Neural retinal regeneration in the anuran amphibian Xenopus laevis post-metamorphosis: transdifferentiation of retinal pigmented epithelium regenerates the neural retina

In urodele amphibians like the newt, complete retina and lens regeneration occurs throughout their lives. In contrast, anuran amphibians retain this capacity only in the larval stage and quickly lose it during metamorphosis. It is believed that they are unable to regenerate these tissues after metamorphosis. However, contrary to this generally accepted notion, here we report that both the neural retina (NR) and lens regenerate following the surgical removal of these tissues in the anuran amphibian, Xenopus laevis, even in the mature animal. The NR regenerated both from the retinal pigment epithelial (RPE) cells by transdifferentiation and from the stem cells in the ciliary marginal zone (CMZ) by differentiation. In the early stage of NR regeneration (5-10 days post operation), RPE cells appeared to delaminate from the RPE layer and adhere to the remaining retinal vascular membrane. Thereafter, they underwent transdifferentiation to regenerate the NR layer. An in vitro culture study also revealed that RPE cells differentiated into neurons and that this was accelerated by the presence of FGF-2 and IGF-1. The source of the regenerating lens appeared to be remaining lens epithelium, suggesting that this is a kind of repair process rather than regeneration. Thus, we show for the first time that anuran amphibians retain the capacity for retinal regeneration after metamorphosis, similarly to urodeles, but that the mode of regeneration differs between the two orders. Our study provides a new tool for the molecular analysis of regulatory mechanisms involved in retinal and lens regeneration by providing an alternative animal model to the newt, the only other experimental model.     

Concentration dependence of inductive activity in the mixture of lens epithelium proteins

As shown elsewhere, the mixture of proteins secreted by lens epithelium cells in the process of microcultivation can selectively induce eye and forebrain tissues in the early gastrula ectoderm (Zemchikhina et al., 2000, 2003). In the present work, the dependence of inductive activity of this protein mixture on its concentration in culture solution has been studied. The test-system was the early gastrula ectoderm of Xenopus laevis frogs. The results of the experiments revealed no direct dependence of the spectrum of induced tissues on the concentration of the protein mixture. At a concentration of 0.5 mg/ml, brain appeared being accompanied by retina, pigmented epithelium, and lentoids, while at 0.031 mg/ml a perfect lens developed along with brain, retina and pigmented epithelium. At 0.125 mg/ml not only brain with accompanying structures but also muscle fibers were equally differentiated. These data suggest a new approach to the problem of dependence of the character of induction on the concentration of inducing factors, and they enable us to suppose that this dependence is not realized as a simple concentration dependence but may de determined by some adaptive, yet not elucidation processes.     

Over-expression of fibroblast growth factors in Xenopus embryos.

A number of forms of fibroblast growth factor (FGF) were over-expressed within Xenopus embryos by injection of synthetic FGF mRNAs into fertilized eggs. Injected embryos showed abnormalities in development which were mainly secondary to a disruption of gastrulation movements. The effects observed after injection of bFGF mRNA, however, were much less severe than those observed after injection of an altered form of bFGF mRNA which differs only by the addition of a signal sequence for secretion, or of another member of the FGF family, kFGF, which is normally efficiently secreted. All forms of FGF caused the induction of mesoderm in animal cap explants isolated from blastulae, but the amount of bFGF mRNA required to induce the formation of significant levels of mesoderm was higher by a factor of over a hundred than that of the FGFs which contain a signal sequence for secretion. Over-expressed bFGF accumulated in the nuclei of blastulae but did not necessarily cause mesoderm formation. These results show that FGFs must be secreted from the cells in which they are synthesised in order to act efficiently as mesoderm inducing factors and suggest that bFGF itself, which does not contain a signal sequence for secretion, is unlikely to be directly involved in mesoderm induction during early embryonic development.     

FGF-mediated induction of ciliary body tissue in the chick eye

Upon morphogenesis, the simple neuroepithelium of the optic vesicle gives rise to four basic tissues in the vertebrate optic cup: pigmented epithelium, sensory neural retina, secretory ciliary body and muscular iris. Pigmented epithelium and neural retina are established through interactions with specific environments and signals: periocular mesenchyme/BMP specifies pigmented epithelium and surface ectoderm/FGF specifies neural retina. The anterior portions (iris and ciliary body) are specified through interactions with lens although the molecular mechanisms of induction have not been deciphered. As lens is a source of FGF, we examined whether this factor was involved in inducing ciliary body. We forced the pigmented epithelium of the embryonic chick eye to express FGF4. Infected cells and their immediate neighbors were transformed into neural retina. At a distance from the FGF signal, the tissue transitioned back into pigmented epithelium. Ciliary body tissue was found in the transitioning zone. The ectopic ciliary body was never in contact with the lens tissue. In order to assess the contribution of the lens on the specification of normal ciliary body, we created optic cups in which the lens had been removed while still pre-lens ectoderm. Ciliary body tissue was identified in the anterior portion of lens-less optic cups. We propose that the ciliary body may be specified at optic vesicle stages, at the same developmental stage when the neural retina and pigmented epithelium are specified and we present a model as to how this could be accomplished through overlapping BMP and FGF signals.     

Cornea-lens transdifferentiation in the anuran, Xenopus tropicalis

Previously, the only anuran amphibian known to regenerate the lens of the eye was Xenopus laevis. This occurs during larval stages through transdifferentiation of the outer cornea epithelium under control of factors presumably secreted by the neural retina. This study demonstrates that a distantly related species, X. tropicalis, is also able to regenerate lenses through this process. A transgenic line of X. tropicalis was used to examine the process of cornea-lens transdifferentiation in which green fluorescent protein (GFP) is expressed in differentiated lens cells under the control of the Xenopus gamma1-crystallin promoter element. Unlike X. laevis, the process of cornea-lens transdifferentiation typically occurs at a very low frequency in X. tropicalis due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening. The inner cornea endothelium serves as a key physical barrier that normally prevents retinal signals from reaching the outer cornea epithelium. If this barrier is circumvented by implanting outer cornea epithelium of transgenic tadpoles directly into the vitreous chamber of non-transgenic X. tropicalis larval eyes, a higher percentage of cases formed lenses expressing GFP. Lenses were also formed if these tissues were implanted into X. laevis larval eyes, suggesting the same or similar inducing factors are present in both species. When pericorneal ectoderm and posteriolateral flank ectoderm were implanted into the vitreous chamber, only in rare cases did pericorneal ectoderm form lens cells. Thus, unlike the case in X. laevis, competence to respond to the inducing factors is tightly restricted to the cornea epithelium in X. tropicalis. As controls, all these tissues were implanted into the space located between the inner and outer corneas. None of these implants, including outer cornea epithelium, exhibited GFP expression. Thus, the essential inductive factors are normally contained within the vitreous chamber. One explanation why this type of lens regeneration is not seen in some other anurans could be due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening once the original lens is removed. These findings are discussed in terms of the evolution of this developmental process within the anurans.     

Spemann's influence on Japanese developmental biology

The discovery of the organizer by H. Spemann and Hilde Mangold, prompted a number of studies of embryonic induction in Japan. C.O. Whitman, N. Yatsu, T. Sato, H. Oka, T. Yamada, and Y.K. Okada were the pioneers in the field of embryonic induction. T. Yamada postulated the double potential theory for embryonic induction. O. Nakamura has modified the fate map of Vogt using newt and Xenopusblastulae. T.S. Okada and G. Eguchi proposed the new concept of "transdifferentiation" based on in vitro experiments in the retina and lens. T.S. Okada is not only an excellent scientist, but he has also nurtured many active developmental biologists. M. Takeichi, from his school, discovered the cell adhesion molecle, cadherin. Nakamura and colleagues tried to determine the origin and formation of the organizer. They performed recombination experiments using the ectoderm, endoderm and mesoderm, and concluded that the phenomenon in which various mesoderm tissues are formed by the recombination of the presumptive ectoderm with endoderm was "regulation of the vegetal-animal gradient". Some groups have also tried to purify specific inducing factors. T. Yamada and colleagues isolated two different types of ribonucleoproteins. I. Kawakami and colleagues showed that the ribosome fraction has neural inducing capacity, and that the extracellular matrix contains mesodermal inducing factors. Finally Asashima and colleagues isolated and identified activin A as a MIF factor. This finding had a great influence not only in the field of developmental biology, but also in molecular biology. Using activin, Asashima's group has successfully generated various organs, tissues, trunk-tail and head structures in vitro using animal caps (undifferentiated cells). Some other important molecules such as BMP, chordin and bFGF are also being studied by young Japanese scientists.     

Lens-Specific Expression of PDGF-A Alters Lens Growth and Development

The vertebrate lens provides anin vivomodel to study the molecular mechanisms by which growth factors influence development decisions. In this study, we have investigated the expression patterns of platelet-derived growth factor (PDGF) and PDGF receptors during murine eye development byin situhybridization. Postnatally, PDGF-A is highly expressed in the iris and ciliary body, the ocular tissues closest to the germinative zone of the lens, a region where most proliferation of lens epithelial cells occurs. PDGF-A is also present in the corneal endothelium anterior to the lens epithelium in embryonic and early postnatal eyes. PDGF-B is expressed in the iris and ciliary body as well as in the vascular cells which surround the lens during early eye development. In the lens, expression of PDGF-α receptor (PDGF-αR), a receptor that can bind both PDGF-A and PDGF-B, is restricted to the lens epithelium throughout life. The expression of PDGF-αR in the lens epithelial cells and PDGF (A- and B-chains) in the ocular tissues adjacent to the lens suggests that PDGF signaling may play a key role in regulating lens development. To further examine how PDGF affects lens developmentin vivo,we generated transgenic mice that express human PDGF-A in the lens under the control of the αA-crystallin promoter. The transgenic mice exhibit lenticular defects that result in cataracts. The percentage of surface epithelial cells in S-phase is increased in transgenic lenses compared to their nontransgenic littermates. Higher than normal levels of cyclin A and cyclin D2 expression were also detected in transgenic lens epithelium. These results together suggest that PDGF-A can induce a proliferative response in lens epithelial cells. The lens epithelial cells in the transgenic mice also exhibit characteristics of differentiating fiber cells. For example, the transgenic lens epithelial cells are slightly elongated, contain larger and less condensed nuclei, and express fiber-cell-specific β-crystallins. Our results suggest that PDGF-A normally acts as a proliferative factor for the lens epithelial cellsin vivo.Elevated levels of PDGF-A enhance proliferation, but also appear to induce some aspects of the fiber cell differentiation pathway.     

Inductive characteristics of proteins secreted by retinal cells

The studies of the development of eye rudiments and formation of adult eye tissues have always been among priorities in developmental biology and then in developmental genetics, which is associated with the peculiarities of the development and structure of the eye. In the late 80s, it was established by the group of developmental factors of the Institute of Gene Biology of RAS that many differentiated tissues are able to produce proteins causing homologous differentiations in polypotent cells of early gastrula ectoderm. The aim of our present study was isolation of proteins secreted by mammalian and fish retinal cells and determination of their inductive properties in early gastrula ectoderm of Xenopus laevis. The sets of proteins secreted by retina induce tissues homologous to the inducer, that is, neural tissue, brain, retina, pigmented epithelium, and also lenses and ear vesicles. The retinal inductive proteins retain their homologous inductive capacity after lyophilization. Biological testing shows that a total mixture of proteins secreted by retinal cells induces in polypotent gastrula ectoderm of X. laevis a narrower spectrum of tissues than the fractions obtained from this mixture. The above-outlined results obtained in thecourse of investigations of inductive peculiarities of retina and its fractions help in the elucidation of questions concerning embryonic induction and factors determining it, as well as questions concerning the maintenance of tissue specifity and regenerative capacity of the tissue studied.     

The ability of the epithelium of diencephalic origin to differentiate into cells of the ocular lens.

After the discovery that in adult salamanders following lentectomy a new, functional lens develops by transdifferentiation (cell-type conversion) of previously depigmented epithelial cells of the iris (Wolffian lens regeneration), this phenomenon has been intensively studied by various experimental approaches. During the last two decades it was shown that pleiomorphic aggregates of atypical lens cells (lentoids) differentiated in reaggregates of dissociated cells of the chick neural retina and in spread cell cultures of the pigmented epithelium of the iris and retina, of the neural retina and the pineal gland of the chick embryo. The neural retina of human fetuses and adults also displayed this capacity. We showed that lentoids developed at a low incidence in renal isografts of rat embryonic shields or isolated embryonic ectoderm and of lentectomized eyes of rat fetuses, as well as in organ cultures of rat embryonic shields in chemically defined media. The addition of transferrin significantly increased the incidence of differentiation of lentoids in explants. In both renal isografts and explants in vitro a continuous transformation of retinal epithelial cells into atypical lens cells was observed. In renal isografts lentoids were also observed to originate from the ependyma of the brain ventricle. All tissues having the capacity to convert into lens cells belong to the diencephalon in a broad sense. Evolutionary aspects of this feature are discussed.     

Role of calcium-dependent and calcium-independent mechanisms in the adhesion of retinal and pigment epithelium cells in the chick embryo

The mechanisms of adhesion of the retinal and pigment epithelium cells, as well of cell interaction within each of these tissues were studied during development. It was shown by means of separation of retina from pigment epithelium in different dissociation media that the adhesion of these tissues in 5-6 day old chick embryos is realized via a Ca2+-independent mechanism. The adhesion of these tissues decreases between days 7 and 16. Starting from day 16, both Ca2+-independent and Ca2+-dependent mechanisms are involved in the interaction of the retinal and pigment epithelium cells. By measuring the output of single cells into the suspension after the treatment of retina and pigment epithelium with different dissociating agents, it was shown that from the 5th day of incubation on the adhesion of pigment epithelium cells is mediated by Ca2+-dependent mechanism. In the retina three types of cells were found: interacting via Ca2+-dependent mechanism only, Ca2+-independent mechanism only, and both the mechanisms. In the course of differentiation, the numbers of the population of cells interacting only via Ca2+-dependent mechanism increase, while those of cells interacting via Ca2+-independent mechanism decrease. It is suggested that at each developmental stage those retinal cell possess Ca2+-dependent mechanism of adhesion which are closest to the definitive state.     

Isolation and effects of inducing factors secreted by the lens epithelium cells

Our previous studies showed that, unlike tissue extracts, the cells of living organs secrete substances capable of inducing the same organ rudiments in the early gastrula ectoderm (EGE). In this work, the molecular nature of these substances was studied. The porcine lens epithelium was chosen for the initial analysis. When cultivated, this epithelium secreted a mixture of proteins, which were separated by gel-filtration. Both the total protein mixture and its individual fractions were tested for their inducing capacity using the early gastrula ectoderm of Rana temporaria. Unexpected results were obtained, which indicated that (a) the mixture of native proteins secreted by lens epithelium has a selective inducing capacity differing from those of individual fractions isolated from this mixture and (b) each fraction has a specific effect, but all of them cause the induction of neural tissue or sensory organs. These results (obtained for the first time) suggest that the inducing capacity of individual protein fractions is wider than that of the total protein mixture secreted by lens epithelium. This fact raises a question concerning the relationships between the mechanisms underlying the corresponding inducing effects.     

Distribution of differentiation potentials and the conditions for their realization in the amphibian neuroectoderm

The X. laevis neuroectoderm (NE) at the mid and late gastrula stages is capable to form mesoderm in vitro after its separation from mesoderm. This capacity is inherent in posterior 2/3 of NE underlied by axial mesoderm in the embryo and forming deuterencephalic and trunk regions of the brain in the normal development. The archencephalic 1/3 of NE of the late gastrula, underlied in the embryo by prechordal plate, is capable of differentiation into archencephalic regions of the brain, rather than into mesoderm. For the typical differentiation of archencephalic NE to be realized, it should be surrounded by the outer ectoderm layer. In the absence of the latter, the whole explant develops into retina and brain only. Inside the closed explants, ectomesenchyme and melanophores arise and the eye material is subdivided into retina and pigmented epithelium. The archencephalic NE, dissociated to individual cells and wrapped into epidermis, forms much more ectomesenchyme and melanophores than the usual NE explants.     

The pattern of DNA methylation in the delta-crystallin genes in transdifferentiating neural retina cultures

Terminally differentiated lens fibre cells are formed in the vertebrate lens throughout life. Lens fibre cells may also be obtained by an in vitro process termed transdifferentiation, from certain tissues of different developmental origin from lens, such as embryo neural retina. delta-Crystallin is the major protein in the chick embryo lens fibre cells, and also in transdifferentiated lens cells obtained from cultured embryonic neural retina. Lens crystallin proteins and mRNA are present at low levels in the intact embryonic neural retina but are no longer detectable in the early stages of neural retina cell culture. However, levels rise steeply in the later stages and crystallins become the major products in terminally transdifferentiating neural retina cultures. We have used this system to test the hypothesis that the patterns of DNA methylation in particular genes are correlated with gene expression. A number of developmentally regulated genes have been found to be undermethylated in tissues where they are expressed, and methylated in tissues where they are not. However this correspondence does not always hold true. Eight-day-old embryonic neural retina was cultured for the period of time during which crystallin gene expression increases 100-fold. DNA methylation in the delta-crystallin gene region was analysed at several stages of cell culture by using the restriction endonucleases HpaII and MspI which cleave at the sequence CCGG. The former enzyme cannot cleave internally methylated cytosine (CmCGG) while the latter cannot cleave externally methylated cytosine (mCCGG). We detect no change in the methylation of CCGG sites within the delta-crystallin gene regions during transdifferentiation. Since dramatic changes in delta-crystallin gene expression occur during this process we conclude that large scale alterations in the pattern of DNA methylation are not a necessary accompaniment to changes in gene activity.     

Inductive effect of the eye tissues of adult clawed toads on the gastrula ectoderm

The inducing influence of adult eye tissues on the early gastrula ectoderm was studied in vitro. Both retina and pigment epithelium induced in the early gastrula ectoderm similar spectra of cell types, including nervous tissue, retina, pigment epithelium, lentoids, ectomesenchyme, and melanophores. It is suggested that the correspondence of these cell types with those arising at a spontaneous transdifferentiation of the isolated retina and pigment epithelium cells in vitro or at the induction of the early gastrula ectoderma by archencephalic endomesoderm during the normal development can be accounted for by that in these eye cells molecular determinants appeared as a result of induction and maintaina the stability of their differentiation and their potencies to transdifferentiation in vitro being reproduced during the lifetime of these cells.     

Mesoderm induction and blood island formation by angiogenic growth factors and embryonic inducing factors

Factors which induce mesoderm, including endothelium lined cavities and primitive blood cells in omnipotent amphibian ectoderm, have been isolated from different sources. Recently it was shown that angiogenic factors, which belong to the protein families of the heparin binding growth factors (acidic and basic fibroblast growth factor) and the transforming growth factors (TGF-beta 1 and -beta 2), also induce mesodermal tissues in amphibian ectoderm. In triturus ectoderm, capillary like endothelial networks are induced preferentially by the transforming growth factors. The relationship between growth factors and inducing factors is discussed.