The cell cycle of cultured iris epithelial cells: Its possible role in cell-type conversion

Cell cycle parameters were estimated in primary cultures of iris epithelial cells, obtained from explanted dorsal and ventral irises of adult newts (Notophthalmus viridescens). No significant difference was found between parameters of dorsal and ventral iris epithelial cell cultures. Compared with the total cell cycle time of iris epithelial cells in situ in the pathway of conversion, that of cultured iris epithelial cells is longer by a factor of 1.88. The results support the working hypothesis that the basic requirement for conversion of iris epithelial cells into lens cells is the passage of a definite number of cell cycles instead of the inductive influence of neural retina.     

Effects of exogenous FGF-1 treatment on regeneration of the lens and the neural retina in the newt, Notophthalmus viridescens

Experiments were designed to compare the effects of recombinant newt fibroblast growth factor-1 (rnFGF-1) and recombinant human glial growth factor (rhGGF) on lens and retina regeneration in the eyes of adult newts. Both eyes were retinectomized and lentectomized. Beginning 3 days after the operation, one eye was given either 0.1 microg of rnFGF-1 or 0.1 microg of rhGGF in 1 microl of phosphate-buffered saline (PBS) per injection, three per week. Contralateral operated eyes served as controls and were treated with PBS alone or were not injected. In eyes that were not injected, injected with PBS alone, or with PBS containing rhGGF, regeneration of both the retina and the lens proceeded normally as described in the literature. In these control eyes, the entire retinal pigmented epithelium (RPE) depigmented/dedifferentiated and a retina rudiment formed from which a new retina regenerated by the end of the experiment at day 41 post-operation. Likewise, only a small area of dorsal iris depigmented/dedifferentiated and formed a lens vesicle from which a lens subsequently regenerated. The vitreous remained relatively free of loose cells.In eyes given rnFGF-1, the RPE depigmented/dedifferentiated and formed what appeared to be a retina rudiment but a new retina did not regenerate. Instead, vesicles were seen associated with the retina rudiment. In eyes given rnFGF-1, both the dorsal iris and ventral iris depigmented/dedifferentiated and lens regeneration occurred but the new lenses had abnormal fiber cells and the lens epithelium was very thin or absent. In addition, ectopic lenses usually regenerated in rnFGF-1-treated eyes. An abundance of loose cells were present in the vitreous of rnFGF-1-treated eyes associated largely with the RPE and the dorsal and ventral irises.The results are consistent with the view that the timely expression of FGFs is involved in the depigmentation/dedifferentiation of the RPE and dorsal iris and is necessary for proper regeneration of the lens and neural retina. Continued presence of FGF results in continued and excessive dedifferentiation, resulting in the lack of retina regeneration and abnormal lens regeneration.     

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.     

Radioautographic study of the cellular proliferation of retinal pigment epithelium in axolotls

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.     

Role of the neural retina in newt (Notophthalmus viridescens) lens regeneration in vitro

Removal of the lens from the eye of an adult newt (Notophthalmus viridescens) is followed by regeneration of a new lens from the dorsal iris epithelial cells at the pupillary margin. This process is dependent upon the neural retina for its normal completion in vivo and in vitro. To examine the relationship between the retina and lens regeneration, we have conducted experiments that delimit the time period during which the retinal presence is critical (in vivo) and have investigated the influence of extracts of the retina on the progress of regeneration (in vitro). In vivo, removal of the retina at day 11 seriously retards further progression of regeneration while removal of the retina at day 15 does not retard regeneration significantly. This defines a "critical period" in regeneration of the lens during which the retina is required. Explantation of regenerates 11 or 12 days after lentectomy to organ culture medium enriched with either crude retinal homogenate or extracts prepared from chick or bovine retinas according to Courty et al. ('85, Biochimie, 67:265-269) reveals that the progress of regeneration can be supported in culture by the crude extract. This is the first demonstration of complete iris-lens transformation in culture in the presence of retinal extract. It is possible that the retina acts indirectly by promoting passage of the iris epithelial cells through the critical number of mitoses required before redifferentiation into lens cells can occur (as proposed by Yamada, '77, Monogr. Dev. Biol., 13:126). It is also possible that the retina acts by directly instructing the iris cells to redifferentiate.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Transformation of Iris into Lens in vitro and its Dependency on Neural Retina

Upon lentectomy of adult newt eyes, the dorsal iris epithelium produces a cell population that develops into a new lens. The tissue transformation can be completed not only in the isolated lentectomized eye cultured as a whole, but also in the isolated newt normal dorsal iris combined with the retina of frog larvae in vitro. In this study, 93% of such cultures produced lens tissue made up of newt cells. Well-differentiated lens fibre cells were formed which showed positive immunofluorescence for gamma crystallins. When the isolated dorsal iris epithelium was cultured under the same conditions, well-differentiated lens tissue was again formed in 95% of the cases, suggesting that iris epithelial cells and not iris stromal cells are responsible for lens formation. In contrast, the combination of newt ventral iris with frog retina did not produce any newt lens tissue. No lens tissue was produced when the dorsal iris was cultured with newt spleen or lung, although a considerable number of iris epithelial cells became depigmented. Isolated normal dorsal iris or normal dorsal iris epithelium cultured alone infrequently produced a population of depigmented cells but failed to form lens tissue. On the basis of the present and earlier data, it is concluded that in Wolffian lens regeneration in situ , interaction of the iris epithelial cells with the retina plays a decisive role. However, it is suggested that the iris epithelial cells may have an inherent tendency towards lens formation, and that the factor(s) from the retina facilitates the realization of this tendency, rather than instructing the cells to produce lens. The reported experiments provide the first direct evidence for the existence of cellular metaplasia by demonstrating transformation of fully differentiated iris epithelial cells into lens cells.     

From lens regeneration in the newt toin-vitrotransdifferentiation of vertebrate pigmented epithelial cells

Through studies to clarify the cellular origin of lens regeneration in the newt, the pigmented epithelial cells of the iris and the retina of many vertebrate species have been shown to possess a dormant potency to transdifferentiate into the lens. The method ofin-vitroculture of pigmented epithelial cells has been optimized to enable detailed studies of the transdifferentiation process by molecular techniques. Growth factors and extracellular matrix components are found to be important in the control of the transdifferentiation process. New systems forin-vitroculture are introduced, while prospects for renewedin-vivostudies using newts are given.     

Transdifferentiation of eye tissues in anuran amphibians: Analysis of the transdifferentiation capacity of the iris of Xenopus laevis larvae

Abstract. Lensectomized Xenopus laevis larvae are capable of regenerating a lens from the cells of the outer cornea. Unlike the outer cornea, the iris of larval Xenopus exhibits a high degree of phenotypic stability, even when it has been damaged to various degrees in order to stimulate its latent transdifferentiative competence. However, when isolated from its surrounding tissues and implanted in an appropriate site, the dorsal iris of larval Xenopus is capable of following a differentiative pathway different to that normally followed in situ. Our results show that, when such an implant is placed in the vitreous chamber of a lensectomized eye, the pigmented epithelial cells of the iris transdifferentiate into neural retina regardless of whether the iris stroma is present or not. Unlike the vitreous chamber, the environment of the anterior chamber of a lensectomized eye does not promote the transdifferentiative process of the iris. We suggest the existence of eye factors that promote retina-forming transformation of the iris and that are distributed in a gradient in lensectomized eyes.     

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.     

Retina and lens regeneration in anuran amphibians

Anuran amphibians can regenerate the retina through differentiation of stem cells in the ciliary marginal zone and through transdifferentiation of the retinal pigmented epithelium. By contrast, the regeneration of the lens has been demonstrated only in larvae of species belonging to the Xenopus genus, where the lens regenerates through transdifferentiation of the outer cornea. Retinal pigmented epithelium to neural retina and outer cornea to lens transdifferentiation processes are triggered and sustained by signaling molecules belonging to the family of the fibroblast growth factor. Both during retina and lens regeneration there is a re-activation of many of the genes which are activated during development of the eye, even though the spatial and temporal pattern of gene expression is not a simple repetition of that found in development.     

Macrophage invasion and phagocytic activity during lens regeneration from the iris epithelium in newts

Following removal of the lens through the cornea, early stages of lens regeneration from the dorsal iris of the adult newt, Notophthalmus viridescens, were studied using light and electron microscopic observations on sectioned, plastic-embedded irises. Specimens were fixed in Karnovsky's fixative every 2 days from 0 to 12 and 15 days after lentectomy. Infiltration of the iris epithelium by macrophages and their phagocytosis of melanosomes and small fragments of iris epithelial cells were observed. These macrophages were characterized by coarse nuclear chromatin, numerous mitochondria, free ribosomes, granular endoplasmic reticulum, Golgi complexes, vesicles, lysosomes, and phagosomes containing ingested melanosomes. Lamellipodia of varying length projected from their surface. Most of the cells lying on or close to the posterior surface of the iris could be identified as macrophages by these criteria. During this period, there was enlargement of the intercellular spaces within the iris epithelium. The iris epithelial cells near the margin of the pupil elongated, lost their melanin pigment and some associated cytoplasm, and acquired abundant free polyribosomes to form a lens vesicle of depigmented cells.     

Hyaluronic acid production and hyaluronidase activity in the newt iris during lens regeneration

The process of lens regeneration in newts involves the dedifferentiation of pigmented iris epithelial cells and their subsequent conversion into lens fibers. In vivo this cell-type conversion is restricted to the dorsal region of the iris. We have examined the patterns of hyaluronate accumulation and endogenous hyaluronidase activity in the newt iris during the course of lens regeneration in vivo. Accumulation of newly synthesized hyaluronate was estimated from the uptake of [3H]glucosamine into cetylpyridinium chloride-precipitable material that was sensitive to Streptomyces hyaluronidase. Endogenous hyaluronidase activity was determined from the quantity of reducing N-acetylhexosamine released upon incubation of iris tissue extract with exogenous hyaluronate substrate. We found that incorporation of label into hyaluronate was consistently higher in the regeneration-activated irises of lentectomized eyes than in control irises from sham-operated eyes. Hyaluronate labeling was higher in the dorsal (lens-forming) region of the iris than in ventral (non-lens-forming) iris tissue during the regeneration process. Label accumulation into hyaluronate was maximum between 10 and 15 days after lentectomy, the period of most pronounced dedifferentiation in the dorsal iris epithelium. Both normal and regenerating irises demonstrated a high level of endogenous hyaluronidase activity with a pH optimum of 3.5-4.0. Hyaluronidase activity was 1.7 to 2 times higher in dorsal iris tissue than in ventral irises both prior to lentectomy and throughout the regeneration process. We suggest that enhanced hyaluronate accumulation may facilitate the dedifferentiation of iris epithelial cells in the dorsal iris and prevent precocious withdrawal from the cell cycle. The high level of hyaluronidase activity in the dorsal iris may promote the turnover and remodeling of extracellular matrix components required for cell-type conversion.     

Influence of chromatographic fractions of extracts derived from bovine neural retina on newt (Notophthalmus viridescens) lens regeneration in vitro

Removal of the ocular lens in adult newts (Notophthalmus viridescens) is followed by a series of cellular events leading to regeneration of a new lens by cell type conversion of pigmented iris epithelial cells at the dorsal pupillary margin (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). Following depigmentation and five to seven cell divisions, iris epithelial cells redifferentiate into lens fiber cells and synthesize crystallin proteins (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). This process is dependent upon neural retina in vivo (Stone, Anat. Rec. 131:151-172, 1958; Reyer, Dev. Biol. 14:214-225, 1966) and in vitro (Yamada et al., Differentiation 1:65-82, 1973). Acting on the hypothesis that the role of the neural retina is to promote passage of iris epithelial cells through the requisite number of cell cycles which will then allow them to redifferentiate as lens fiber cells (Yamada, in: Cell Biology of the Eye. Academic Press, New York, 1982), we undertook testing of the effects of eye-derived mitogenic substances, as well as other mitogens, on regeneration of lens from iris in organ culture. We have previously defined a critical period for the retinal influence in vivo and in vitro, and have shown that crude extracts of retina can enhance regeneration of lenses in culture (Connelly et al., J. Exp. Zool., 240:343-351, 1986). In this paper, we report on the lens regeneration enhancing activity (LRA) of more highly purified fractions of the retinal extracts. Heparin-sepharose chromatography of the crude retinal extract yields three fractions (Courty et al., Biochemie 67:265-269, 1985) called EDGF I, II, and III. EDGF I and II have affinity for heparin, while EDGF III does not. In our bioassay, LRA appears only in the EDGF III fraction. Dialysis of EDGF III against 0.1 N acetic acid yields a fraction which has affinity for cibacron blue sepharose (eluting at 2.15 M salt) and also has significant LRA. Because insulin at high doses has a marginal effect on lens regeneration in culture (Williams and McGlinn, Am. Zool. 19:923, 1979; Connelly, Differentiation 16:85-91, 1980), we tested IGF-I. Because of the putative neurotrophic effects of transferrin (Tf) (Mescher and Munaim, J. Exp. Zool., 230:485-490, 1986), we tested Tf for its ability to enhance regeneration of the lens in culture. IGF-I seems to have an enhancing effect on lens regeneration; Tf does not.     

Thrombin activation of S-phase reentry by cultured pigmented epithelial cells of adult newt iris

Following local injury or tissue removal, regeneration in urodele amphibians appears to be dependent on cell cycle reentry and dedifferentiation of postmitotic, terminally differentiated cells in the remaining tissues. Regeneration of the lens of the eye occurs by the dedifferentiation of pigmented epithelial cells (PEC) of the iris and their subsequent transdifferentiation into lens cells. A key question is how cell cycle reentry is regulated. Here we demonstrate that thrombin activates S-phase reentry of newt PEC in vitro. Based on these findings, and on previous experiments showing that newt skeletal myotubes reenter the cell cycle following thrombin stimulation, we suggest that thrombin is a critical signal for initiation of vertebrate regeneration.     

Direct Action of Light in Naturally Pigmented Muscle Fibers : I. Action spectrum for contraction in eel iris sphincter

Contraction due to light in excised eel irises appears to follow a simple first order law. The action spectrum for contraction has a maximum which agrees with the eel rhodopsin absorption maximum. Inasmuch as rhodopsin is the rod pigment-opsin complex and the iris sphincter pupillae evolves from the pigment epithelium of the retina in the region of the iris, the muscle pigment might be the same as the visual pigment. In the human eye the contraction of the iris sphincter is activated only by light incident on the retina and the pupil diameter varies inversely with the square root of the light intensity. The inverse first power relation observed in the present experiments suggests a more primitive origin for the light reaction in eel irises. Relaxation is a much slower process and can be approximated as the sum of two first order processes.     

The role of calcium in depigmentation of iris epithelial cells during cell-type conversion

During the conversion of newt iris epithelial cells into lens cells, melanosomes disappear from the cytoplasm. In this “depigmentation,” exocytosis of melanosomes is involved. The role of Ca²⁺ in this process has been the subject of this work. The intracellular Ca²⁺ concentration of cultured iris epithelial cells was increased by three methods: microinjection of 10^?3, M CaCl₂ into the cytoplasm, fusion of phospholipid vesicles containing 10^?3, M CaCl₂ with the cell membrane, and exposure to the calcium ionophore A23187. Each of these treatments caused an increase in the release of melanosomes. Further experiments suggest that cAMP stimulates exocytosis probably by liberating Ca²⁺ from intracellular stores.     

GROWTH OF LENS AND OCULAR ENVIRONMENT: ROLE OF NEURAL RETINA IN THE GROWTH OF MOUSE LENS AS REVEALED BY AN IMPLANTATION EXPERIMENT

The lens of 6-day-old normal mouse was implanted into the lentectomized eye of adult mouse to examine the effect of retina upon the growth of the implanted lens in vivo. The implanted lens grew normally and its transparency was kept for more than 5 months after implantation. The connection between the implanted lens and the ciliary part of the recipient iris was well established with the regeneration of zonular fibers from the recipient. In young lenses implanted reversely into adult eyes, the epithelial cells facing the retina elongated and a new epithelium was formed on the corneal side of the lens within 5 days. Young lenses implanted either in normal or reverse orientation into eyes from which the retina was previously removed did not grow. The cells of the original lens epithelium of these lenses were randomly accumulated beneath the posterior lens capsule, while the anterior portion of the implanted lenses became an epithelial structure without cell elongation. These results suggest that the growth of the implanted lens may be dependent on the retina of the adult eye.