Lens Regeneration Research Articles

The antitumor drug 3-nitrobenzothiazolo(3,2-a)quinolinium chloride (NBQ): effects on lens regeneration and interaction with DNA of Notophthalmus viridescens

Abstract We have studied the effect of 3-nitrobenzothiazolo(3,2-a)quinolinium (NBQ) on the regeneration of the lens in adult newt Notophthalmus viridescens. NBQ has marked cytotoxic effects in tumor cells, intercalates DNA, and was found to enhance lens regeneration. Newt liver DNA was isolated, and the thermal denaturation temperature (T m ) determined to be 76.6%±0.8%. The G–C content was determined to be 44.0%±0.4% and 45.0%±0.1%. Parameters of NBQ binding to newt DNA were determined by spectrophotometric methods and compared with those obtained for calf thymus and Micrococcus lysodeikticus. The association constant, K(o), was found to be 1.1 × 10 +5 M −1 with a site-size parameter, n, of 8.7 nucleotides. No explanation is apparent for the paradoxical stimulation of lens regeneration.

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 (nonlens-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 35–40. 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.

Effect of optic nerve denervation on lens regeneration in adult newts, Triturus viridescens.

This experiment was designed to study the effect of optic nerve denervation on lens regeneration. Adult newts, Triturus viridescens, were divided into 4 groups: A) lentectomized newts, which served as controls, B) lentectomized newts with the optic nerve exposed and lightly palpated, C) newts with lens removed and the optic nerve cut simultaneously, and D) newts with lens removed first and optic nerve cut 7 days and 14 days later. Control animals required about 21 days to produce a new lens, and animals in group B demonstrated the same regeneration rate. In groups C and D, the regeneration rate was greatly retarded during the first 14 days. However, most of these animals regenerated a perfect new lens by 21 days postlentectomy.

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)

Lens regeneration from cultured newt irises stimulated by retina-derived growth factors (EDGFs)

It has been shown that lens regeneration from the iris of the newt Notophthalmus viridescens is dependent on the presence of neural retinal tissue in organ culture and in vivo. The recent discovery of various eye-derived growth factors (EDGFs) in the bovine retina [14] prompted us to investigte whether one of these factors may be involved in the stimulation of lens regeneration. Dorsal irises were cultured for 20 days in serum-supplemented diluted Eagle's medium. Growth factors from bovine retina of various degrees of purification were added. Lens regeneration was assessed on the basis of morphological lens-regeneration stages and by immunofluorescent detection of a lensspecific marker protein, α-crystallin. Crude isotonic retinal extract at 80–800 μg/ml significantly augmented lens regeneration. Very similar results were obtained when EDGF III, the nonretained retinal factor after heparin-affinity chromatography, was present at 2–20 μg/ml. Lens regeneration was also significantly increased when EDGF II, the retinal form of acidic fibroblast growth factor (aFGF) at 50–500 ng/ml was added to the cultures. On the other hand, EDGF I at 4–40 ng/ml and brain basic FGF at 5–50 ng/ml did not seem to significantly stimulate lens regeneration under the conditions used. Our results suggest that at least two retina-derived growth factors (EDGF II and III) can stimulate lens regeneration. These growth factors may be the putative signal that is naturally produced by the retina during lens regeneration in the newt.

Chapter 2 Instability in Cell Commitment of Vertebrate Pigmented Epithelial Cells and their Transdifferentiation into Lens Cells

Publisher Summary Although the cell type conversion of pigmented epithelial cells (PECs) can be observed in the process of Wolffian lens regeneration and of neural retina regeneration in a limited number of species, the results of cell culture works since 1973 permit to conclude that the population of PECs capable of transdifferentiation is widely conserved in vertebrates including humans, at least in the embryonic period. However, this multipotential property is well stabilized in physiological conditions in situ . The finding that close cell–cell contact may be one of the requisites for the stabilization of PECs is revealed by observations of a cohesive epithelial cell sheet supported by a continuous collagen substrate. This chapter establishes a culture system in which a pure population of dedifferentiated cells (dePECs) can be maintained by introducing phenylthiourea (PTU), hyaluronidase (HUase), and dialyzed fetal bovine serum in an appropriate way. With this system, the cellular properties of dePECs can be investigated in detail. Preliminary efforts to elucidate the mechanisms of transdifferentiation in terms of the regulation of gene expression have been started. It has been demonstrated that δ-crystallin mature mRNA can be detected in some nonlenticular tissues, such as pigment epithelium, neural retina, and others, in early chick embryos in situ . To further understand the multipotent dedifferentiated state of PECs at the level of gene expression, it is necessary to extend investigation to the transcription of genes that are specifically expressed in the melanogenic cell and to determine whether the transcripts are also present in dePECs.

Comparison of newt lens regeneration stimulating activity in preparations of mammalian thyrotropin and fibroblast growth factor purified by various methods

Irises of the newt Notophthalmus viridescens will regenerate a new lens in organ culture in the presence of the bovine thyrotropin preparation NIH-TSH-B8. It is not certain, however, whether thyrotropin itself is responsible for this stimulatory effect. To elucidate this problem further we compared the lens regeneration stimulating activity of thyrotropin preparations from several species, prepared by various methods. The lowest effective concentrations were approximately 3·0 μg ml −1 for the bovine NIH-TSH-B8 and 1·4 μg ml −1 for the ovine NIAMDD-oTSH-9 preparations. At those lowest concentrations. lenses with elongating lens fiber cells (stage 6) and enlarged lens fiber core (stage 8) were obtained, respectively, and the lens-fiber-specific protein γ-crystallin was present in both cases. The crude bovine thyrotropin fraction. Sigma-TS-10. stimulated lens regeneration only at the highest concentration, 1400 μg ml −1. Bovine Pierce-bTSH, the purest thyrotropin preparation, stimulated lens regeneration sporadically at the lower concentration of 0·04 μg ml −1 up to the advanced stage 9 with large lens fiber core and flattened lens epithelium in one of nineteen irises. The pituitary fibroblast growth factor is a known contaminant of thyrotropin preparations. The preparation CR-FGF-40002 at concentrations between 0·001 and 0·1 μg ml −1 did not promote lens regeneration. Therefore, the lens regeneration stimulating activity in thyrotropin preparations is not attributable to the fibroblast growth factor. and may also be independent of thyrotropin because the lens regeneration stimulating activity is not proportional to the thyrotropic activity in the preparations examined.

Conversion of iris epithelial cells as a model of differentiation control

It has been shown that, upon lentectomy or in culture, iris epithelial cells (IECs) of adult newts become converted into lens cells, and this conversion is the basic event of lens regeneration in newts. Whether in situ or in cell culture, the conversion requires the passage of a specific number of cell cycles. The progeny of IECs which fails to traverse this cell-cycle number redifferentiates as IECs in situ. The passage through cell cycles of IECs is associated with progressive alterations of cytoplasm and cell surface, during which the original state of differentiation disappears (dedifferentiation). It is speculated that the altered state of cells caused by proliferation leads to the appearance of factors which interact with the genome and switch the gene activation pattern to that of the lens cell. In this model, developmental controls are geared to the cell-cycle progression and not directly to the activation of lens-characteristic genes. A number of points are raised which speak against the long-held idea that a factor from neural retina induces lens differentiation in IECs. It is proposed that the retinal factor plays the role of growth factor which is essential in the conversion in situ, but not required in the conversion in cell culture. The proposed model is compared with reprogramming of differentiation of some cell lines by cytidine analogs and with ontogenic systems of differentiation control.

Retinal Pigment Epithelium: Proliferation and Differentiation during Development and Regeneration

Publisher Summary This chapter describes the proliferation and differentiation of retinal pigment epithelium (RPE) during development and regeneration. RPE is a pigmented monolayer of hexagonal cells of neural origin located between the neural retina and the choroid. It is one of the most important structures in the visual system because of the role it plays in the viability and function of the neural retina. The comparison of RPE development in three species (rats, chicks, and humans) showed that the pattern and schedule of RPE growth and differentiation causally related to cell proliferation are significantly different. The chapter discusses the proliferation of pigment epithelium cells in the amphibian retina and iris in the process of retinal and lens regeneration. The importance of analyzing the proliferative activity of pigmented epithelia of the amphibian eye is because of the amphibian's striking potential for the regeneration of the neural retina and lens. Cell proliferation is not only closely related to cell multiplication and the reduction of cell volume but is also essential for RPE melanotic differentiation and for transdifferentiation into other cell types.

Synthesis of Sulfated Glycosaminoglycan in Newt Iris during Lens Regeneration

Lens regeneration in newts involves the dedifferentiation of pigmented iris epithelial cells and their conversion into lens fibers at the dorsal pupillary margin. We have investigated the pattern of incorporation of 35SO 4 into sulfated glycosaminoglycan (GAG) within the iris during this process. Newts were injected with [ 35S]H 2SO 4 at varying intervals after lens removal. Iris and lens tissues were excised and the accumulation of label into total sulfated GAG was measured from the radioactivity incorporated in the ethanol-insoluble, cetylpyridinium chloride-precipitable fractions of pooled tissue homogenates. The proportions of label in GAG of the chondroitin sulfate, dermatan sulfate, and heparin/heparan sulfate types were determined from the reduction of label after chondroitinase AC, chondroitinase ABC, and nitrous acid treatments. Compared with the normal iris of the unoperated newt, uptake of label into sulfated GAG was increased dramatically after lens removal. Labelling rose to a peak at 15 days after lentectomy and declined slightly from its maximum by day 30. At all times heparin/heparan sulfate accounted for approximately 60% of total label uptake while chondroitin sulfate and dermatan sulfate each contributed 15–25%. Incorporation of 35SO 4 was nearly equivalent in dorsal (lens-forming) and ventral (non-lens-forming) halves of the iris at all stages examined. Only negligible quantities of label were associated with either mature (normal) or developing (30 day) lenses. It is concluded that lentectomy stimulates an elevation in the total rate of incorporation of 35SO 4 into newly synthesized sulfated GAG within the newt iris, while preserving the relative labelling rates of the different types of GAG produced. We suggest that an enhanced synthesis of GAG may be associated with the dedifferentiation of iris epithelial cells following lentectomy and the retrieval pathway of redifferentiation into pigment cells.

Lens regeneration from the dorsal iris in Eurycea bislineata, the two-lined salamander.

Following lens removal from the eye of adultEurycea bislineata, the northern (USA) 2-lined salamander, it was found that this salamander has the capacity for lens regeneration. Its widespread distribution and high percentage of regenerative success suggests it as a suitable organism for the study of this differentiative phenomenon.

α-, β- and γ- Crystallins in the regenerating lens of Notophthalmus viridescens

Removal of the lens from the eye of adult Notophthalmus viridescens, the Eastern spotted newt, is followed by regeneration of another lens from the dorsal iris. This cell-type conversion of iris epithelial cells into lens cells is accompanied by the subsequent synthesis of α-, β- and γ-crystallins, proteins specific for the normal vertebrate lens. The immunofluorescence technique was employed to determine the ontogeny and localization of the crystallin classes in the regenerating lens rudiments. Antibodies specific for α-, β- and γ-crystallins were applied to tissue sections through regenerate Stages III–XI Yamada 1967. The first positive reaction for α-crystallins did not occur until Stage VII, in a few cells of the developing lens fiber region; and in the external layer (presumptive lens epithelium) at Stage VIII, at which time secondary lens fibers have begun to form. β-Crystallins were first detectable in the thickening internal layer of the Stage V lens (vesicle) and in the external layer at Stage VIII. The γ-crystallins also first appeared, albeit erratically, in a few cells of the internal layer of the Stage V regenerate, and were not detectable in the external layer/lens epithelium at any time. Thus the crystallins are not present in the earliest stages, but are indicative of cellular differentiation in succeeding stages of lens regeneration.

Inhibition of lens regeneration in larval Xenopus laevis

The present research aims at showing the role played by the lens in inhibiting lens-forming transformations of the outer cornea of Xenopus laevis tadpoles. Two types of experiment were carried out: 1) lentectomy and insertion of a Millipore filter disk at the side of the lens, and 2) lentectomy and insertion of a Millipore disk with a central hole. Results indicate that when the lens is replaced by a mechanical obstacle capable of preventing direct communication between vitreous and anterior chambers, definite inhibition of the lens-forming transformations of the outer cornea occurs. These data strongly suggest that the inhibition exerted by the lens on lens-forming transformations of the outer cornea in Xenopus laevis eyes is mechanical rather than chemical in nature.

Dedifferentiation of iris epithelium during lens regeneration in newt larvae.

Early stages in lens regeneration from the pigmented epithelium of the dorsal iris were studied in larval Notophthalmus viridescens by means of transmission electron microscopy. Normal iris epithelium is composed of two layers of low cuboidal cells. packed with melanosomes and surrounded by a basal lamina. Scattered desmosomes attach adjacent cells. Following lens removal, the intercellular spaces enlarge and the epithelial cells increase in size. Some irregular microvilli from these cells extent into the intercellular spaces. Macrophages invade the iris epithelium and phagocytize melanosomes discharged from the pigmented cells. These invading macrophages have numerous microprojections and are often separated from the surface by a very thin layer of iris epithelial cell cytoplasm. In the iris cells, nucleoli become more prominent and granular, polyribosomes increase greatly in number, melanosomes gradually disappear, mitochondria become more numerous, and mitotic activity is greatly augmented. Fine cell processes of adjacent interdigitate near the external surface, where numerous micropinocytotic vesicles can be seen. Over the external surface, the basal lamina may be disrupted or duplicated in places where pseudopodia project from iris cells or a macrophage has entered an intercellular space. It is lacking on the lumenal surface. Sloughed membranes are often found in these intercellular spaces.

Lens regeneration from the pupillary margin of the eyecup in newt embryos.

Early stages in lens regeneration from the dorsal margin of the pupil, following removal of the young lens from the embryonic eyecup, were studied with transmission electron microscopy in Notophthalmus viridescens. At the stage of operation, the eyecup cells have an undifferentiated, embryonic appearance with numerous free ribosomes and scattered mitochondria. In normal embryonic eyes containing a lens, the iris epithelium differentiates from the edge of the optic cup by growth and flattening of the cells to form a two-layered cuboidal epithelium. There is extensive melanogenesis in both inner and outer layers. In lentectomized eyes, this flattening does not occur and melanogenesis takes place mainly in the outer layer of epithelium. Mitoses are abundant and a vesicle of unpigmented columnar cells, enclosed by a basal lamina, develops from the dorsal pupillary margin of the eyecup. Macrophages are present and phagocytize some of the melanin but less than during dedifferentiation of pigmented iris epithelium. During this regeneration of a lens vesicle, there is an increase in the number of polyribosomes, granular endoplasmic reticulum, and mitochondria in preparation for synthesis of the lens proteins. Micropinocytosis occurs at the cells surfaces.

Secondary lens formation from the cornea following implantation of larval tissues between the inner and outer corneas of Xenopus laevis tadpoles

Secondary lens formation from the cornea of larval Xenopus laevis has been used as a measure of the lens-inducing capacities of various larval Xenopus tissues. The experimental design employed involved implantation of selected body tissues between the inner and outer cornea of stage-50 tadpole eyes, in such a way that the integrity of the inner cornea and eye cup was not disrupted. Implantation of retina, pituitary, limb blastema or limb bud resulted in secondary lens formation from the outer cornea. Such lenses were similar in appearance to stage-5 lens regenerates described by Freeman (1963). No secondary lenses were observed in eyes receiving either heart or hind brain implants or in eyes which underwent corneal separation but which received no implant. It is concluded that the retina is the natural source of a stimulatory factor which initiates and maintains corneal transformation to lens during lens regeneration following lensectomy. Influences emanating from pituitary, limb blastema and limb bud, but apparently not from heart or hind brain, are able to act on cornea in a way similar to the retinal factor. Furthermore, our findings support the contention that in the normal eye, the inner cornea is a barrier to the passage of retinal factor and so maintains the single lens structure of the eye. When this barrier is by-passed by lens-inducing tissue, as in the present experimental design, lens formation from the cornea is able to take place. Electronmicroscopical studies have shown that the inner cornea, in the stage-50 tadpole eye, consists of a dense meshwork of collagen fibrils and a basal layer of cohesive elongated mesenchymal cells well suited for this barrier function.

Experimental analysis of the lens-forming competence of the cornea, iris, and retina in Xenopus laevis tadpoles.

The present research was carried out using Xenopus laevis tadpoles at stage 50-51 to ascertain whether eye territories other than the outer cornea and the pericorneal epidermis have lens-forming competence, particularly the iris and/or retina. Five experiments were performed: simple lentectomy; removal of lens, outer cornea, and pericorneal integument; removal of lens, outer cornea, and pericorneal integument, and simultaneous incision of retina; removal of lens, outer cornea and pericorneal integument, and simultaneous incision of dorsal iris; removal of lens and simultaneous lesion of iris and retina. Results show that when lentectomy is performed without damaging any territories other than the outer and inner cornea, lens regeneration is always from the outer cornea, and the iris and retina do not exhibit any lens-forming competence. The iris and retina show no clear-cut lens-forming competence even after they have been stimulated by comparatively extensive lesions.

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.

Intracellular Levels of Adenosine 3′:5′-Cyclic Monophosphate in the Dorsal Iris of the Adult Newt, During Lens Regeneration

The intracellular levels of adenosine 3':5'-cyclic monophosphate (cAMP) were measured in the dorsal iris of the adult newt, during the first 20 days of lens regeneration. It was found that by day 2 after lens removal there is a significant drop in the levels of cAMP. After day 2 the levels of the nucleotide increase and by day 3 they are higher than those detected on day 0. The levels of cAMP remain high up to day 8. From day 8 to day 9 there is a second drop. From day 9 to day 20 the levels of cAMP did not differ significantly from the value obtained for day 0, except for days 10, 12, and 15. The period of high levels of cAMP coincides with the period of depigmentation of iris epithelial cells, the key event of lens regeneration.