Radioautographic study of cell proliferation in the pigment epithelium of tritons after transplantation into the cavity of a lens-free eye

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%.     

Cultivation of the retinal pigment epithelium in the cavity of the lentectomized eye of newts

A study was made of proliferative activity and transdifferentiation of the cells of retinal pigment epithelium (RPE) cultivated in the cavity of the lensectomized eye of adult newt. Implantation of the newt RPE together with vascular membrane and scleral coat resulted in the regeneration of retina. In this process the character of changes in the proliferative activity of RPE and differentiation of retinal cells were the same as in the regeneration of retina in situ. RPE implanted with the vascular membrane alone, despite a high level of proliferation during the first ten days of cultivation, no differentiated retina was formed. Possible causes of these differences are discussed, and the comparison is made of the data obtained with those on RPE cultivation in vitro. After lens removal, with RPE implants present in the eye cavity, in addition to the regenerated lens, 2-3 extra lenses and retina were formed from the cells of the inner layer of the recipient's dorsal iris. Also some cases were revealed of lens formation from the cells of ventral iris. With a complete detachment of the recipient's retina (an after-effect of transplantation) a second differentiated retina regenerated in situ from the recipient's RPE cells.     

Radioautographic study of the cell proliferation of the pigment epithelium of the retina in albino clawed frogs

The proliferative activity of pigment epithelium was studied by means of 3H-thymidine autoradiography after the removal of retina, lens and iris with the ciliary-terminal zone in the adults. The cell population of pigment epithelium was shown to be heterogeneous on the level of proliferative activity. A low level of proliferation is characteristic of the cells of epithelial monolayer and the cells leaving it and forming aggregates. An intensive local proliferation leading to the formation of expansions was found in the pigment epithelium layer in 7% of cases. On the 20th day after the operation, the index of labelled nuclei in the expansions amounted to 43.4--59.3% and the mitotic index to 1.4--2.1%. On the 75th day elements of atypical retinal differentiation, besides the high proliferative activity, were observed in one expansion.     

Effect of experimental arrest of eye growth on the proliferation and polyploidization of the retinal pigment epithelium in rats

Following the lens removal from the left eye of the newborn rats, animals were obtained with one normal (control) and another microphtalmic eye. The animals were sacrificed on the 2nd, 3rd, 5th, 7th and 9th days of postnatal development after four injections of 3H-thymidine during 19 hrs. The number of labelled nuclei and mono- and binuclear cells in the central zone of the eye fundus was counted on the autographs. After the initial increase of the index of labelled nuclei in the operated eyes (on the 2nd, 3rd and 5th days) it fell below the control level (on the 7th and 9th days). The number of binuclear cells in the operated eyes, as well as in the control, attains on the 5th day 50% of the total number of cells and remains at this level up to the end of the experiment, whereas in the control eyes the number of binuclear cells increases up to 60% on the 7th and 80% on the 9th day. The results obtained have shown that in rats the factors of total eye growth participate in the control of proliferative activity and polyploidization of the pigment epithelium cells in the retina.     

Expression of regulatory and tissue-specific genes controlling regenerative potencies of the eye tissues in vertebrates

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 regulatory and tissue-specific genes controlling regenerative potencies of eye tissues in vertebrates

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.     

Transdifferentiation of Adult Frog Iris in Retina or Lens by Exogeneous Influences

The mechanisms of transdifferentiation of iris epithelial cells of Rana temporaria (Anura) in culture depending on influences from different sources were studied. In terminally differentiated iris cells, the process of transdifferentiation is initiated by dedifferentiation. Melanosomes are shed from iris cells due to cell surface activity. After depigmentation, iris epithelial cells become capable of proliferating and competent to react to the influences of various exogenous factors. Under the influence of retinal factors secreted by lentectomized tadpole eyes, both dorsal and ventral irises are converted to neural retina. Under the influence of factors from eye vesicles, the irises are converted to neural retina as well. Similar results were obtained in transfilter experiments, in which a 3-day period of transfilter interaction between the irises and eye vesicles ensured depigmentation of the iris followed by transdifferentiation into complete NR with visual receptor. Lentoid formation occurred under the influence of adult frog lens epithelium. Immunofluorescent analysis confirmed the lens nature of the lentoids. In control experiments under the conditions of the tadpole eye orbit, in which programming influences were absent, iris epithelial cells remained unaffected.
The problem of true cell-reprogramming to new differentiation in contrast to expression of inherent properties of the iris epithelial cells is discussed.     

Electrophoretic and immunofluorescent study of lactate dehydrogenase isoenzymes during eye regeneration in adult tritons]

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.     

Fibronectin distribution during the transdifferentiation and proliferation of eye cells after retinal detachment and removal of the crystalline lens in tritons

Expression of fibronectin (Fn) during eye tissue regeneration in the newt after retinal detachment and lens removal was studied by immunohistochemistry. Proliferation of cells involved in eye tissue regeneration was studied using autoradiography. Fn was detected around the cell membranes of undifferentiated proliferating and migrating cells in ciliary body of the iris and growth zone of the retina. Redistribution of Fn was observed in proliferating cells of the dorsal iris participating in lens regeneration. Fn appeared on the apical surface of proliferating redifferentiating pigment epithelium (PE) cells at the periphery of the eye and over the whole surface of proliferating PE cells in the central part of the eye. The Fn level in the Bruch's membrane decreased in the area of transdifferentiating cells detachment from PE layer (in the lower part of the eye) but continued to be stable in the area of PE cell redifferentiation (at the periphery of the eye). The role of Fn is discussed in relation to transdifferentiation, proliferation and migration of cells in the regenerating eye.     

Proliferation of the cells of iris and pigment epithelium of the retina in the early periods after removal of the lens and iris in Spanish newts]

It has been shown by means of autoradiography that following the simultaneous removal of lens and retina in the eyes of adult ribbed newts (Pleurodeles waltlii) the proliferative processes related to the regeneration of retina, rather than lens, are most active at the early stages of eye restoration. During the lens regeneration in the absence of retina, the proliferation of the cells of pars iridica of the dorsal iris zone, a source of lens regeneration, is delayed, possibly due to the increase of the duration of mitotic cycle of these cells.     

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.     

Proliferative activity of the pigment epithelium and regenerating retinal cells in Ambystoma mexicanum

Cellular sources of retinal regeneration and proliferative activity of the cells taking part in retina restoration have been studied in axolotls using 3H-thymidine. The cells of ciliary-terminal zone proved to be the main source of retinal restoration. Besides these cells, the pigmented cells of the iris inner and outer layers and pigment epithelium cells can take part in this process. Morphological stages of retinal regeneration have been established and regular changes in the level of proliferation in different zones of regenerating retina have been found with respect to the stage of retina restoration. The high level of proliferative activity of the pigment epithelium cells found soon after the operation favoured the restoration of disturbed integrity of the pigment epithelium layer, the increase of cell density in it, the elongation of the pigment epithelium layer, the formation of processes, and, sometimes, the replenishment of regenerating retina.     

Melanin granule metabolism in the iris and retinal pigment epithelium in adult tritions after completion of eye regeneration

It was concluded that the newly synthesized melanin granules were replaced in the pigmented tissues of the newt eye on the basis of redistribution of the cells of pigment epithelium of retina and iris labelled by 3H-DOPA 2.5 and 6.5 months after the isotope injection. The replacement of melanin granules and, correspondingly, melanin synthesis proceed more actively in the peripheral zones of the pigment epithelium of retina. The depigmentation of cells preceding the melanin synthesis appears to be realized with the participation of macrophages.     

Cellular analysis on localization of lens forming potency in the newt iris epithelium

The localization of a lens forming potency in the iris epithelium was studied by autoradiographic analysis of the distribution of ³H-thymidine labelled cells to be participated in lens regeneration in newts. DNA synthesis started from the dorsal portion of the iris epithelium around 4 days after lentectomy. 5 days after lentectomy, a large number of labelled cells were mostly found in the dorsal sector, showing strong contrast to the ventral and lateral sectors of iris, which contained a few labelled cells. The labelled index (the number of labelled cells/the number of cells in the definite pigmented area of the iris epithelium) of the dorsal sector attained the highest value, 29.7 ± 2.35, on day 7 after lentectomy, and dropped temporarily. This was followed by the second peak on day 12. The dorso-ventral ratio of the labelled index reached to the highest value, 6.87 ± 0.67, on day 5. This ratio decreased rapidly after the completion of a lens rudiment, and it became about 1. In “chase” experiments by diluting the radio-isotope with excess cold thymidine, it was obviously shown that most of the cells labelled with the radio-isotope and distributed in the dorsal marginal iris 5 days after lentectomy participated in the formation of a lens regenerate during the period of chasing. From these results, the following conclusion was drawn. The iris epithelium consists of at least 2 different cell populations; one is capable of transformation into lens cells and is distributed mostly in the dorsal portion of the iris epithelium, while the other has no potency for transformation and is able to grow to compensate a loss of the dorsal marginal cells which transformed into lens cells during the process of lens regeneration.     

A novel role of the hedgehog pathway in lens regeneration

Lens regeneration in the adult newt is a classic example of replacing a lost organ by the process of transdifferentiation. After lens removal, the pigmented epithelial cells of the dorsal iris proliferate and dedifferentiate to form a lens vesicle, which subsequently differentiates to form a new lens. In searching for factors that control this remarkable process, we investigated the expression and role of hedgehog pathway members. These molecules are known to affect retina and pigment epithelium morphogenesis and have been recently shown to be involved in repair processes. Here we show that Shh, Ihh, ptc-1, and ptc-2 are expressed during lens regeneration. The expression of Shh and Ihh is quite unique since these genes have never been detected in lens. Interestingly, both Shh and Ihh are only expressed in the regenerating and developing lens, but not in the intact lens. Interfering with the hedgehog pathway results in considerable inhibition of the process of lens regeneration, including decreased cell proliferation as well as interference with lens fiber differentiation in the regenerating lens vesicle. Down-regulation of ptc-1 was also observed when inhibiting the pathway. These results provide the first evidence of a novel role for the hedgehog pathway in specific regulation of the regenerating lens.     

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.     

3H-thymidine incorporation into the macrophages in the process of eye regeneration in adult tritons]

Changes in the number of labelled macrophages in the regenerating eye cavity of the adult common newts were studied within 2 to 12 days following the injection of 3H-thymidine and removal of retina and lens from the eyes. A small number of labelled macrophages was found in the eye cavity (0,2--5.9%) at all regeneration stages under study upon the pulse 3H-thymidine incorporation. Their number rapidly increased and attained by the end of observation (12 days) 73%. These results suggest that mainly mature non-dividing forms of macrophages which completed the mitotic cycle S-phase in the places of their active reproduction migrate in the eye cavity. The sharp increase of the number of labelled macrophages in the eye cavity is determined by the migration of those macrophages the precursors of which were labelled as a result of both the pulse 3H-thymidine incorporation and reutilization of the labelled precursors of DNA synthesis. New portions of labelled macrophages migrate in the eye cavity within 2 to 4 days.     

Lactate dehydrogenase isoenzymes during regeneration of the lens and retina in adult tritons

The range of lactate dehydrogenase (LDG) isozymes has been studied at the consecutive stages of retina regeneration from pigmented epithelium cells and lens regeneration from iris margin in adult crested newts. It was shown that the spectra of LDG isozymes peculiar to pigment epithelium cells and iris and characterized by the predominance of slowly migrating forms are replaced in the lens and retina regenerates by spectra characterized by the predominance of rapidly migrating isozymes which are peculiar to definitive lens and retina.     

Transdifferentiation of ocular tissues in larval Xenopus laevis

Transdifferentiation phenomena offer a useful opportunity to study experimentally the mechanisms on which cell phenotypic stability depends. The capacities of vertebrate eye tissues to reprogram cell differentiation are well known in avian and mammalian embryos, and in larval and adult newt. From research into the capacity of anuran eye tissues to reprogram differentiation into a new pathway, considerable data have accumulated concerning the transdifferentiative capacities of eye tissues in larval Xenopus laevis. This work reviews the data concerning the transdifferentiative phenomena of eye tissues in that species and, based on these, aims to establish the extent of our knowledge about the mechanism controlling these processes. In larval Xenopus laevis the outer cornea can regenerate a lens by a lens-transdifferentiation process triggered and substained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye phenotypic stability of the outer cornea is guaranteed by the presence of the inner cornea and lens, which prevent the spread of retinal factor(s). The stimulus for lens transdifferentiation of the outer cornea can be supplied by other tissues as well, but this capacity is not widely distributed. The iris and retinal pigmented epithelium can transdifferentiate into neural retina if isolated from the surrounding tissues and implanted in the vitreous chamber. As for lens transdifferentiation of the outer cornea, retinal transdifferentiation of the iris can be stimulated by certain nonocular tissues as well.     

Early postnatal proliferation of the retinal pigment epithelium cells in the mouse

A burst of proliferative activity with a maximum of DNA-synthesizing cells on the first day after birth was found in the central zone of the retinal pigment epithelium (RPE) in albino mice from the moment of birth to 9 days of life using radioautography with 3H-thymidine pulse labelling. During this period the central RPE zone, which consists in newborns of mononuclear cells by 95%, gradually transforms in a population with predominance of binuclear cells and fluctuations in the index of labelled nuclei (after the kinetics of cell population in the central RPE zone is similar in mice and rats both in accumulation of binuclear cells and fluctuations in the index of labelled nuclei (after pulse labelling), except that in mice the peak of the index of labelled nuclei is observed earlier than in rats.