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.     

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.     

Stimulation of lens regeneration from the newt dorsal iris when implanted into the blastema of the regenerating limb

Dorsal iris from the eyes of adult Notophthalmus viridescens was transplanted into the blastema of regenerating limbs, subcutaneously in the limb or shoulder region, into the dorsal fin of larval newts and into the hindbrain of larval Ambystoma maculatum. The iris implants into the blastema regenerated lens vesicles or lenses with fibers in 40–75% of the cases. Multiple lenses were found in a few instances. No lenses developed from iris implants into the dorsal fin. Twenty percent of subcutaneous implants of iris formed lenses or lens vesicles, but lens regeneration from implants into the brain occurred only rarely. Denervation of the limb at the time of iris transplantation into the blastema greatly reduced the number of lenses regenerated. Studies on nerve fiber distribution in dorsal fin, subcutaneous areas, and denervated and innervated regenerating limbs, using the Bodian method, showed a general correlation between density of nerve fibers in the implant site and the incidence of lens regeneration from iris implants into that site. These results provide some evidence for a trophic action of nerve fibers on lens regeneration from the iris.     

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.     

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.     

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.     

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.     

The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.     

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.     

Developmental analysis of ocular morphogenesis in alpha A-crystallin/diphtheria toxin transgenic mice undergoing ablation of the lens.

The role of the lens in early eye development was examined in transgenic mice carrying the cytotoxic diphtheria toxin A gene driven by hamster alpha A-crystallin promoter sequences. Mice hemizygous for this construct are microphthalmic and contain a vacuolated and highly disorganized lens, whereas adult homozygous mice are completely ablated of the lens and lack a pupil, aqueous and posterior chamber, vitreous humor, iris, and ciliary body and show extensive convolution of the sensory retina. Developmental analysis of animals homozygous for the transgene revealed that the optic cup and lens vesicle form normally and that ablation of the lens occurs as a gradual degenerative process beginning between Days 12 and 13 of gestation. Degeneration of the lens vesicle coincides with retarded growth and development of the neuroretina, sclera, and cornea. The anterior lip of the optic cup fails to differentiate into the normal epithelium of the iris and ciliary body and the vitreous body does not develop. Although the retinal layers apparently form normally, retinal folding becomes prominent following lens degeneration. These results suggest that development of a functional lens from Embryonic Day 12.5 onward is critical for formation of the ciliary epithelium, iris, and vitreous body, as well as for appropriate growth, development, and maintenance of morphology of the retina, cornea, sclera, and optic nerve. Our results also provide information on the time course of DT-A-mediated cell destruction in vivo and are discussed in context with previous lens ablation studies and the importance of developmental analysis for interpretation of the extent to which morphogenetic aberrations are concurrent with or secondary to genetic ablation of the target tissue.     

A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens

The anterior segment of the vertebrate eye includes the cornea, iris, ciliary body, trabecular meshwork, and lens. Although malformations of these structures have been implicated in many human eye diseases, little is known about the molecular mechanisms that control their development. To identify genes involved in anterior segment formation, we developed a large-scale in situ hybridization screen and examined the spatial and temporal expression of over 1000 genes during eye development. This screen identified 62 genes with distinct expression patterns in specific eye structures, including several expressed in novel patterns in the anterior segment. Using these genes as developmental markers, we tested for the presence of inductive signals that control the differentiation of anterior segment tissues. Organ culture recombination experiments showed that a chick lens is capable of inducing the expression of markers of the presumptive iris and ciliary body in the developing mouse neural retina. The inducing activity from the lens acts only over short ranges and is present at multiple stages of eye development. These studies provide molecular evidence that an evolutionarily conserved signal from the lens controls tissue specification in the developing optic cup.     

Secreted frizzled-related protein disrupts PCP in eye lens fiber cells that have polarised primary cilia

Planar cell polarity (PCP) signaling polarises cells along tissue axes. Although pathways involved are becoming better understood, outstanding issues include; (i) existence/identity of cues that orchestrate global polarisation in tissues, and (ii) the generality of the link between polarisation of primary cilia and asymmetric localisation of PCP proteins. Mammalian lenses are mainly comprised of epithelial-derived fiber cells. Concentrically arranged fibers are precisely aligned as they elongate along the anterior-posterior axis and orientate towards lens poles where they meet fibers from other segments to form characteristic sutures. We show that lens exhibits PCP, with each fiber cell having an apically situated cilium and in most cases this is polarised towards the anterior pole. Frizzled and other PCP proteins are also asymmetrically localised along the equatorial-anterior axis. Mutations in core PCP genes Van Gogh-like 2 and Celsr1 perturb oriented fiber alignment and suture formation. Suppression of the PCP pathway by overexpressing Sfrp2 shows that whilst local groups of fibers are often similarly oriented, they lack global orientation; consequently when local groups of fibers with different orientations meet they form multiple, small, ectopic suture-like configurations. This indicates that this extracellular inhibitor disrupts a global polarising signal that utilises a PCP-mediated mechanism to coordinate the global alignment and orientation of fibers to lens poles.     

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.     

Nerve growth factor-induced stimulation of dorsal root ganglion/spinal cord co-grafts in oculo: enhanced survival and growth of CGRP-immunoreactive sensory neurons

Intraocular co-grafts of rat fetal spinal cord and dorsal root ganglia were used to examine the enhanced survival, growth, and differentiation of sensory neurons by nerve growth factor. E14 lumbar spinal segments were implanted into the anterior eye chamber of capsaicin-pretreated rats. Two weeks later, an E14 dorsal root ganglion was implanted beside the spinal cord graft. Nerve growth factor or vehicle was injected weekly for 4 weeks into the anterior eye chamber. Co-grafts were examined weekly and, at 6 weeks, processed for calcitonin gene-related peptide (CGRP) immunofluorescence. No differences in overall size were determined for the grafts. Co-grafts treated with nerve growth factor contained many more CGRP neurons (19.4 cells/20 microm) that were significantly larger (mean 764 microm2) than neurons from control co-grafts (8.6 cells/20 microm; mean 373 microm2). In co-grafts treated with nerve growth factor, CGRP-immunoreactive fibers were extensive in the dorsal root ganglion, adjacent iris, and spinal cord compared to control co-grafts. A few CGRP-positive motoneurons were observed in the spinal cord, but no differences in number or size of motoneurons were found. The current report demonstrates that spinal cord and dorsal root ganglia can be co-grafted in oculo for long periods of time. Many dorsal root ganglion neurons survive and send peripheral processes into the iris and central processes into the spinal cord under the influence of exogenous nerve growth factor. The intraocular graft paradigm can be of use to further examine the role of neurotrophic factors in regulating or modulating dorsal root ganglion and spinal cord neurons.     

Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus

Dorsal or ventral blastomeres of the 16- and 32-cell stage animal hemisphere were labeled with a lineage dye and transplanted into the position of a ventral, vegetal midline blastomere. The donor blastomeres normally give rise to substantial amounts of head structures and central nervous system, whereas the blastomere which they replaced normally gives rise to trunk mesoderm and endoderm. The clones derived from the transplanted ventral blastomeres were found in tissues appropriate for their new position, whereas those derived from the transplanted dorsal blastomeres were found in tissues appropriate for their original position. The transplanted dorsal clones usually migrated into the host's primary axis (D1.1, 92%; D1.1.1, 69%; D1.1.2, 100%), and in many cases they also induced and populated a secondary axis (D1.1, 43%; D1.1.1, 67%; D1.1.2, 63%). Bilateral deletion of the dorsal blastomeres resulted in partial deficits of dorsal axial structures in the majority of cases, whereas deletions of ventral midline blastomeres did not. When the dorsal blastomeres were cultured as explants they elongated. Notochord and cement glands frequently differentiated in these explants. These studies show that the progeny of the dorsal, midline, animal blastomeres: (1) follow their normal lineage program to populate dorsal axial structures after the blastomere is transplanted to the opposite pole of the embryo; (2) induce and contribute to a secondary axis from their transplanted position in many embryos; (3) are important for the normal formation of the entire length of the dorsal axis; and (4) autonomously differentiate in the absence of exogenous growth factor signals. These data indicate that by the 16-cell stage, these blastomeres have received instructions regarding their fate, and they are intrinsically capable of carrying out some of their developmental program.     

The comparative characteristics of the effect of retinoic acid on the regeneration of the crystalline lens and the extremity in adult tritons]

Effect of retinoic acid (RA) on morphogenesis and proliferation of regenerating extremity and lens cells was studied using 3H-thymidine autoradiography and morphometry. The 3H-thymidine incorporation into the inner layer of dorsal and ventral iris was 1.5-3 times reduced by the 8th day following the RA administration. The applied RA dose (0.25 mg per animal) exerted no significant effect on the morphogenesis of regenerating lens with the exception of the case of forming an additional lens from dorsal iris. The RA effect on the regeneration of extremity corresponds to available data of literature and manifests itself in the decelerated regeneration and the appearance of additional structures along the proximodistal axis.     

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.