V. I. Mitashov

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.

Expression of regulatory homeobox genes during retina regeneration in adult newts

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 relationship 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.

Multipotent and Stem Cells in the Developing, Definitive, and Regenerating Vertebrate Eye

This is a review of the experimental studies on the vertebrate retina neurogenesis. Data are provided on the distribution and localization of multipotent and stem cells in the developing, definitive, and regenerating eye. At the early stages of retina development, the neuroepithelial cells divide synchronously, thus leading to the accumulation of a certain number of the retinal rudiment cells. Synchronous divisions precede the asynchronous ones, when the differentiation of the retinal cells is initiated. The neuroepithelial cells are multipotent: the neuroblast is a source of the cells of different types, for example, neurons and glial cells. The proliferating multipotent cells are preserved in the ciliary-terminal zone of the retina of amphibians, fish, and chickens during their entire life. The differentiated pigment epithelium cells also proliferate in this area of the eye. The multipotent cells of the retinal ciliary-terminal zone and cells of the pigment epithelium in the eye periphery provide for the growth of amphibian and fish eyes during the entire life of these animals. In adult mammals, clonable and self-renewable cells were found among the pigmented differentiated cells in the ciliary folds. In a culture, the stem cells form spheroids consisting of depigmented and proliferating cells. Upon transdifferentiation, the cells of spheroids form rods, bipolar cells, and ganglion and glial cells, thus suggesting the possible regenerative potencies of the stem cells in the ciliary body of the mammalian eye. The main event of retinal regeneration in newts is the transdifferentiation of the pigment epithelium cells. The results of comparative analysis suggest that the stem cells of the ciliary body in the mammalian eye and pigment epithelium cells in lower vertebrates exhibit similar potencies and use similar mechanisms during the formation of the cells of the neural series.

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.

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 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.

Quantitative Estimation of HRP-Labeled Sensory and Motor Neurons during Nerve-Dependent and Nerve-Independent Periods of Urodele Limb Regeneration

The relationship between urodele regeneration and the possibility of regeneration in mammals is unclear, but the idea of possible regeneration of neural elements in man is being studied because of its potential clinical importance. One of the great challenges is to gain sufficient knowledge about the basic biology of animal regeneration and to use it for the betterment of the mankind. It is known that the initial stages of urodele limb regeneration depend on the presence of intact nerve fibers connected to their cell bodies. The nerve fibers severed at the level of limb amputation regrow and penetrate the blastema, providing blastema cells with indispensable factors. These factors are produced in the perikarya of neurons and transported via their axons to the blastema. Numerous studies have been performed to elucidate the quantitative relationships between nerve fibers and limb regeneration. However, there are no reports dealing with the individual nerve cells at work. The aim of this investigation was to analyze the quantitative participation and qualitative distinctions of different nerve cells innervating the regenerating parts of the urodele limb and their possible roles in the nerve-dependent and nerve-independent periods of regeneration. The cells under study are housed in the dorsal ganglia (sensory neurons) and in the ventral part of the spinal cord gray matter (motor neurons). The direct involvement of these neurons in different regeneration periods was visualized by means of horseradish peroxidase (HRP) labeling. A total of 34 animals (21 experimental and 13 control) were used to study fluctuations in the numbers of labeled nerve cells. The results are summarized as follows: (a) the first nerve cells incorporating HRP within 5 days after amputation are found in the dorsal ganglia, whereas motor neurons in the gray matter are labeled within 7 days; (b) the number of labeled perikarya increases during the nerve-dependent regeneration period (0–21 days after amputation), with the percentage of implicated sensory neurons exceeding that found in the control series; and (c) during the next, nerve-independent period, the number of participating labeled neurons decreases gradually. Such fluctuations in the number of labeled neurons might represent the metabolic status of these cells in their effort to provide the blastema cells with the factors needed at the appropriate time. The current findings support previous observations that the periods of dependence and independence of urodele limb regeneration on the integrated control of brachial nerves reflect changes in the metabolism of individual sensory and motor neurons.

Transdifferentiation Potential of Ciliary and Pigment Epithelial Cells in Lower Vertebrates and Mammals

Cellular composition of the peripheral region of the eye in amphibians and mammals as well as embryonic fissure in amphibians was studied. Different distributions of proliferating cells in retinal pigment epithelium have been revealed in adult amphibians (newt, axolotl, and Xenopus). Single cells incorporated [3H]thymidine in the newt and Xenopus; 0.4% cells, in the axolotl. An embryonic fissure was observed in the eye of the axolotl. Pigment epithelial cells in the embryonic palpebral region actively proliferated: about 20% cells incorporated [3H]thymidine. Proliferating cells were also localized in the ciliary marginal zone of the retina in all studied amphibians, particularly, in the axolotl. In newborn hamsters, [3H]thymidine-labeled cells have been revealed in the pigment epithelium as well as in the outer pigmented and inner unpigmented layers of the ciliary body. Proliferative activity of the peripheral regions of the eye is due to eye growth in adult amphibians and newborn hamsters. After retinectomy, the retina is regenerated from the cells of the growth ciliary marginal zone in all amphibians, pigment epithelial cells in the newt, and pigment epithelial cells of the embryonic fissure in the axolotl. Heterogeneous composition of the pigment epithelium in the newt and axolotl reflects high transdifferentiation potential of these regions. Structural comparison of the peripheral region of the eye in amphibians and mammals demonstrate that the ciliary body of mammals containing stem cells is homologous to the ciliary marginal zone of amphibians containing multipotent cells.