Regulated lens regeneration from isolated pigmented epithelial cells of newt iris in culture in response to FGF2/4

When a lens is removed from the newt eye, a new lens is regenerated from the pigmented epithelial cells of the dorsal iris, whereas the ventral iris never shows such an ability. It is important to clarify the nature of signaling molecules which act directly on the iris cells to accomplish lens regeneration from the iris and also to gain insight into the mechanism of dorso-ventral difference of the regeneration potential. To examine the effects of exogenous factors, we established an in vitro culture of reaggregates made from dissociated pigmented epithelial cells of dorsal or ventral halves of newt iris. Foci of depigmented cells appeared within the cell reaggregates, regardless of their origins, when the cell reaggregates were cultured with FGF2 or FGF4. In contrast, only the depigmented cells in the dorsal iris cell reaggregates underwent extensive proliferation and developed a lens with the synthesis of lens-specific crystallins, recapitulating the normal lens regeneration. On the other hand, neither FGF8, FGF10, EGF, VEGF, nor IGF promoted lens development from iris cell reaggregates. Consistent with the FGF-specific action, FGFR-specific inhibitor SU5402 suppressed the lens development from the cultured cell reaggregates. These results demonstrated that FGF2 or FGF4 is essential for the in vitro lens regeneration from the pigmented cells of the dorsal iris. In addition, these findings indicated that unequal competence in the dorsal and ventral iris to FGF2/4 contributes to the difference in lens forming ability between them.     

Analysis of a unique molecule responsible for regeneration and stabilization of differentiated state of tissue cells.

In Wolffian regeneration in the newt, a functional lens can be regenerated through cellular transdifferentiation of the pigmented epithelium of the mid-dorsal marginal iris. A novel monoclonal antibody, 2NI-36 mAb, generated in our laboratory has been utilized as a highly useful probe to study newt lens regeneration. The antigen molecule against this 2NI-36 mAb (2NI-36) became temporarily undetectable only at the site of lens regeneration. Moreover, the ventral iris pieces expressed the ability to differentiate a lens when pretreated with this monoclonal antibody and implanted in lentectomized eyes (Eguchi, Cell Differ. Dev. 25, Suppl., 1988). We have investigated the distribution of 2NI-36 in newt tissues. 2NI-36 was not specific to iris pigmented epithelium and distributed in many different kinds of mesodermal tissues, including dermis, blood vessel, mesonephros and so forth. 2NI-36 was also detected in either cell surface or intercellular spaces of cultured pigmented epithelial cells when they organized an epithelial cell sheet. Western blot analysis showed that 2NI-36 had the molecular weight of 50-200kD and was completely digested by trypsin, suggesting that 2NI-36 was a glycoprotein with many carbohydrate chains. It was also revealed by Western blot analysis that all the tissues in which 2NI-36 could be detected expressed this molecule similar to that in the iris epithelium. We expect that 2NI-36 is a glycoprotein expressed by various newt tissues and is functional to stabilize the differentiated state of each tissue cell in the same way as observed in the iris pigmented epithelial cells.     

In vitro analysis of cellular metaplasia from pigmented epithelial cells to lens phenotypes: a unique model system for studying cellular and molecular mechanisms of "transdifferentiation"

Pigmented epithelial cells (PECs) were dissociated from eyes of 8- to 9-day-old chick embryos and were cultured in EdF medium (Eagle's MEM supplemented with dialyzed fetal bovine serum) containing phenylthiourea (PTU) and testicular hyaluronidase (HUase). The PECs rapidly lost melanosomes as they proliferated and dedifferentiated in culture. These dedifferentiated PECs (dePECs) which did not manifest any identifiable specificity could be directed to one of two different differentiated phenotypes; viz., lens or pigment cells, depending upon subsequent culture conditions. Almost all dePECs began to synthesize melanin and redifferentiated to PECs by Day 10 of culture with EdF medium containing ascorbic acid (AsA). In contrast, the sister population of dePECs, when cultured at extremely high cell density with EdF medium containing PTU, HUase and AsA, synthesized delta-crystallin which is specific for lens. This transdifferentiation into lens cells occurred by Day 15 of culture. Using this culture system we are able to produce a homogeneous cell population with the potential for synchronous differentiation into either lens or pigment cell phenotype. The system is useful for studying mechanisms involved in cellular metaplasia.     

Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster

In Wolffian lens regeneration, lentectomized newt eye can produce a new lens from the dorsal marginal iris, but the ventral iris has never shown such capabilities. To investigate the difference of lens regenerating potency between dorsal and ventral iris epithelium at the cellular level, a transplantation system using cell reaggregates was developed. Two methods were devised for preparing the reaggregates from pigmented iris epithelial cells. One was rotating cells in an agar-coated multiplate on a gyratory shaker and the other was incubating cells in a microcentrifuge tube after slight centrifugation. Reaggregates made of dorsal iris cells that had been completely dissociated into single cells were phenotypically transformed into a lens when placed in the pupillary region of the lentectomized host eye. None of the ventral reaggregates produced a lens. Even dorsal reaggregates could not transdifferentiate into lens when they were placed away from the pupil. The produced lenses from the reaggregates were morphologically and immunohistochemically identified. To obtain evidence whether produced lenses really originated from singly dissociated cells, we labeled dissociated cells with a fluorescent dye (PKH26) before reaggregate formation and then traced it in the produced lens.     

Direct evidence for transformation of differentiated iris epithelial cells into lens cells

Although it is generally assumed that the lens regenerated in the newt eye after complete lentectomy is formed by cells derived from the dorsal iris epithelium, experimental evidence so far obtained for this transformation does not rule out participation of cells from the dorsal iris stroma. When the normal dorsal iris epithelium of adult Notophthalmus (Triturus) viridescens was isolated and cultured in the presence of frog retinal complex, newt lens tissue was produced in 88% of cultures. These lens tissues were positive for immunofluorescence for lens-fiber-specific gamma crystallins as well as for total lens protein. On the basis of a study of stromal cells contaminating the samples of dorsal iris epithelium and a test for the lens-forming capacity in vitro of the dorsal iris stroma in the presence of frog retinal complex, it is concluded that lens formation observed in the above experiment is not dependent on the contaminating stromal cells. This implies that, in Wolffian lens regeneration, fully differentiated adult cells completely withdrawn from the cell cycle are transformed into another cell type. An additional culture experiment demonstrated that lens-forming capacity is not restricted to the dorsal half of the iris epithelium, but extends into its ventral half.     

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.     

Determinative role of Wnt signals in dorsal iris-derived lens regeneration in newt eye

We have previously shown that lens regeneration from the pigmented epithelium of the dorsal iris in the adult newt eye proceeds in two steps after lens removal or intraocular FGF2 injection. The FGF2-dependent proliferation of iris pigmented epithelium and activation of early lens genes that occur over the entire circumference of the iris comprise the first step, while subsequent dorsally confined lens development marks the second step. Here, we investigated the expression of Wnt and Wnt receptor Frizzled genes in lens-regenerating iris tissues. Wnt2b and Frizzled4 were activated only in the dorsal half of the iris in synchrony with the occurrence of the second step, whereas Wnt5a and Frizzled2 were activated in both halves throughout the period of the first and second steps. Cultured explants of the iris-derived pigmented epithelium in the presence of FGF2 underwent dorsal-specific lens development fully recapitulating the in vivo lens regeneration process. Under these conditions, Wnt inhibitors Dkk1, which specifically inhibits the canonical signal pathway, and/or sFRP1 repressed the lens development, while exogenous Wnt3a, which generally activates the canonical pathway like Wnt2b, stimulated lens development from the dorsal iris epithelium and even caused lens development from the ventral iris epithelium, albeit at a reduced rate. Wnt5a did not elicit lens development from the ventral epithelium. These observations indicate that dorsal-specific activation of Wnt2b determines the dorsally limited development of lens from the iris pigmented epithelium.     

A critical role for thrombin in vertebrate lens regeneration

Lens regeneration in urodele amphibians such as the newt proceeds from the dorsal margin of the iris where pigment epithelial cells (PEC) re-enter the cell cycle and transdifferentiate into lens. A general problem in regeneration research is to understand how the events of tissue injury or removal are coupled to the activation of plasticity in residual differentiated cells or stem cells. Thrombin, a pivotal regulator of the injury response, has been implicated as a regulator of cell cycle re-entry in newt myotubes, and also in newt iris PEC. After removal of the lens, thrombin was activated on the dorsal margin for 5-7 days. Inactivation of thrombin by either of two different inhibitors essentially blocked S-phase re-entry by PEC at this location. The axolotl, a related species which can regenerate its limb but not its lens, can activate thrombin after amputation but not after lens removal. These data support the hypothesis that thrombin is a critical signal linking injury to regeneration, and offer a new perspective on the evolutionary and phylogenetic questions about regeneration.     

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.     

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%.     

Possible demonstration of multipotential nature of embryonic neural retina by clonal cell culture.

The possible multipotential nature of the neural retina of early chick embryos was examined by the technique of clonal cell culture. Cultures were prepared from cells dissociated from freshly excised neural retinas of 3.5-day-old chick embryos or from cells harvested from primary highdensity cultures. The following four colony types were obtained: colonies differentiating into “lentoid bodies”; colonies with pigment cells; colonies with both “lentoid bodies” and pigment cells; and colonies comprised entirely of unidentifiable cells. Neuronal differentiation occurred frequently in the early stages of culture (up to about 10 days). In some of these neuronal colonies, “lentoid bodies” and, rarely, both “lentoid bodies” and pigment cells differentiated after a further culture period of up to 30 days. Secondary colonies established from primary colonies after 9–10 days demonstrated that these original colonies fell into four different categories: those giving rise to secondary colonies containing only “lentoid bodies,” those giving rise to pigmented colonies only, those developing both lentoid and pigmented colonies, and finally those which gave rise to secondary colonies of all three types, lentoid, pigmented, and mixed colonies. When primary pigmented colonies were recloned at about 30 days after inoculation, the differentiated pigment cells transdifferentiated into lens. Whether multispecific colonies were really of clonal origin or not is discussed. The possible presence of a multipotent progenitor cell able to give rise to multispecific clones in the neural retina of 3.5-day-old chick embryos is suggested. A sequence of differentiation starting from multipotent neural retinal cells to be terminated with lens through the differentiation of neuronal and pigment cells is hypothetically proposed.     

The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells

A monoclonal antibody (MC/1) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that MC/1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomal matrix. A single protein band with an apparent molecular weight of 115,000 was labelled by MC/1 in Western blotting. The 115 kDa polypeptide identified by MC/1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable.     

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.     

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.     

Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration

The analysis of newt lens regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of lens regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into lens cells in newt lens regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene-transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in lens regeneration, Pax6, which is suggested to be involved in normal eye development and lens regeneration, was transfected. Ectopic expression of lens-specific crystallins was obtained in cells that show no such activity in normal lens regeneration. These results made it possible for the first time to analyze the molecular mechanism of lens regeneration in the adult newt.     

Lens formation by pigmented epithelial cell reaggregate from dorsal iris implanted into limb blastema in the adult newt

In newt lens regeneration, the dorsal iris has lens forming ability and the ventral iris has no such capability, whereas there is no difference in the morphological criteria. To investigate the real aspects of this characteristic lens regeneration in the newt at the cellular level, a useful model system was constructed by transplanting the dorsal and ventral reaggregate derived from singly dissociated pigmented epithelial cells of the iris into the blastema of the forelimb in the newt. The lens was formed from the dorsal reaggregate with high efficiency, but not from the ventral one. No lens formation was observed in the implantation of the reaggregate into the tissue of the intact limbs. In detailed examination of the process of lens formation from the reaggregate, it was shown that tubular formation was the first step in the rearrangement of cells within the reaggregate. This was followed by depigmentation, vesicle formation with active cell growth, and the final step was lens fiber formation by transdifferentiation of epithelial cells composing the lens vesicle. The process was almost the same as in situ lens regeneration except the reconstitution of the two-layered epithelial structure was embodied as flattened tubular formation in the first step. The present study made it possible for the first time to examine lens forming ability in the reaggregate mixed with dorsal and ventral cells, because the formation of a reaggregate was started from singly dissociated cells of the dorsal and ventral cells of the iris. Mixed reaggregate experiments indicated that the existence of the dorsal cells in a cluster within the reaggregate is important in lens formation, and ventral cells showed an inhibitory effect on the formation. The present study demonstrated that the limb system thus constructed was effective for the analysis of lens formation at the cellular level and made it possible to examine the role of dorsal and ventral cells in lens regeneration.     

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)     

In VitroCulture System for Iris-Pigmented Epithelial Cells for Molecular Analysis of Transdifferentiation

Dissociated cells of the iris-pigmented epithelium (IPE) from a 1-day-old chick grew in monolayer culture and stably maintained their differentiated state when cultured with standard culture medium. After replacement of the control medium by EdFPH medium, which is effective in inducing dedifferentiation of retinal pigmented epithelium (RPE) cells, all cells rapidly lost pigment granules, proliferated intensively, and dedifferentiated. By further addition of ascorbic acid, dedifferentiated cells accumulated and formed a large number of lentoids. This system provides a useful opportunity for analyzing cellular and molecular mechanism involved in each step of transdifferentiation. Furthermore, Northern blot data indicates that the up-regulation of pax-6 gene could be an important event during lens regeneration as well as during normal lens development.     

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.