β- and γ-Crystallin Synthesis in Rat Lens Epithelium Explanted with Neural Retina

Culturing the dorsal iris epithelium of a newt with a pituitary gland in organ culture greatly enhances the ability of the iris epithelium to produce advanced lens regenerates in vitro. In an attempt to elucidate the mechanism by which the pituitary enhances lens regeneration irido-corneal complexes from adult newts were cultured in medium to which various substances had been added either singly or in numerous combinations. Prolactin, insulin, hydrocortisone, and thyroxine failed to enhance the production of advanced lens regenerates in any of the doses or combinations tested. Similarly, addition of 50 μg/ml of sodium or calcium ascorbate had no effect on the progress of lens regeneration in vitro. Addition of dibutyryl cyclic-AMP caused an inhibition of depigmentation and regeneration at high doses. The results of these experiments show that the effects of the pituitary cannot be duplicated by hormones which other authors have asserted to be beneficial to limb or tail regenerates in vitro. The results with cyclic AMP suggest that prolonged exposure to high doses of cyclic AMP inhibit regeneration and indicate that further studies on the fluctations in cyclic AMP levels throughout the process of lens regeneration must be done.     

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.     

Influence of the pituitary on Wolffian lens regeneration

Removal of the pituitary 3 days before lentectomy retards Wolffian lens regeneration in the adult newt, Notophthalmus viridescens, by two stages over a 21-day period. Hypophysectomy 5 or 10 days after lentectomy does not alter the progress of regeneration during the subsequent 10-day period. Hypophysectomy 3 days before lentectomy also significantly decreases the incorporation of [³H]thymidine by iris epithelial nuclei 5 days after lentectomy but has no statistically significant effect on the incorporation 7 days after lentectomy.Pituitary tissue from newts or frogs enhances the regenerative activity of newt iris epithelial cells in vitro and in many cases promotes lens fiber formation. To a lesser extent, other tissues, such as nerve ganglion, also enhance the production of lens fiber cells from iris epithelium in vitro, whereas muscle tissue does not; and under certain conditions iris epithelial cells were found to depigment and redifferentiate into lens cells in the absence of other tissues in vitro.     

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)     

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.     

An increase of pituitary 3', 5' cyclic adenosine monophosphate produced by estradiol benzoate in vitro: possible implication of this increase in the secretion of luteinizing hormone.

In an attempt to study the site and mechanism of action of estrogen in producing positive feedback control, porcine anterior pituitary slices were incubated in vitro in the presence of estradiol benzoate (EB). EB elevated pituitary cyclic AMP concentration within 5 min and augmented pituitary release of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone (LH-RH) elevated pituitary cyclic AMP concentration and stimulated pituitary release of LH. The magnitude of increase of cyclic AMP and LH release was inversely related to the doses of LH-RH used. EB and LH-RH were additive in increasing cyclic AMP. Progesterone and clomiphene citrate interfered with an increase of pituitary cyclic AMP produced by EB, but did not significantly affect the basal level of pituitary cyclic AMP. Testosterone propionate, human chorionic gonadotropin and hexestrol were without effect on either basal or stimulated level of pituitary cyclic AMP. Since cyclic AMP and dibutyryl cyclic AMP (DBC) stimulated LH release, it is suggested that EB directly stimulates the release of LH by augmenting cyclic AMP synthesis in the anterior pituitary.     

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.     

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.     

Lens regeneration in axolotl: new evidence of developmental plasticity

Background

Among vertebrates lens regeneration is most pronounced in newts, which have the ability to regenerate the entire lens throughout their lives. Regeneration occurs from the dorsal iris by transdifferentiation of the pigment epithelial cells. Interestingly, the ventral iris never contributes to regeneration. Frogs have limited lens regeneration capacity elicited from the cornea during pre-metamorphic stages. The axolotl is another salamander which, like the newt, regenerates its limbs or its tail with the spinal cord, but up until now all reports have shown that it does not regenerate the lens.

Results

Here we present a detailed analysis during different stages of axolotl development, and we show that despite previous beliefs the axolotl does regenerate the lens, however, only during a limited time after hatching. We have found that starting at stage 44 (forelimb bud stage) lens regeneration is possible for nearly two weeks. Regeneration occurs from the iris but, in contrast to the newt, regeneration can be elicited from either the dorsal or the ventral iris and, occasionally, even from both in the same eye. Similar studies in the zebra fish concluded that lens regeneration is not possible.

Conclusions

Regeneration of the lens is possible in the axolotl, but differs from both frogs and newts. Thus the axolotl iris provides a novel and more plastic strategy for lens regeneration.

     

Macrophage mobilization and morphology during lens regeneration from the iris epithelium in newts: studies with correlated scanning and transmission electron microscopy

The lens was removed from both eyes of adult newts (Notophthalmus viridescens), and the eyes were fixed in Karnovsky's fixative every 2 days 0-20 days after operation. Anterior half-eyes were prepared by standard procedures for scanning electron microscopy of the surface. Before fixation, the posterior iris surface was cleaned of adhering vitreous mechanically with forceps or by treatment with bovine testicular hyaluronidase or with hyaluronidase and collagenase. Some specimens were cryofractured in buffer or ethanol transverse to the mid-dorsal iris, and the fractured surface viewed with scanning electron microscopy (SEM). Cells with various combinations of ridges, blebs, filopodia, and lamellipodia were observed adhering to the posterior surface of the iris by 6 days after lentectomy. These cells, which exhibited the surface characteristics of macrophages, became more numerous in specimens fixed after longer intervals. Invasion of the iris epithelium was observed in a cryofractured specimen. After observations with SEM, selected specimens were embedded in plastic and sectioned for study with transmission electron microscopy (TEM). The cells on the iris surface had the cytological characteristics of macrophages, and other macrophages were located within the iris epithelium. In specimens fixed 16 or more days after lentectomy, a bulging lens vesicle was regenerating from the dorsal pupillary margin of the iris. Macrophages were absent or few on the surface of this developing lens but remained scattered over the adjoining iris. Roles that might be played by these macrophages during the transdifferentiation of iris epithelium into lens are discussed.     

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.     

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.     

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%.     

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.     

Effect of bovine pituitary hormone preparations on newt lens regeneration in vitro: stimulation by thyrotropin

The anterior lobe of the pituitary gland can stimulate lens regeneration from the dorsal iris in the newt Notophthalmus viridescens. We have studied the effect of pituitary hormone preparations on this process. Dorsal irises were cultured for 20 days in diluted Medium 199 supplemented with 10% fetal calf serum. Bovine thyrotropin TSH-B8 at concentrations of 30 to 3000 μg/ml significantly stimulated lens regeneration in these dorsal irises. Well-developed lenses, up to stage 9, were formed, in which γ-crystallin, a protein specific for lens fibers of young lenses, was detected by immunofluorescence. Additionally, the mitotic index was 5.5 times elevated in these explants when compared to their controls. Lutropin LH-B10 at concentrations of 30 to 3000 μg/ml, prolactin PRL-B4 at concentrations of 23 to 1600 μg/ml, and porcine adrenocorticotropin ACTH-6002 at concentrations of 3 to 300 μg/ml did not stimulate lens regeneration. A weak stimulation of lens formation was observed in iris cultures with 2700 μg/ml of follitropin FSH-B1 or 3000 μg/ml somatotropin GH-B18, but not at concentrations of 30 μg/ml. Our results suggest that the inherent ability of the dorsal iris to form lens can be activated by the bovine thyrotropin preparation TSH-B8.     

Glucosaminidase and Dedifferentiation of Newt Iris Epithelium

To try to understand the mechanism of the dedifferentiation process which occurs during metaplastic transformation of iris epithelial cells into lens cells in newt lens regeneration, the activity of N -acetylglucosaminidase in iris and iris epithelium was studied as a function of time after lentectomy. The activity was found to increase during the dedifferentiation phase of the iris epithelium. The dorsal iris, where definite dedifferentiation occurs side by side with incomplete dedifferentiation, shows significantly greater enhancement of the activity than the ventral iris, where only incomplete dedifferentiation takes place. When the cells complete dedifferentiation and engage in redifferentiation into lens cells, the level of activity drops, approaching that of the normal lens. Evidence is also presented for release of the enzyme into the ocular fluid during dedifferentiation. The possibility that the enzyme is involved in surface alterations of iris epithelial ceils engaged in dedifferentiation is discussed.     

Determinative roles of FGF and Wnt signals in iris-derived lens regeneration in newt eye

Total regeneration of experimentally excised lens from the dorsal part of the iris-pigmented epithelium of newts has been a key model of tissue regeneration via cells originating from a foreign tissue. Due to the strict spatial restriction of the lens origin in the newt iris, it has often been assumed that only the dorsal iris cells are endowed with an intrinsic potential to give rise to lens tissues. However, our reinvestigation of the process revealed completely different mechanisms underlying lens regeneration and its spatial restriction, comprising the following two steps: (i) Fibroblast growth factor (FGF) 2-dependent proliferation of iris-pigmented epithelium and activation of early lens genes ( Pax6, Sox2, MafB ) over the entire circumference of the iris; and (ii) dorsal iris-restricted activation of the canonical Wnt signals (involving Wnt2b and Frizzeld4) that leads to localized expression of late lens genes ( Prox1, Sox1, β-crystallin ). Injection of FGF2 into normal eyes specifically elicited the second lens development from the dorsal iris, and the administration of recombinant Wnt3a to the cultured iris-pigmented epithelium caused even ventral iris-derived lens development. Thus, it is concluded that the regulation of FGF2 and Wnt signals is a determinative of the iris-derived lens regeneration in the newt eye.     

Cellular Studies of X-Ray Induced Inhibition of Lens Regeneration

Whole-body X-irradiation of adult newts 0 to 3 days after lentectomy inhibits transformation of the dorsal iris epithelium into a lens in all cases. The first question raised was whether irradiation affects infiltration of the iris area by macrophages, and the phagocytic activities of these cell types in the iris epithelium (prominent phenomena in this system). The number of macrophages infiltrating into the iris epithelium, and their phagocytic activities (indicated by uptake of melanosomes) were not affected by irradiation under those conditions. The second group of experiments concerns the possible effects of irradiation on DNA replication of iris epithelial cells, which become transformed into lens cells in the non-irradiated system. Autoradiographic studies of iris epithelial cells in vivo revealed a significant suppressive effect of irradiation on the frequencies of cells incorporating ³H-thymidine 7 and 14 days after lentectomy. When autoradiography was applied to the primary pure culture of iris epithelial cells at different time intervals after the start of culture and irradiation in vitro , significant and persistent reduction of cell labelling due to irradiation, was demonstrated. Multiplication of spread cells in the iris epithelial culture was strongly and persistently inhibited throughout a period of 2 months. Inhibition of cell labelling and of cell multiplication was always accompanied by reduction in the extent of de-pigmentation of iris epithelial cells. De-pigmentation is one of the requirements for the cells become transformed into lens cells. The possible mechanism of radiation-induced inhibition of lens regeneration 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.