ABSTRACT
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.
Subject(s)
Embryonic Stem Cells/metabolism , Eye Proteins/biosynthesis , Eye/embryology , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/biosynthesis , Octamer Transcription Factor-3/biosynthesis , Pluripotent Stem Cells/metabolism , Cell Differentiation/physiology , Cell Nucleus/genetics , Cell Nucleus/metabolism , Embryonic Stem Cells/cytology , Eye/cytology , Eye Proteins/genetics , Homeodomain Proteins/genetics , Humans , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Pluripotent Stem Cells/cytology , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
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.
Subject(s)
Eye Proteins/biosynthesis , Gene Expression Regulation, Developmental/physiology , Lens, Crystalline/embryology , Retina/embryology , Tubulin/biosynthesis , Humans , Lens, Crystalline/cytology , Retina/cytologyABSTRACT
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.
Subject(s)
Eye Proteins/biosynthesis , Eye/embryology , Homeodomain Proteins/biosynthesis , Transcription Factors/biosynthesis , Humans , Protein Isoforms/biosynthesis , RNA, Messenger/biosynthesis , Homeobox Protein PITX2ABSTRACT
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.
Subject(s)
Genes, Regulator/physiology , Pigment Epithelium of Eye/metabolism , Regeneration/physiology , Retina/metabolism , Animals , Eye Proteins/metabolism , Fibroblast Growth Factor 2/metabolism , Homeodomain Proteins/metabolism , Nerve Tissue Proteins/metabolism , Otx Transcription Factors/metabolism , PAX6 Transcription Factor , Paired Box Transcription Factors/metabolism , Repressor Proteins/metabolism , Salamandridae , Homeobox Protein SIX3ABSTRACT
Comparative analysis of the early transformations of differentiated cells of the pigment epithelium, ciliary fold epithelium, and Muller glia in the eye of lower vertebrates and mammals during retina regeneration and cultivation was performed for the first time. Dedifferentiation and proliferation of cells and formation of progenitor multipotent cells, which are a source of retina regeneration in adult newts, were characterized using cell, molecular, and genetic markers. Neurospheres were formed during cultivation of the differentiated cells, in which progenitor multipotent cells were found that transformed into neurons of retina and brain and into glial cells. Comparative analysis of changes in the pigment epithelium cells during retina regeneration and during cultivation of differentiated cells of the pigment and ciliary epithelia and Muller glia suggests similar cell transformations at the early stages of transdifferentiation.
Subject(s)
Cell Differentiation/genetics , Eye , Gene Expression Regulation, Developmental/physiology , Ocular Physiological Phenomena , Regeneration/genetics , Vertebrates , Animals , Cells, Cultured , Ciliary Body/cytology , Ciliary Body/embryology , Ciliary Body/metabolism , Ciliary Body/physiology , Eye/cytology , Eye/embryology , Eye/metabolism , Pigment Epithelium of Eye/cytology , Pigment Epithelium of Eye/embryology , Pigment Epithelium of Eye/metabolism , Pigment Epithelium of Eye/physiology , Retina/cytology , Retina/embryology , Retina/metabolism , Retina/physiologySubject(s)
Cornea/physiology , Melanocytes/physiology , Pleurodeles/physiology , Animals , Bromodeoxyuridine/metabolism , Cell Movement , Cornea/blood supply , Cornea/cytology , DNA/biosynthesis , Epithelium, Corneal/cytology , Melanocytes/cytology , Pleurodeles/anatomy & histology , Regeneration/physiologyABSTRACT
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.
Subject(s)
Eye Proteins/biosynthesis , Eye/embryology , Homeodomain Proteins/biosynthesis , Paired Box Transcription Factors/biosynthesis , Repressor Proteins/biosynthesis , Transcription Factors/biosynthesis , Cell Differentiation , Cornea/cytology , Cornea/embryology , Cornea/metabolism , Eye/cytology , Eye/metabolism , Humans , Lens, Crystalline/cytology , Lens, Crystalline/embryology , Lens, Crystalline/metabolism , Organ Specificity , PAX6 Transcription Factor , Polymerase Chain Reaction , Retina/cytology , Retina/embryology , Retina/metabolism , Tumor Suppressor Proteins , Homeobox Protein PITX2ABSTRACT
Studies have been considered, which concern identification of regulatory genes in adult newts and their expression during retinal and lens regeneration. B.L. Astaurov repeatedly urged to join efforts of geneticists and embryologists in studies of the mechanisms underlying biological phenomena. This was also true for studies of regeneration. Such studies became possible only after introduction of molecular biology methods. Studies of the mechanisms underlying regeneration have been recently carried out jointly by geneticists and developmental biologists. This review presented at the conference dedicated to the 100th anniversary of B.L. Astaurov deals with these aspects in studies of regeneration.
Subject(s)
Gene Expression Regulation , Lens, Crystalline/physiology , Pigment Epithelium of Eye/physiology , Regeneration/genetics , Salamandridae/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors , Cell Differentiation/genetics , DNA-Binding Proteins/genetics , Genes, Homeobox/genetics , Lens, Crystalline/cytology , Pigment Epithelium of Eye/cytology , Salamandridae/physiology , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Cell Differentiation , Cell Proliferation , Retina/cytology , Retina/embryology , Cells, Cultured , Glial Fibrillary Acidic Protein/genetics , Humans , Immunohistochemistry , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recoverin/genetics , Tubulin/genetics , Vimentin/geneticsABSTRACT
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.
Subject(s)
Gene Expression Regulation/physiology , Homeodomain Proteins/biosynthesis , Pigment Epithelium of Eye/physiology , Regeneration/physiology , Retina/physiology , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Proliferation , Gene Expression Profiling , Gene Expression Regulation/genetics , Homeodomain Proteins/genetics , In Situ Hybridization , Lens, Crystalline/physiology , Pleurodeles , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Regeneration/geneticsSubject(s)
Gene Expression Regulation , Homeodomain Proteins/genetics , Lens, Crystalline/physiology , Pleurodeles/physiology , Regeneration , Retina/physiology , Animals , Genes, Homeobox , In Situ Hybridization , Pleurodeles/genetics , Polymerase Chain Reaction , RNA Probes , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Tumor Suppressor ProteinsABSTRACT
Results obtained from nine experiments performed onboard Russian biosatellites have shown that microgravity promotes tissue regeneration in the newt, Pleurodeles waltl. The effect has been reproduced in all flights and on a clinostat as well for eye tissues (lens and retina), limbs and tail. The effect was demonstrated in 1.5- to 2-fold increase in cell proliferation in the early stages of regeneration in space flight. Animals "flown" intact and operated after flight regenerated faster than control ones and showed long-lasting micro-"g" effect. The most recent experiment flew aboard the Bion-11 biosatellite. This test was performed for study on microgravity effect on neural retina regeneration after optic nerve lesioning in the newt. Obtained results confirmed our previous information about intensification of regenerative processes in detached neural retina in urodela exposed to simulated weightlessness (Grigoryan et al., 1998). In particular, we found the increase and activation of cell populations participating in neural retina restoration and maintenance of retinal structure. Our findings suggest that promoting effect of microgravity upon regeneration could be influenced by several factors, largely influenced by a response of the whole organism to changed gravity vector. We hypothesized the synthesis of the specific range of stress proteins induced by micro-"g" and their regulative role in cell proliferation. Such a hypothesis for the existence of "altered gravity stress proteins" is discussed.
Subject(s)
Pleurodeles/physiology , Regeneration/physiology , Retina/physiology , Space Flight , Weightlessness , Animals , Cell Division , Glial Fibrillary Acidic Protein/metabolism , Models, Animal , Neuroglia/metabolism , Neuroglia/physiology , Optic Nerve/physiology , Optic Nerve/surgery , Retina/cytology , Retina/metabolismABSTRACT
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.
Subject(s)
Gene Expression Regulation , Genes, Homeobox , Regeneration/genetics , Retina/physiology , Age Factors , Animals , DNA, Complementary , Eye Proteins , Homeodomain Proteins/genetics , Nerve Tissue Proteins/genetics , PAX6 Transcription Factor , Paired Box Transcription Factors , Polymerase Chain Reaction/methods , Repressor Proteins , Salamandridae , Tumor Suppressor Proteins , Homeobox Protein SIX3ABSTRACT
The development of the eye is compared in Drosophila and vertebrates. Although the structure of the compound eye of invertebrates and of the cameral eye of vertebrates differs morphologically, there are striking similarities at the molecular level. Three groups of genes control morphogenesis of the eye: the genes responsible for eye rudiment formation, neurogenic genes, and proneural genes. In the eye rudiments of Drosophila and vertebrates, the homologous regulatory homeobox-containing genes ey/Pax6, so/Six3, rx/Rx, and optix/Optx2 are expressed. Transcription factors encoded by these conservative genes are involved in specific interactions with DNA. Another set of homologous genes, eya/Eya and dac/DACH/Dac, is also expressed during this developmental period. These genes encode nuclear transcription factors that are devoid of DNA-binding domains but are involved in the protein-protein interactions that control gene expression. Transcriptional complexes, which are products of homeobox-containing genes and nuclear factors, control morphogenesis of the eye in Drosophila and vertebrates. A similar set of homologous regulatory and nuclear genes controls morphogenesis during formation of ectopic eyes in Drosophila and vertebrates. Molecular biology approaches have allowed eye development to be examined at the level of developmental mechanisms. It has become evident that progress in understanding the mechanisms of eye development is due to studies carried out on Drosophila. Supporters of the idea of the polyphyletic origin of the compound and cameral eyes have noted significant differences in their structure and have failed to find distinct common features in their development. The hypothesis of the monophyletic origin of eyes has been substantiated by the results of molecular investigations.
Subject(s)
Drosophila/growth & development , Drosophila/genetics , Eye/growth & development , Gene Expression Regulation, Developmental , Vertebrates/growth & development , Vertebrates/genetics , Animals , MorphogenesisABSTRACT
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 ganglionic 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.
Subject(s)
Multipotent Stem Cells/physiology , Regeneration , Retina/cytology , Retina/embryology , Vertebrates/embryology , Animals , Cell ProliferationABSTRACT
Spatial localization and time of expression of glial, neurospecific, and extracellular antigens were studied during retina regeneration in adult newts using antibodies against the glial acidic fibrillar protein (GFAP), neurospecific molecules (N-CAM and PSA-N-CAM), and tenascin (Tn). At the early stages of regeneration (5th day after the operation), slightly differentiated cells in the retina growth area, which are not cellular sources of regeneration, were brightly stained by the antibodies. The one-two-layered retina rudiment formed from the pigment epithelium layer a week later was also intensely stained by the antibodies to GFAP. The retina rudiment cells belong to slightly differentiated precursors of the regenerating retina. It was first shown that the antibodies to GFAP were not only markers of the glial cells, but also markers of slightly differentiated precursors of the regenerating retina. Expression of neurospecific antigens was found in depigmented cells of the retina rudiment. It appeared to have been initiated by cell interactions in the regenerate. An expression of tenascin was found in the cells migrating in the eye cavity and contacting with the retina rudiment cells. The role of tenascin in interaction with the retina rudiment is unknown.
Subject(s)
Antigens/metabolism , Extracellular Matrix/metabolism , Neural Cell Adhesion Molecule L1 , Neuroglia/metabolism , Regeneration , Retina/physiology , Animals , Glial Fibrillary Acidic Protein/metabolism , Neural Cell Adhesion Molecules/metabolism , Retina/cytology , Retina/metabolism , Salamandridae , Sialic Acids/metabolism , Tenascin/metabolismABSTRACT
The work brought up initial information on the impacts of space flight (SF) on regeneration of nerve tissues in vertebrata. Summarized are data of analysis of the retinal regeneration following section of the ocular nerve and blood vessels in space-flown adult newts (Pleurodeles waltlii). Two weeks in SF were found not to impede the regeneration of retina as its growth was fully dependent on the same cell sources as in the condition of 1 g. In the newts which had been operated 2 wk prior to launch, recovery of retina in SF proceeded more intensively (phases V-VI) compared with the synchronous controls (phase IV). According to the morphometric analysis, differentiation of regenerates' layers in the space animals was also a more rapid process. The proliferative activity of cells in regenerates estimated with the 3H-timidine radioautography turned to be higher, too: the labeled nuclei index in early non-differentiated regenerates was in 1.2 to 1.5 times higher than in the control. Immunohistochemical array with the help of GFAP antibodies performed at the late phases of regeneration revealed an activating effect of SF on the Muller glia cells. These findings indicate that microgravity can stimulate general retinal regeneration and activate regenerate cells, specifically those involved in morphogenesis.
Subject(s)
Nerve Regeneration/physiology , Optic Nerve/physiology , Pleurodeles/physiology , Retina/physiology , Spacecraft , Animals , Autoradiography , Immunohistochemistry , Optic Nerve/cytology , Optic Nerve/surgery , Retina/cytology , Thymidine/metabolism , Time Factors , TritiumABSTRACT
Intensity of osteoclastic resorption and calcium content were investigated in intact limb bones of the newts flown on board of a biosatellite Cosmos-2229 after amputation of their forelimbs and tail. Using X-ray microanalysis it was shown an increase in calcium content in the bones on 20th day after operation. Histological study revealed an activation of osteoclastic resorption on endosteal surface of long bones. The newts exposed after surgery on a biosatellite had the same level of bone mineralisation as operated ground control ones, but the increase in number of polynuclear osteoclasts was lower.