Retina and lens regeneration in anuran amphibians.

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

Neurogenesis during optic tectum regeneration in Xenopus laevis.

The regenerative neurogenesis of the optic tectum of larval Xenopus laevis has been studied analyzing the proliferative and morphogenetic phases of the regeneration process after removal of one optic lobe. To this end, short-term and long-term pulses were carried out using the thymidine analog BrdU, selectively incorporated into cells during the S phase of the cell cycle. Results indicate that while in early larvae (stage 49/50, according to Nieuwkoop & Faber 1967) regeneration occurs mainly at the expense of the stem cells present in extensive proliferation zones ("matrix areas") of the midbrain, in late larvae (stage 55/56) regeneration occurs at the expense of stem cells present in very limited matrix areas of the brain and of quiescent cells, which re-enter the cell cycle following trauma. Moreover, in early larvae, morphogenesis of the optic tectum is carried out according to a precise spatio-temporal order from rostro-caudal to latero-medial. By contrast, in late larvae, the topographical order of the regenerative morphogenesis of the optic lobe is completely altered. As a consequence, the regenerated optic tectum in early larvae has an apparently normal structure, while the regenerated optic tectum in late larvae lacks stratification.

The optic vesicle promotes cornea to lens transdifferentiation in larval Xenopus laevis.

The outer cornea and pericorneal epidermis (lentogenic area) of larval Xenopus laevis are the only epidermal regions competent to regenerate a lens under the influence of the retinal inducer. However, the head epidermis of the lentogenic area can acquire the lens-regenerating competence following transplantation of an eye beneath it. In this paper we demonstrate that both the outer cornea and the head epidermis covering a transplanted eye are capable of responding not only to the retinal inducer of the larval eye but also to the inductive action of the embryonic optic vesicle by synthesizing crystallins. As the optic vesicle is a very weak lens inductor, which promotes crystallin synthesis only on the lens biased ectoderm of the embryo, these results indicate that the lens-forming competence in the outer cornea and epidermis of larval X. laevis corresponds to the persistence and acquisition of a condition similar to that of the embryonic biased ectoderm.

The lens-regenerating competence in the outer cornea and epidermis of larval Xenopus laevis is related to pax6 expression.

After lentectomy, larval Xenopus laevis can regenerate a new lens by transdifferentiation of the outer cornea and pericorneal epidermis (lentogenic area). This process is promoted by retinal factor(s) accumulated into the vitreous chamber. To understand the molecular basis of the lens-regenerating competence (i.e. the capacity to respond to the retinal factor forming a new lens) in the outer cornea and epidermis, we analysed the expression of otx2, pax6, sox3, pitx3, prox1, betaB1-cry (genes all involved in lens development) by Real-time RT-PCR in the cornea and epidermis fragments dissected from donor larvae. The same fragments were also implanted into the vitreous chamber of host larvae to ascertain their lens-regenerating competence using specific anti-lens antibodies. The results demonstrate that there is a tight correlation between lens-regenerating competence and pax6 expression. In fact, (1) pax6 is the only one of the aforesaid genes to be expressed in the lentogenic area; (2) pax6 expression is absent in head epidermis outside the lentogenic area and in flank epidermis, both incapable of transdifferentiating into lens after implantation into the vitreous chamber; (3) in larvae that have undergone eye transplantation under the head or flank epidermis, pax6 re-expression was observed only in the head epidermis covering the transplanted eye. This is consistent with the fact that only the head epidermis reacquires the lens-regenerating competence after eye transplantation, forming a lens following implantation into the vitreous chamber; and (4) in larvae that have undergone removal of the eye, the epidermis covering the orbit maintained pax6 expression. This is consistent with the fact that after the eye enucleation the lentogenic area maintains the lens-regenerating competence, giving rise to a lens after implantation into the vitreous chamber. Moreover, we observed that misexpression of pax6 is sufficient to promote the acquisition of the lens-regenerating competence in flank epidermis. In fact, flank epidermis fragments dissected from pax6 RNA injected embryos could form lenses when implanted into the vitreous chamber. The data indicate for the first time that pax6 is a pivotal factor of lens-regenerating competence in the outer cornea and epidermis of larval X. laevis.

Relationships between Presence of the Eye Cup and Maintenance of Lens-Forming Capacity in Larval Xenopus laevis.

The lentectomized eye of larval Xenopus laevis can regenerate a lens by a process of lens-transdifferentiation of the cornea and pericorneal epidermis. These tissues can form the lens only when they become in direct communication with the environment of the vitreous chamber (neural retina) indicating that the eye cup plays a fundamental role in this process. In this work the role of the eye cup in the maintainance of the lens-forming capacity of the cornea and pericorneal epidermis was studied by allowing these tissues to cover the enucleated orbit for different periods, and then implanting them into the vitreous chamber of the contralateral eye. Under these experimental conditions the maintainance of the lens-forming capacity of the cornea and pericorneal epidermis showed no significant correlation with the time from enucleation to implantation.

Experimental analysis of lens-forming capacity in Xenopus borealis larvae.

Previously, the only anuran amphibians known to have the capacity to regenerate a lens after lentectomy were Xenopus laevis and Xenopus tropicalis. This regeneration process occurs during the larval life through transdifferentiation of the outer cornea promoted by inductive factors produced by the retina and accumulated inside the vitreous chamber. However, the capacity of X. tropicalis to regenerate a lens is much lower than that of X. laevis. This study demonstrates that Xenopus borealis, a species more closely related to X. laevis than to X. tropicalis, is not able to regenerate a lens after lentectomy. Nevertheless, some morphological modifications corresponding to the first stages of lens regeneration in X. laevis were observed in the outer cornea of X. borealis. This suggested that in X borealis the regeneration process was blocked at early stages. Results from histological analysis of X. borealis and X. laevis lentectomized eyes and from implantation of outer cornea fragments into the vitreous and anterior chambers demonstrated that: (i) in X. borealis eye, the lens-forming competence in the outer cornea and inductive factors in the vitreous chamber are both present, (ii) no inhibiting factors are present in the anterior chamber, the environment where lens regeneration begins, (iii) the inability of X. borealis to regenerate a lens after lentectomy is due to an inhibiting action exerted by the inner cornea on the spreading of the retinal factor from the vitreous chamber towards the outer cornea. This mechanical inhibition is assured by two distinctive features of X. borealis eye in comparison with X. laevis eye: (i) a weaker and slower response to the retinal inducer by the outer cornea; (ii) a stronger and faster healing of the inner cornea. Unlike X. tropicalis and similar to X. laevis, in X. borealis the competence to respond to the retinal factor is not restricted to the corneal epithelium but also extends to the pericorneal epidermis.

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis.

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.

Lens-forming competence in the epidermis of Xenopus laevis during development.

In larval X. laevis the capacity to regenerate a lens under the influence of inductive factors present in the vitreous chamber is restricted to the outer cornea and pericorneal epidermis (Lentogenic Area, LA). However, in early embryos, the whole ectoderm is capable of responding to inductive factors of the larval eye forming lens cells. In a previous paper, Cannata et al. (2003) demonstrated that the persistence of lens-forming competence in the LA is the result of early signals causing lens-forming bias in the presumptive LA and of late signals from the eye causing cornea development. This paper analyzes 1) the decrease of the lens-forming capacity in ectodermal regions both near LA (head epidermis) and far from LA (flank epidermis) during development, 2) the capacity of the head epidermis and flank epidermis to respond to lens-competence promoting factors released by an eye transplanted below these epidermal regions, and 3) the eye components responsible for the promoting effect of the transplanted eye. Results were obtained by implanting fragments of ectoderm or epidermis into the vitreous chamber of host tadpoles and by evaluating the percentage of implants positive to a monoclonal antibody anti-lens. These results demonstrated that the lens-forming competence in the flank region is lost at the embryonic stage 30/31 and is weakly restored by eye transplantation; however, lens-forming competence in the head region is lost at the larval stage 48 and is strongly restored by eye transplantation. The authors hypothesize that during development the head ectoderm outside the LA is attained by low levels of the same signals that attain the LA and that these signals are responsible for the maintenance of lens-forming competence in the cornea and pericorneal epidermis of the larva. In this hypothesis, low levels of these signals slacken the decrease of the lens-forming competence in the head ectoderm and make the head epidermis much more responsive than the flank epidermis to the effect of promoting factors released by a transplanted eye. Results obtained after transplantation of eyes deprived of some components indicate that the lens and the retina are the main source of these promoting factors. The immunohistochemical detection of the FGFR-2 (bek variant) protein in the epidermis of stage 53 larvae submitted to eye transplantation at stage 46 showed that the eye transplantation increased the level of FGFR-2 protein in the head epidermis but not in the flank epidermis, indicating that the lens-forming competence in X. laevis epidermis could be related to the presence of an activated FGF receptor system in the responding tissue.

Analysis of morphogenetic potential of caudal spinal cord in Triturus carnifex adults (Urodele amphibians) subjected to repeated tail amputations.

The present research was aimed at testing whether the extraordinary morphogenetic and histogenetic potential exhibited in the regenerating new tail remains constant even after repeated amputation or whether it changes as a result of the mechanisms responsible for the regenerative process. Particular attention was focused on regeneration of the spinal cord and ganglia. For this purpose, tail regeneration in adult specimens of Triturus carnifex subjected to repeated amputation (up to 7 times) was compared with that of control animals subjected to a single amputation. Results show that although it slowed down the morphogenetic and differentiative phase, repeated amputation did not significantly alter either the morphogenetic or the histogenetic potential of the ependymal layer of the regenerating spinal cord. The latter result leads to hypothesized that the cells of the ependymal layer of the stump, which are responsible for the formation of the apical ampulla and the ependymal tubule inside the regenerative blastema, do not derive from undifferentiated reserve elements triggered after tail amputation but rather from differentiated ependymal elements that dedifferentiate after the trauma and re-acquire embryonic potential. If this regeneration were actually to take place at the expense of the reserve elements, the continual regenerative processes induced by the repeated amputation would lead to the increasing depletion of these elements and a consequent reduction in regenerative capacity.

Tissue interactions and lens-forming competence in the outer cornea of larval Xenopus laevis.

After lentectomy through the pupillary hole, the outer cornea of larval Xenopus laevis can undergo transdifferentiation to regenerate a new lens. This process is elicited by inductive factor(s) produced by the neural retina and accumulated into the vitreous chamber. During embryogenesis, the outer cornea develops from the outer layer of the presumptive lens ectoderm (PLE) under the influence of the eye cup and the lens. In this study, we investigated whether the capacity of the outer cornea to regenerate a lens is the result of early inductive signals causing lens-forming bias and lens specification of the PLE, or late inductive signals causing cornea formation or both signals. Fragments of larval epidermis or cornea developed from ectoderm that had undergone only one kind of inductive signals, or both kinds of signals, or none of them, were implanted into the vitreous chamber of host larvae. The regeneration potential and the lens-forming transformations of the implants were tested using an antisense probe for pax6 as an earlier marker of lens formation and a monoclonal antibody anti-lens as a definitive indicator of lens cell differentiation. Results demonstrated that the capacity of the larval outer cornea to regenerate a lens is the result of both early and late inductive signals and that either early inductive signals alone or late inductive signals alone can elicit this capacity.