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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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., (c2002 COSPAR. Published by Elsevier Science Ltd. All rights reserved.)

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.

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.

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.

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.

Data on forelimb and eye lens regeneration in urodeles under spaceflight conditions (SFC) have been obtained in our previous studies. Today, evidence is available that SFC stimulate regeneration in experimental animals rather than inhibit it. The results of control on-ground experiments with simulated microgravity suggest that the stimulatory effect of SFC is due largely to weightlessness. An original experimental model is proposed, which is convenient for comprehensively analyzing neural regeneration under SFC. The initial results described here concern regeneration of neural retina in Pleurodeles waltl newts exposed to microgravity simulated in radial clinostat. After clinorotation for seven days (until postoperation day 16), a positive effect of altered gravity on structural restoration of detached neural retina was confirmed by a number of criteria. Specifically, an increased number of Mullerian glial cells, an increased relative volume of the plexiform layers, reduced cell death, advanced redifferentiation of retinal pigment epithelium, and extended areas of neural retina reattachment were detected in experimental newts. Moreover, cell proliferation in the inner nuclear layer of neural retina increased as compared with control. Thus, low gravity appears to intensify natural cytological and molecular mechanisms of neural retina regeneration in lower vertebrates.

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.

Tail-amputated adult Triturus vulgaris, fettered in cuvettes of a fast-rotating clinostat were exposed to simulated weightlessness (60 rpm; equiv. to 10(-3)-10(-4) g), during a 14-day period. To feed and clean the animals rotation was stopped once a day for approx. 10 min. To test the influence of the fettering stress, a second series of animals was kept separately under normal earth conditions without rotation. A further control series was kept in a dark container without any handicap. While tail regeneration of the rotated animals was markedly accelerated, the fettered-only animals showed a considerably less marked acceleration effect. At the end of the 14-day period, all regenerates were reamputated together with an additional 5 mm of the tail stump. Although this second level of amputation was distant from the first, the regenerative growth rate of the rotated series was accelerated 123% in contrast to both the control and the fettered-only series. Our results demonstrate that the growth acceleration is induced by clinorotation. Fettering stress has no comparable influence. The growth promoting effect is not limited to the regenerating area.

This review is a comparative analysis of retina regeneration in different amphibians. Special attention is given to the newt, which, unlike other vertebrates, retains the capacity for the regeneration of eye structures for all life. The review focuses on the sources of the cells which contribute to retina regeneration, proliferative activity of cells participating in regeneration, the factors which control the process, and the genes expressed during the course of regeneration.

Differential expression of homeobox genes of the Six family, homologues of Drosophila gene sine oculis, was revealed during limb regeneration of ribbed newt Pleurodeles waltlii and common frog Rana temporaria. Expression of these genes correlated to regeneration capacity in the studied animals. The primary structure of the most conservative region in the identified genes has been determined. We assessed the differences in the primary structure of Six family genes obtained from regenerating limbs of the newt and frog (86 and 94.9% similarity at the nucleotide and amino acid levels, respectively). Comparison of the obtained and published sequences of Six family genes has demonstrated that they are most similar to mouse gene Six1 (84.2 and 82.8% nucleotide sequence similarity in the frog and newt, respectively, and 99.2 and 94.1% amino acid sequence similarity in the frog and newt, respectively.

A method of construction of amplified cDNA libraries was developed on the basis of selective inhibition of cDNA amplification and modified for the studied models for analysis of expression of the genes containing LeR-1 and VeR-1 sequences. Time-related changes in expression of these genes were studied during regeneration of the adult lens and during embryogenesis of newts. The pattern of expression of the LeR-1 and VeR-1 genes proved to be not tissue-specific. A fragment adjoining 5'-area of the LeR-1 gene was obtained using a new approach: rapid amplification of cDNA ends by polymerase chain reaction (5'-RACE-PCR). Analysis of the LeR-1 clone primary structure using Gene Bank did not show essential homology with the known nucleotide sequences. Sequence LeR-1 is characterized by evolutionary conservation of genomic DNA in Pleurodeles waltlii, Xenopus laevis, and Rana temporaria.

The intensity of osteoclastic resorption and calcium contact were investigated in intact limb bones of the newts flown onboard of biosatellite Cosmos-2229 after amputation of their forelimbs and tail. X-ray microanalysis has shown an increase of calcium content in the bones on the 20th day after operation. Histological study revealed activated osteoclastic resorption on the inner surface of long bones. The newts exposed to weightlessness after the operation had the same level of bone mineralization as the operated ground control ones, but the number of polynuclear osteoclasts increased to a lesser extent.

The most important data obtained in studies of the influence of space flight factors on the limb and lens regeneration in the newt Pleurodeles waltlii have been summarized. Regeneration of these organs under the conditions of space flight and after its termination proceeded just as on the ground. However after the 7th, 13th and 16th days of space flight, a trend was shown toward acceleration of the limb and lens regenerates development according to the regeneration stages and synchronization of the achieved stages, which was accompanied in some cases by an increased proliferative activity of the regenerate cells. It was shown that in two groups of animals operated before the 16-day space flight (I) and just after its termination (II) accelerated development of the limb and lens regenerates was observed. In group II, at the large bud stage, the index of 3H-thymidine labeled blastema cells was more than twice that in the control. The size of regenerates in groups I and II exceeded reliably those in the control. These results suggest a prolonged effect of space flight factors on the limb and lens regeneration. It was also shown that the capacity of the muscle minced 14 days before the flight for regeneration was not suppressed, although somewhat decreased. An electron microscopic study showed degenerative-atrophic changes in the intact hind limb muscle after the 16-day space flight. We believe that the phenomenon of synchronization and accelerated regeneration of the limb and lens observed in the most experiments are due to the effect of microgravity on the animals.

This review is a comparative analysis of retina regeneration in amphibians, fish, birds, and mammals. Special attention is given to the newt, which, unlike other vertebrates, retains the capacity for the regeneration of eye structures during all life. The review focuses on the sources of the cells which contribute to retina regeneration, the factors which control the process, and the genes expressed during the course of regeneration.

Over the past century extensive experimental materials have been accumulated concerning cell sources of lens and retina regeneration, successive transformations of the cells, regulatory factors, and gene expression during restitution of these eye structures. The use of nuclear and cytoplasmic markers provided convincing evidence that the removed lens is restituted from the dorsal iris cells in vivo or from embryonic cells of the pigment epithelium and retina in vitro. The removed or destroyed retina is restituted as a result of transdifferentiation of the pigment epithelium cells in amphibians, fish, birds, and mammals during embryogenesis, in larvae of some anuran amphibians, and in adult newts. Cell precursors of rods are a cell source of retina regeneration in adult fish. A subpopulation of randomly distributed cells, which are a cell source of rod formation during the normal development of the eye was found in the external nuclear layer with the use of electron microscopy and nuclear and cytoplasmic markers. These cells are not only a source of regeneration of rods, but also of cones and cells of the internal nuclear layer after destruction of the corresponding retina layers. There is a peripheral growth area in the retina of vertebrates, where multi- and unipolar cells are localized, which provide for the retina growth during ontogenesis. A paradox of retina regeneration consists in that these little differentiated cells are not a source of complete restitution of the removed or destroyed retina. They make only a small contribution to its regeneration corresponding to the growth potential of cells of this eye region, while restitution of the retina proceeds only at the expense of cells of another type of differentiation. A factor controlling the differentiated state of the cell was found in the dorsal iris during studies of lens regeneration. Removal of this factor in the early stages of cell transformations leads to the initiation of lens regeneration. The factor is not specific and was identified in many cells of vertebrates, including the pigment epithelium and limb tissues, which, as is known, may be fully restituted. Studies of gene expression during lens and retina regeneration are now at the initial stage. The greatest advances were achieved on the model of transdifferentiation of the pigment epithelium cells of chick embryos into lentoids. Expression of genes MMP115 and pP344 was established in the pigment epithelium cells, which characterize the pigmented phenotype of the initial cells. Expression of the alpha-, beta-, and delta-crystallin genes was found in the lentoids, which characterize the phenotype of regenerating structures. The gene activity appears to be switched at an intermediate stage during cell dedifferentiation. Expression of the gamma-crystallin genes during lens regeneration in adult newts is initiated after completion of dedifferentiation and cell proliferation in the dorsal iris. The genes specifically expressed in the dorsal and ventral iris and in the retina rudiment have been identified by the method of gene subtraction. Expression of homeobox-containing genes from the family of PAX genes was found during lens regeneration in adult newts and retina regeneration in adult fish. The role of growth factors (FGF) as morphogenetic factors was proved, which are involved in a yet unknown way of altering the differentiation pathway of the initial cells during formation of the neuroepithelium rudiment in chick embryos, adult newts, and fish.

Two species of newts (Urodela) and two types of clinostats for fast clinorotation (60 rpm) were used to investigate the influence of simulated weightlessness on regeneration and to compare results obtained with data from spaceflight experiments. Seven or fourteen days of weightlessness in Russian biosatellites caused acceleration of lens and limb regeneration by an increase in cell proliferation, differentiation, and rate of morphogenesis in comparison with ground controls. After a comparable time of clinorotation the results obtained with Triturus vulgaris using a horizontal clinostat were similar to those found in spaceflight. In contrast, in Pleurodeles waltl using both horizontal and radial clinostats the results were contradictory compared to Triturus. We speculate that different levels of gravity or/and species specific thresholds for gravitational sensitivity could be responsible for these contradictory results.

In this paper most important data obtained in studies on the effect of space flight conditions on regeneration in the adult newt are summarized. We demonstrate a phenomenon of synchronization of limb and lens regeneration and increase in its rate during and after space flight. We also describe a peculiarities of cell proliferation in lens, limb and tail regenerates and of the process of minced muscle regeneration.

Dynamic features of neural retina regeneration in the adult newt Pleurodeles waltl were analyzed using immunohistochemical studies. Antibody to Glial Fibrillary Acidic Protein (GFAP) was used as a marker of the retinal glial supportive system in order to obtain an overview of the retinal reorganization pattern. Unexpectedly, retinal progenitor cells displayed GFAP staining, as did later Müller glial processes and astrocytes supporting ganglional axons. To study changes of plasticity during retinal restoration, the expression patterns of highly- (PSA) and weakly-sialylated N-CAM were examined by double staining. In the retina of adult newts, a sustained expression of total-N-CAM and PSA-N-CAM was detected. However, while an intense distribution of N-CAM was observed throughout the retina, PSA labeling was especially seen in the outer retinal layers. During retinal regeneration, similar widespread staining patterns were observed with the two antibodies, but labeling appeared higher with anti-total-N-CAM antibody than with anti-PSA-N-CAM antibody. On the other hand, tenascin (Tn) expression was analyzed for the first time during retinal regeneration. At the early stages, brightly stained matrix fibers of abundant Tn accumulating in the eye cavity were seen close to the retinal rudiment cells, which suggested that Tn was secreted from these cells. Tn expression was seen nearly throughout the retinal regenerate during neurite migration and then became restricted to the plexiform layers. In the light of the functions attributed to N-CAM and Tn in histogenetic events, the putative roles played by these morphoregulatory molecules in adult newt retinal regeneration were discussed.

During lens regeneration in Pleurodeles waltl, the dorsal iris zone is the cell source of the lens regeneration, while the ventral iris zone can serve as the cells' source of lens regeneration only under certain experimental conditions. The method of subtractive hybridization was used for the identification of genes responsible for the different proliferative potential of these zones. Differential screening of the enriched cDNA libraries, which were obtained as a result of subtractive hybridization of the cDNA samples of the ventral and dorsal iris zones 14 days after lens removal, revealed four clones specific to the dorsal iris and six clones specific to the ventral iris. Two of these, LeR-1 and VeR-1, were structurally characterized. Comparison of their primary structure with data from the Gene Bank showed no essential homology with the known sequences. Time-related changes in LeR-1 and VeR-1 expression were shown during lens regeneration. LeR-1 and VeR-1 expression was activated at the early stages of lens regeneration. The peaks of LeR-1 and VeR-1 expression were observed on the 14th day of lens regeneration in the dorsal and ventral iris zones, respectively. Furthermore, LeR-1 is activated during retina regeneration. The results of Southern hybridization suggest the presence of sequences complementary to LeR-1 in the genomes of Pleurodeles waltl and Rana temporaria. We propose that the activation of LeR-1 expression is related to the triggering of lens regeneration, while the activation of VeR-1 expression accompanies the inhibition of proliferative activity in the ventral iris zone.

This paper constitutes a review of the methodical approaches allowing analysis of the mechanisms underlying development and differentiation. Progress in investigation of the mechanisms underlying embryogenesis is related to the discovery of genic families in the Drosophila genome, which are responsible for different periods of embryogenesis. The true revolution in studies of developmental mechanisms began with the application of molecular-genetic methods for analysis of Drosophila mutant lines. The clarification and analysis of the genes controlling regeneration is one of the most effective paths toward an understanding of the mechanisms underlying regeneration. No mutations affecting regeneration are, and the development of alternative (i.e., not based on mutation analysis) methods of discovery of the genes controlling regeneration is necessary for investigation of the genetic mechanisms of regeneration. The advantages and drawbacks of the two main approaches for discovery of the genes controlling regeneration are considered. The first approach is based on the production of a bank of sequences expressed in the regenerating structures and subsequent screening of the bank by the known probes. This approach also involves analysis of the structure, function, and expression pattern of the obtained homologs. The second approach is based on subtractive hybridization, which allows identification of the genes specifically expressed in the regenerating structures. This approach was made it possible to identify, for the first time, new genes specifically expressed during lens and retina regeneration in amphibians.

It has been already established that a tendency towards synchronization and acceleration of the forelimb and lens regeneration is observed in Pleurodeles waltlii under the effect of space flight factors. Here we present the results obtained after 16-day space flight of two groups of newts. In animals of group I forelimbs were amputated and lenses were removed 14 and 7 days before the space flight, respectively. Intact animals of group II were operated on the day of the sputnik landing. Regenerates of the flight and corresponding control animals were fixed at the same time after the operation. For evaluation of the regeneration rate morphological criteria were used: morphological stages of regeneration were compared in the experiment and the control. For quantitative assay of the regeneration rate we determined the index of nuclei labelled with 3H-thymidine in the blastema and lens rudiment cells and used morphometry of the lens regenerates. Acceleration of forelimb and lens regeneration was observed in both groups of animals. In group II more than two-fold increase of the index of labelled nuclei was found in the blastema cells at the comparable stages of development. The size of lens regenerates in flight groups I and II exceeded reliably those in the control animals. The results obtained suggest a prolonged effect of the space flight factors on forelimb and lens regeneration. Under the conditions of space flight the lens regenerates reached more advanced stages of regeneration, as compared with the control animals operated after the space flight. These results also suggest acceleration of regeneration in lower vertebrates.

Comparative analysis of experimental studies on regeneration of bone and muscle tissue under the influence of space flight factors (SFF) during restoration of experimental fracture in rats and epimorphic limb regeneration in adult newt is presented SFF-induced retardation of regeneration in mammals and its acceleration in tailed amphibians results from changed calcium metabolism in conditions of microgravity. Bidirectional effect of SFF on bone regeneration is due to specific control of calcium homeostasis in these taxa. Upon termination of the space flight (SF) the Wistar SPF rats proved to have increased functional activity of parathyrocytes and blocked calcitonin secretion to the blood by the C-cells of thyroid gland. Hence, inhibition of posttraumatic bone regeneration in rats is due to decreased formation rate and enhanced resorption of bony tissue. Sharp decrease of the calcitonincytes population following the initial hypertrophy of ultimobranchial gland (UBG) is observed in adult newt Pleurodeles waltlii Michah during SF. Progressing demineralization of skeletal elements including the amputated limb stump accelerates the first stages of its regeneration in conditions of SF.

The lens removal from the newt eye stimulates regeneration of the latter from the dorsal iris cells. We have undertaken an attempt to stimulate lens regeneration from teh ventral iris pigmented cells by growth factors: basic and acidic forms of the fibroblast growth factor and the epidermal growth factor. In the presence of the growth factors, the mitotic activity of the ventral iris cells increased 1.5-to 3-fold on the 30th (18) and 70th (58) days after the operation (implantation). Depigmentation of the pupal margin and separation of the ventral iris sheets was observed on the 70th day in teh same animals, what was considered as stage III of lens regeneration. No such stimulation of proliferation was observed in the dorsal iris, but by the 70th day additional lenses have been found to form from the dorsal iris cells.

Effect of retinoic acid (RA) on morphogenesis and proliferation of regenerating extremity and lens cells was studied using 3H-thymidine autoradiography and morphometry. The 3H-thymidine incorporation into the inner layer of dorsal and ventral iris was 1.5-3 times reduced by the 8th day following the RA administration. The applied RA dose (0.25 mg per animal) exerted no significant effect on the morphogenesis of regenerating lens with the exception of the case of forming an additional lens from dorsal iris. The RA effect on the regeneration of extremity corresponds to available data of literature and manifests itself in the decelerated regeneration and the appearance of additional structures along the proximodistal axis.

Tests of the morphogenetic potency of the urodele limb regeneration blastema by a variety of transplantation experiments indicate that blastema is a self-organizing system. The cells of blastema inherit a memory of their position on the three cardinal axes of the limb. This memory is called positional memory, and it is fundamental to recognizing loss of structure, triggering cellular interactions to replace missing structures, and specifying the level from which new structures can begin. Retinoic acid (RA) modifies the positional memory in all three cardinal axes of the limb. The vitamin proximalizes positional memory in the proximodistal axis, posteriorizes it in the anteroposterior axis, and ventralizes it in the dorsoventral axis. In vivo and in vitro assays of interactions between cell derived from different proximodistal limb levels, before and after treatment with RA, suggest that the cellular basis of positional memory resides in recognition/affinity property of the cell surface. RA probably modifies positional memory by modulating transcription, via a nuclear retinoid receptor. It is suggested that the genes involved in specifying positional memory, and whose transcription is therefore affected in regenerating limbs, are homeo box genes and genes that encode for molecules involved in determining the composition and architecture of the cell surface and extracellular matrix.

The proliferative activity of the pigment epithelium cells in the axolotl eyes was studied using 3H-thymidine in two types experiments: after the removal of lens, iris and retina and upon the cultivation of the pigment epithelium pieces in the cavity of lens-less eye. Irrespective of the operation type, the level of proliferation of the pigment epithelium cells changed regularly with respect to the time of observation. In the intact eye, the level of proliferation of the pigment epithelium cells was not high: the index of labelled nuclei equaled 0.5%, no mitoses were found. The highest values of the index of labelled nuclei (12.6-32.1%) and of the mitotic index (0.54-1.07%) were registered on the 10-20th days after the operation. After 40 days, the indices of proliferative activity of the pigment epithelium cells approached gradually those for the intact eye. The cultivation of the pigment epithelium cells in the cavity of a lens-less eye for 50 days did not result in their transdifferentiation into retina cells. The layered retina found in 7.7% of cases after the removal of lens, iris and retina could regenerate either from the cells of the retina growth zone localized in the region of embryonic split, or due to transdifferentiation of the pigment epithelium cells.

The spectrum of LDH isozymes was studied at the successive stages of retinal regeneration from the pigment epithelium and lens cells from the iris margin in the adults Pleurodeles waltlii. The combination of two methods, electrophoresis and immunofluorescence, has revealed the slow and rapid LDH isozymes with different intensity of histochemical staining in cells of the tissues under study (pigment epithelium, retina, iris and lens). During the regeneration the spectra of LDH isozymes peculiar to the pigment epithelium and iris and characterized by the predominance of slow forms were substituted by those peculiar to the retina and iris and characterized by the predominance of rapid forms. The rearrangement is realized in the proliferative phase during the transformation of one cell type into another.

The late labelling of the actively proliferating cells of iris and pigment epithelium in the regenerating eye was found in the adult smooth newts within 2 to 12 days following the intraperitoneal injection of 3H-thymidine. This late labelling is due to the long-term presence of the labelled precursors of DNA synthesis in the organism and to their reutilization. The index of labelled nuclei upon reutilization in the pigment epithelium is twice and in the iris 3 to 4 times higher than in the experiments with pulse labelling what cannot be accounted for by the label dilution only in the process of proliferation. The intensity of labelling upon reutilization is 10 to 20 times weaker than upon the pulse 3H-thymidine incorporation. This phenomenon is of general biological value and it is to be taken into consideration when determining the parameters of cell cycles and studying the cell migration. It can also be utilized as a technique for determination of the maximum number of labelled cells and the rate of their entrance into S phase of the cell cycle. Mitoses in the iris cells of the regenerating eye appeared on the 4--6th day following the removal of retina and lens, i.e. long before the formation of retinal rudiment. These data do not confirm the hypothesis by Reyer (1971) and Yamada (1977) on the role of retinal factor in the transition of the iris cells from G2-block to mitosis.

The proliferation of pigmentary-epithelium cells after they have given rise to cells of the regenerating retina is associated with the growth of the pigmentary epithelium and not with additions to the regenerating retina. The proliferative activity of pigmentary-epithelium cells during this period takes place against a background of simultaneous synthesis of specific proteins.

The proliferative activity of the pigment epithelium cells transplanted in the lens-less eyes was studied in the adult crested newt. The cells of transplanted pigment epithelium incorporated 3H-thymidine injected intraperitoneally. Within 10 days after explantation, the index of labelled nuclei equaled 27.8-34.0% and within 20 days the number of labelled cells doubled. By that time the proliferating transplant cells were depigmented and formed 2-3 rows of cells of retinal rudiment. In response to the removal of lens from the of recipients eyes their regeneration proceeded. Irrespective of participation (dorsal iris) or nonparticipation in lens regeneration (ventral iris), the index of labelled nuclei in these regions of iris had similar values. The eyes of recipients were also characterized by a local proliferation of pigment epithelium cells in the zones of retinal detachment. In these zones the index of labelled nuclei in the pigment epithelium equaled 11.0-31.3%.

It was shown by means of 3H-thymidine autoradiography that the cells of almost all layers of intact eyes had proliferative potencies both in albino and normal strains of Xenopus laevis, as it is the case in the Urodela. The removal of retina and lens in X. laevis, unlike in the Urodela, did not result in the intensification of proliferation of the pigment epithelium cells. The cells of ciliary region represent the source of retina regeneration of X. laevis. The regenerating retina is incomplete by its structure and volume.