglionic layers that correspond to Pitx2 protein localization in the native retina. Protein Ptix2 has also been found in the nucleus of less differentiated cells of the peripheral region of regenerative and native retina. It was demonstrated that protein Pitx2 is expressed not only in retina but also in other tissues of the posterior sector of the eye (pigment epithelium, choroid) using immune-histochemical and Western blot hybridization. It is supposed that transcriptional factor Pitx2 has been involved in the control of subsequent stages of retina regeneration from pigment epithelium cells.

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.

FGF2 signaling pathway components in tissues of the posterior eye sector in the adult newt Pleurodeles waltl

The FGF2 signaling pathway components in tissues of the posterior wall in the normal and regenerating eye of the adult Pleurodeles waltl newt were detected for the first time. The fgf2 gene expression was found in the retina, retinal pigment epithelium, and choroid using polymerase chain reaction (PCR). A high homology of the mRNA nucleotide sequence of the most conservative fgf2 gene region in the P. waltl with the fgf2 orthologs in other vertebrates was proved. The Fgf2 protein amino acid sequence of the P. waltl newt demonstrates even more homology with this growth factor in other vertebrates. The Fgf2 protein with a molecular weight 35 kDa was found in the studied eye tissues using Western blot hybridization. Localization of the Fgf2 protein and its Fgfr receptors was immunohistochemically studied in the pigment epithelium, choroid, central and growth retina regions of the newt native eye, and in the connective cilium of photoreceptors. Using real-time PCR and immunohistochemistry methods, it was found that the fgf2 gene down-regulation and a decrease in the intensity of the immunochemical reaction of its protein product (Fgf2) occur in the early period after the retina removal (in 4–8 days) (as compared with those in the same department of the unoperated eye).

Identification of the Pitx1 embryogenesis regulatory gene in a regenerating newt retina

421 The newt retina is a unique model system since there is a possibility of its complete regeneration through transdifferentiation of retinal pigment epithe lium cells, involving the peripheral growth zone of the retina [1]. The process of retinal regeneration is per formed under coordinated control of signal molecules and transcription factors. So far, the following regula tory genes also participating in eye formation during development were identified in a regenerating newt retina: Fgf2, Notch 1, Shh, Wnt, Tgfb, Pax6, Prox1, Six3, Otx2, Mitf, Rx, Chx10, and Musashi 1 [2–6]. Due to the poor knowledge of newt regeneration molecular mechanisms and newt genome in general, identification of genes probably involved in the control of cellular processes leading to retina regeneration still remains an open question. Of special interest is the study of the role of genes of the Pitx family in retina regeneration. The gene family encodes multifunc tional transcription factors of bicoid type. Homeobox containing genes of the Pitx family (Pitx1, Pitx2, and Pitx3) take part in eye development control [7–10]. The expression of Pitx1, Pitx2, and Pitx3 was detected at early stages of embryo development in the central part of the neural plate and later in the forming optic cup [8, 9, 11]. Pitx1 is located in migrating mesenchy mal cells that surround the optic anlage and seem to be the source of the corneal endothelium and stroma, iris stroma, ciliary muscles, vessel endothelium, and sclera [7].

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.

Identification of the gene encoding nucleostemin in the eye tissues of Pleurodeles waltl

Nucleotide sequences were identified in the eye tissues (lens, retina, and retinal pigment epithelium) of the adult newt Pleurodeles waltl by the polymerase chain reaction with primers for the Ns gene. Sequencing showed that these nucleotide sequences belong to the Ns gene of the newt P. waltl, which encodes the nucleolar protein nucleostemin. Structural analysis revealed a high homology of Ns nucleotide sequences of P. waltl with those of newts Cynops pyrrhogaster and Notophthalmus viridescens. The expression of the Ns gene of P. waltl, identified in the specialized eye cells of adult newts of the studied species, indicates that these differentiated cells retain some of the molecular characteristics inherent to the undifferentiated cells.