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Transdifferentiation of ocular tissues in larval Xenopus laevis
Differentiat ion
Differentiation (1988) 3 9 : 4 1 5
Ontogeny and Neoplasia
0 Springer-Verlag 3 988
Review article Transdifferentiation of ocular tissues in larval Xenopw Iaevis Luigi Bosco Department of Animal and Human Biology, University La Sapienza, Via A. Borelli 50,I-00161 Rome, Italy
Abstract. Transdifferentiation phenomena offer a useful opportunity to study experimentally the mechanisms on which cell phenotypic stability depends. The capacities of vertebrate eye tissues to reprogram cell differentiation are well known in avian and mammalian embryos, and in larval and adult newt. From research into the capacity of anuran eye tissues to reprogram differentiation into a new pathway, considerable data have accumulated concerning the transdifferentiative capacities of eye tissues in larval Xenopus laevis. This work reviews the data concerning the transdifferentiative phenomena of eye tissues in that species and, based on these, aims to establish the extent of our knowledge about the mechanism controlling these processes. In larval Xenopus laevis the outer cornea can regenerate a lens by a lens-transdifferentiation process triggered and substained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye phenotypic stability of the outer cornea is guaranteed by the presence of the inner cornea and lens, which prevent the spread of retinal factor(s). The stimulus for lens transdifferentiation of the outer cornea can be supplied by other tissues as well, but this capacity is not widely distributed. The iris and retinal pigmented epithelium can transdifferentiate into neural retina if isolated from the surrounding tissues and implanted in the vitreous chamber. As for lens transdifferentiation of the outer cornea, retinal transdifferentiation of the iris can be stimulated by certain nonocular tissues as well. Introduction
Among vertebrates, the transdifferentiative phenomena of ocular tissues have been observed in the Amphibia, Aves and Mammalia. Many investigations carried out in avian and mammalian embryos, both in vivo and in vitro, have shown different possibilities of reprogramming in the eye tissues of these animals (see reviews by Eguchi [13], Moscona [28] and Okada [30, 31, 321. As regards the Amphibia, it is known that some completely differentiated ocular tissues can reprogram cell differentiation during regeneration of specific parts of the eye. Larval and adult newt can regenerate lens and neural retina by a process of cellular metaplasia (cell-type conversion), which requires dedifferentiation, proliferation, and new differentiation [37, 45, 461. The conversion of pigmented epithelium into neural retina has also been demonstrated in larval and adult anuran amphibians [lo, 26, 411.
The above mentioned phenomena of cellular metaplasia are distinguished from other examples of reprogramming differentiation, seen in cells that have not completed their differentiation, designated tissue metaplasia. In the latter case the new cell specificity results from changes in the pathway of differentiation in cells that are normally destined to realize a specific cell phenotype, but have not yet achieved it [45]. At present, lens-forming transformation from the outer cornea, which occurs during lens regeneration in larval Xenopus laevis [19], cannot be ascribed to any of the above-mentioned categories of reprogrammative phenomena, since it has not yet been established whether the neoformation of the lens is effected by cells that have already completed this pathway of differentiation or not. Thus, the transformation processes in the ocular tissues of larval Xenopus laevis, which are the scope of this review, can be better grouped under the term transdifferentiation. This term can be applied to a much-wider range of modulation in cell expression than that denoted by cell-type conversion (cellular metaplasia). More precisely, cellular and tissue metaplasia can be considered subcategories of a diversity of reprogrammative phenomena, which as a whole can be correctly indicated by “ transdifferentiation”. Lens transdifferentiation of the outer cornea Among the Anura, lens regeneration has been fully demonstrated only in larval Xenopus laevis. Freeman [19] showed that lensectomised larvae of this species can regenerate a lens from the deep layer of the outer cornea or, if the cornea is removed, from the pericorneal epidermis. The the lensforming capacities of these ocular tissues decrease during the larval period and disappear at metamorphosis. Freeman, besides describing in detail the successive phases of the regenerative process (Figs. 1 4 ) showed, by means of homoplastic grafts of cornea labeled with [3H]-thymidine into the eye cup of unlabeled larvae, that the regenerated lens effectively originates from the outer cornea by lensforming transformation of that epithelial tissue. In the same work, Freeman produced evidence that lens regeneration is contingent upon the presence of the eye cup: the outer cornea of enucleated larvae never regenerates a lens. After the publication of Freeman’s data, lens regeneration in larval Xenopus Zuevis became the object of numerous investigations, and the data now available permit delineation of the morphogenetic processes and tissue interactions that are effected during this phenomenon.
5
st.1
st.2
middle st.3
st. 4
st.5
Fig. 1. Diagram of lens regeneration in larval Xenopus luevis [18]; st.1, 36 hours after lensectomy: the cells of the inner layer of the outer cornea change from squamous to cuboidal; st.2, 3 days after lensectomy: the cells of the inner layer of the outer cornea form an aggregate; middle st.3, 5 days after lensectomy : the aggregate continuous with the inner layer of the outer cornea becomes a pedunclated lens-vesicle; lute st.3, 6 days after lensectomy : the pedunculated lens-vesicle grows in size; st.4, 8 days after lensectomy : the lens fibers start to form in the posterior region of the lensvesicle; st.5,ll days after lensectomy : the primary lens-fiber nucleus has formed, and formation of the secondary lens fibers is underway
Campbell and Jones [S] cultivated in vitro fragments of the outer cornea and of the pericorneal epidermis in different combinations : the outer cornea alone, the outer cornea and surrounding pericorneal epidermis, or pericorneal epidermis alone. When the outer cornea was cultivated in vitro alone or with the surrounding pericorneal epidermis, it generated a new lens, whereas the pericorneal epidermis alone did not show this capacity. From the results obtained, Campbell and Jones concluded that the outer cornea of the normal eye in larval Xenopus laevis has an intrinsic capacity to transform itself into lens and the fact that this does not occur in a normal eye must be attributable to an inhibitory action exerted by the lens itself, while the pericorneal epidermis, to manifest its lens-forming potential, must first transform itself into cornea under the influence of the eye cup. Moreover, they attributed the difference between the in vivo and in vitro results to the difference in availability of nutrients to the outer cornea under the two different experimental conditions. Since enucleation deprives the outer cornea of necessary nutritional factors (supplied by aqueous humor by way of the inner cornea), it should thus inhibit its intrinsic lens-forming capacity. Further experiments showed that this explanation, based on the results obtained from in vitro experiments, does not apply to lens regeneration in vivo. In fact, after removing the lens through an incision in the dorsal eye wall and simultaneously damaging either the outer cornea alone or both corneas (outer and inner cornea; Fig. 5), the outer cornea and pericorneal epidermis undergo lens-forming transformation only when they directly communicate with the vitreous-chamber environment (Figs. 6 , 7) [2]. This means that the factor(s) responsible for lens-forming transformation of the outer cornea cannot reach the
Figs. 2-4. Lens regeneration from the outer cornea in larval Xenopus luevis lensectomized at stage 50 according to Nieuwkoop and Faber [29]. x 280. (after Filoni et al. [15]) Fig. 2 Six days after lensectomy: The formation of primary lens fibers starts in the posterior part of the lens-vesicle (early stage 4) Fig. 3 Eight days after lensectomy: the primary nucleus of the lens fibers is completely formed Fig. 4 Ten days after lensectomy: the secondary fibers are added to the primary fibers (st.5)
6
" 0 "0
Fig. 5. Diagram of various types of experiment. A , simple lensectomy; B, lens removal from the dorsal region of the eye; C. lens removal from the dorsal region of the eye and simultaneous incision of the outer cornea; D,lens removal from the dorsal region of the eye and simultaneous incision of the outer and inner cornea. (After Bosco et al. [2])
outer cornea in this form under normal conditions and thus cannot be considered merely nutritional. The intact inner cornea may be thought of as a barrier preventing the spread of, or metabolically inactivating, the active factor(s). On the other hand, the presence of the lens does not prevent lens-forming transformation of the outer cornea if direct communication between the outer cornea and the vitreouschamber environment is established experimentally. Reeve and Wild [38], after removing the original lens and replacing it in the pupillary space, observed lens transformation of
the outer cornea or of fragments of outer cornea implanted in the vitreous chamber. Although these data indicated that the presence of the lens cannot prevent lens-forming transformation of the outer cornea, some criticism could be made. In most cases the regrafted lenses had a normal histological appearance; in other cases, they were damaged. In the authors' opinion, there was no correlation between lens damage and lens transformation of the cornea. However, as the operated eyes were examined some days after the operation, the possibility that some damage occurred, even in the lenses that appeared normal, could not be excluded. Furthermore, it was not indicated whether lens replacement preserving the original polarity was carried out, and the lens may well have differential activity at each pole. Therefore it was necessary to approach this problem in such a way as to exclude the possibility that the presence of any neoformed lens was due to morphological and functional damage suffered by the original lens and/or its reconstitution from the epithelio-capsular fragments. To this purpose, my coworkers and I carried out three different experiments (Fig. 8) in larval Xenopus lueuis [3] : A. Simple lensectomy B. Incision of outer and inner cornea in the region opposite the dorsal iris, without lens removal C . Incision of outer and inner cornea and perforation of the dorsal iris, without lens removal The results obtained showed that the outer cornea underwent lenstransdifferentiation, even in the presence of the intact old lens, only when direct communication was set up between the outer cornea and the environment of the vitreous chamber (Fig. 9). These data, furthermore, strongly suggested that under normal conditions the inhibitory role played by the lens is mechanical in nature, i.e.,
Figs. 6, 7. Regeneration of the lens after lensectomy from the dorsal region of the eye and simultaneous incision of the outer cornea. Examples show lens regeneration from the pericorneal epidermis. x 220 Fig. 6. Ten days after operation: the regenerated lens is connected to pericorneal epidermis by epithelial clusters (mraws) Fig. 7. Fifteen days after operation the basement membrane of the epidermis is continuous with the lens capsule (urrows)
7
Fig. 8A-C. Diagram of various types of experiment. A Simple lensectomy. B Incision of the outer and inner cornea at dorsal iris without lensectomy. C Incision of the outer and inner cornea and perforation of the iris without lensectomy. (After Bosco et al. [ 3 ] )
Fig. 10. Diagram of various types of experiment. A , simple lensectomy; B, removal of the lens and neural retina; C, removal of the anterior complex (inner cornea, iris and ciliary body) and the lens; D,removal of the anterior complex, the neural retina and lens. (After Filoni et al. [16])
Fig. 9. The neoforming lens-vesicle (arrow) is located in front of the perforation of the iris. D.I., dorsal iris; O.L., old lens. x 320. (After Bosco et al. [3])
the lens occupying the pupillary space prevents spread of the factor(s) from the vitreous chamber, and thus prevents lens transdifferentiation of the outer cornea. This last conclusion is also supported by the results obtained from experiments in which the lens was replaced by a Millipore Filter disk to prevent direct communication between the vitreous and anterior chambers. Under these experimental conditions, lens transdifferentiation of the outer cornea is inhibited [9]. The above-reported data all suggest that in a normal eye of larval Xenopus laevis the phenotypic stability of the outer cornea is ensured by the presence of two barriers, both of which prevent the factor(s) necessary to trigger lens transdifferentiation of the outer cornea from spreading from the vitreous chamber towards the anterior chamber. At present there are some experimental data that indicate the neural retina is the tissue that produces the stimulating factor(s) needed for lens transdifferentiation of the outer cornea. Filoni et al. [16] effected various types of experiment (Fig. 10):
A. Simple lensectomy B. Removal of the lens and neural retina C. Removal of the anterior complex (inner cornea, iris, ciliary body) and lens D. Removal of the anterior complex, neural retina and lens In Experiment A, lens regeneration occurred from the outer cornea. In Experiment B, the removal of the neural retina prevented any lens transdifferentiation of the outer cornea; this result indicates that, in lens regeneration from the outer cornea in vivo, an essential role is played by the neural retina, and also indicates that lens transformation of the outer cornea cannot be sustained by pars ciliaris and pars iridea retinae alone. The removal of the anterior complex and lens effected in Experiment C did not prevent lens regeneration from the outer cornea (Figs. 11, 12). On the contrary, when the neural retina was removed together with the anterior complex and lens (Fig. lOD), lens regeneration from the cornea was completely inhibited. In these experiments a very slight degree of regeneration in the retina, left in situ, was observed. On average, the total volume of the retina ranged from 2.6% (day 3 after the operation) to 5% (day 15 after the operation) of the retinal volume of a normal eye in larvae at stage 50 (according to Nieuwkoop and Faber [29]). This small amount of regenerated retina was not able to promote lens regeneration from the outer cornea. Though the exact nature of the retinal factor(s) has not yet been determined, preliminary data indicate they have a protein nature, as pellets of whole protein complement of the eye cup induce lens-forming transformation of the outer cornea when implanted between the outer and inner cornea [17].
8
Figs. 11, 12. Lens regeneration from the outer cornea after removal of the anterior complex and lens. Fig. 11. Five days after operation: at the end of the epithelial cluster of corneal origin a lens vesicle at stage 4 (arrow) can be seen. C., outer cornea; R.,neural retina. x 320 Fig. 12. Seven days after operation: a lens-vesicle at stage 5 can be seen. x 350
My coworkers and I [6] to ascertain whether lens transdifferentiation of the outer cornea in vivo needed the continuous presence of the stimulus provided by the retinal factor(s) or whether, once triggered by this factor(s), the process could continue in the presence of nutritional factors alone recently carried out experiments based on implantation, between the outer and inner cornea of the host larvae, of the outer cornea taken from lensectomised donors at fixed intervals after lensectomy. The results showed that in vivo lens transdifferentiation of the implanted outer cornea required the continuous presence of retinal factor(s). Furthermore, on its own, the nutritional factor(s) supplied to the outer cornea by the aqueous humour could not sustain continued lens differentiation. Moreover, it was also incapable alone of maintaining the lens-transformation stage, attained by the lens-forming structures, that had previously been induced in the implanted outer cornea by retinal factor(s), whatever that stage was. Absence of the retinal factor(s) determined the arrest of transdifferentiation and then the regression of newly-formed structures. These data strongly suggested that the lens-transdifferentiation process of the outer cornea is not a single-step process, but a morecomplex one, which requires a sequence of interactions, extending over a long period of time, during which the retinal factor(s) must be present until complete lens transdifferentiation of the outer cornea is achieved. There is evidence that crystallin synthesis is regulated at all the generally recognized levels, i.e., transcription of RNA, post-transcriptional changes in RNA, and translation of proteins [27, 341. However, recent studies on the regulation of crystallin expression, carried out by transferring the chicken gene into mouse cells, indicate that the ~
~
level of crystallin synthesis is controlled mainly at the transcriptional level and that there are two steps in the process of transdifferentiation of the lens from neural retina in the chick: acquisition of the capacity to express crystallin genes, and derepression of the endogenous crystallin genes. It is probable that these two steps are involved in other cases of cell differentiation [21, 221. The retinal factor(s) of larval Xenopus luevis might act at any one of these levels. Thus, in vivo the lens-transdifferentiation process of the outer cornea is triggered and sustained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye the phenotypic stability of the outer cornea is guaranteed by the presence of the lens and inner cornea, which represent insurmountable barriers to the retinal factor(s) present in the vitreous chamber. This factor is necessary for the entire lens-transdifferentiation process of the outer cornea, and each of these barriers is able to prevent any direct communication between the outer cornea and the environment of the vitreous chamber. Although during lens regeneration the stimulus for lens transdifferentiation of the outer cornea is provided by the retinal factor(s), there is evidence that it can also be supplied by other tissues. Waggoner [47] grafted fragments of the outer cornea of larval Xenopus luevis into the stump of an amputated hindlimb and observed that in this heterotopic site lens transdifferentiation of the outer cornea is possible. Reeve and Wild [39] implanted pituitary, limb bud, or limb-bud blastema between the outer and inner cornea and demonstrated that these structures, deprived of any nervous connection, are able to trigger and support lens transdifferentiation of the outer cornea. Filoni et al. [18], implanting lumbar ganglia innervating normal and regener-
9
Fig. 14. Implant of tentacle blastema, 10 days after implanting. In the newly formed lens (arrow) the primary lens fiber nucleus has formed and formation of secondary lens fibers is underway (st.5). x 380. (After Bosco et al. [ 5 ] )
Fig. 13. Implant of tentacle blastema; 5 days after implanting. A small cell aggregate at stage 3 (arrow)has formed from the outer cornea. ox., outer cornea; ic., inner cornea. x700 (After Bosco et al. IS])
ating limbs between the outer and inner cornea, observed that these structures can induce lens transdifferentiation of the outer cornea, and that ganglia innervating regenerating limbs are more efficient in this action. These data were interpreted as indicating that the neurotrophic factor(s) that the nerves are thought to emit from their resected terminations during amphibian limb regeneration [14, 40, 481, and which appears to stimulate the division of blastemal cells, can replace the retinal factor(s) during lens transdifferentiation of the outer cornea. Recent investigations of limb regeneration in the newt, carried out with monoclonal antibodies to the cells of a regenerating limb, have provided new evidence about the origin of blastemal cells from Schwann cells and myofibers, and identified a subset of blastemal cells whose division is effectively dependent on the nerve supply [ 141. On the basis of these data, my coworkers and I undertook a series of experiments to establish whether the lensinducing capacity on the outer cornea was present in many other larval tissues or whether it was more restricted, and in the latter case whether one or more common features could be singled out [5]. Fragments of iris ring, spleen, kidney, tail, tail blastema, tentacle, tentacle blastema and spinal cord were implanted between the outer and inner cornea of a normal eye. Of these tissues, only the tail blastema, tentacle blastema and spinal cord showed the capacity to give rise to lens-forming transformation of the cornea (Figs. 13, 14), although to different degrees (Table 1). The other tissues induced no appreciable response in the outer cornea. All these data reinforced the hypothesis previously
advanced, on the basis of results obtained after implanting spinal ganglia between the outer and inner cornea, that lens transdifferentiation of the cornea could be stimulated by the neurotrophic factor produced by the ganglion cells. Since this hypothesis could be applied to lens transdifferentiation induced by spinal-cord neurons and also by dedifferentiated cells of tail blastema, Singer [40] proposed that all undifferentiated embryonic cells produced a neurotrophic factor. A comparison of data obtained in larval Xenopus luevis with those regarding lens transdifferentiation of iris epithelial cells of Urodela shows that tissues able to stimulate the newt iris (retina, spinal ganglia, pituitary) [12, 35, 361 correspond to those which stimulate lens transdifferentiation of the outer cornea in larval Xenopus luevis. In the newt it has been shown that the neurotrophic and pituitary factors promote proliferation, and a correlation has been established between proliferative activity of iris cells and their lens-type conversion [45, 461. Considering the fore-mentioned data, it is possible that, in larval Xenopus laevis also, proliferation of epithelial cells in the outer cornea is the direct target of lens-transdifferentiation-promoting factors. Lens transdifferentiation of the iris and retina Lens regeneration in anuran tadpoles has been the focus of numerous investigations. Some researchers have reported cases of lens regeneration, in larvae of various species, occurring through a process of Wolffian lens regeneration (cellular metaplasia or cell-type conversion), while others have obtained negative results, often in the same species. A series of investigations effected in our laboratory has shown that many cases reported as Wolffian regeneration can be attributed to reorganization of epitheliocapsular fragments of the lens remaining in situ following inadequate lensectomy. At present the available data concerning lens
10 Table 1. Summary of the results obtained following implantation of various larval tissues Experiment
I (Iris)
I1 (Spleen)
I11 (Kidney) IV (Tail) V (Tail blastema) VI (Tcntacle) VII (Tentacle blastema)
No. of cases operated
No. of cases dead/ discarded
No. of cases examined
No. of newly forming lenses
10 10 10
1
5 I 10
10 10 10
-
9 10 9 10 10 9
-
I 10
5
10 10 10
-
Days after operation
5
I 10
1
1
10 10 9 10 10 11
-
-
-
5 I 10
11 10 12
5
10 9 9
9
I 8
4 2 3
10 10 10
10 10 10
4 1 1
7 5
7 10
1 1
regeneration in anuran amphibian tadpoles lead us to consider that this phenomenon is much more restricted than previous data have shown [l]. As I mentioned in the previous paragraph, lens regeneration in anuran tadpoles has been demonstrated only in Xenopus luevis, in which the new lens is originated by a process of lens transdifferentiation. However, until some time ago it was not completely clear, even in this species, whether the lens-forming capacity was present, in situ, in other eye tissues as well. The possibility that lens transdifferentiation of the dorsal iris and retina occurs in Xenopus luevis larvae has been claimed to exist by Campbell [7] and by Overton and Freeman [33]. In particular, Campbell [7] reported that in lensectomised Xenopus luevis larvae the lens can originate from the pupillary edge of the iris (12% of the cases), from the outer cornea (12%), or from the retina near the ciliary body (56%). Nevertheless, the data we obtained in some investigations showed that, when lensectomy was performed without damaging the iris and retina, lens regeneration always occurred via lens transdifferentiation of the outer cornea [2, 151. It was, thus, interesting to re-examine experimentally the possibility of lens transdifferentiation of the iris and retina in situ. To this purpose five experiments were performed (Fig. 15) [4] : I. Simple lensectomy 11. Removal of the lens, outer cornea, and pericorneal integument 111. Removal of the lens, outer cornea, and pericorneal integument plus simultaneous incision of the retina IV. Removal of the lens, outer cornea, and pericorneal integument plus simultaneous incision of the dorsal iris V. Removal of lens and simultaneous lesion of iris and retina
5
-
10 10 15
10 10 14 10 10 11
4
-
5 I 10
-
3
-
10
1
2
Percentage of lens-forming transformations (total)
-
10 10 12
5
I
10
VIII (Spinal cord)
1
Lens regenerating stage
-
-
6 4 4 -
-
II
Ill
IV
Fig. 15. Diagram of various types of experiment; I, simple lensectomy; II, removal of lens, outer cornea, and pericorneal integument plus simultaneous incision of retina; ZII, removal of lens, outer cornea, and pericorneal integument plus simultaneous incision of retina; IV, removal of lens, outer cornea and pericorneal integument plus simultaneous incision of dorsal iris; V , removal of lens and simultaneous lesion of iris and retina. (After Bosco et al. [4])
11
Figs. 16-19. Removal of lens and simultaneous lesion of iris and retina. The series of figures shows a case of apparent lens regeneration from iris. The regenerating lens (st.5) is close up against the iris. Continuity with the cornea is visible in the last two photographs (arrow). c., cornea; di.,dorsal iris. x 600. (After Bosco et al. [6])
The results obtained showed that when lensectomy is performed without damaging eye territories other than the outer and inner cornea, lens regeneration takes place at the expense of the outer cornea only. The absence of the lens transdifferentiation capacity of the iris and retina under these experimental conditions cannot be explained on the basis of inhibition by lens-forming structures of the outer cornea, as there was still no lens transdifferentiation even when the cornea and pericorneal epidermis were removed. The iris and retina did not show in situ lens transdifferentiation capacity, even after they had been deliberately
stimulated, by comparatively extensive damage. The presence of regenerated or regenerating lens was sometimes observed under these experimental conditions, but at the same time it was possible to identify the territory of origin as the pericorneal epidermis or the cornea (Figs. 1 6 1 9 ) . Although these results show that the iris and retina have no lens-forming capacity in situ, the possibility that these tissues of larval Xenopus laevis can demonstrate this capacity under different experimental conditions cannot be excluded. Hoperskaya and Zviadadze [20] showed that pieces of the iris of adult Rana temporaria, wrapped in lens epithe-
12
len.
Fig. 20. Schematic representation of experiment performed. The isolated iris epithelium was combined with lens epithelium; precultivation of sandwiches in vitro for 3 days and implantation into a tadpole orbit. (After Hoperskaya and Zviadadze [20])
lium and cultivated for 3 days in a protein-free medium before implantation into the tadpole orbit, transform into lens in 32% of cases (Fig. 20). Lopashov [20, 211 cultivated sandwiches of lens epithelium of adult Rana temporaria with retinal pigmented epithelium from larvae of the same species in a protein-free medium before implantation into the tadpole orbit. He observed that under these conditions pigmented epithelium transformed into lentoids in 85% of cases. Thus, an event involving lens transdifferentiation of the iris (as in Wolffian regeneration) and of the retinal pigmented epithelium can occur in frogs.
Fig. 21. Diagram showing the two types of implantation of dorsal iris taken from a lensectomised eye. a, autoplastic implant of a dorsal iris fragment in the vitreous chamber of a lensectomised eye; b, autoplastic implant of a dorsal iris fragment in the anterior chamber of a lensectomised eye
Retinal transdifferentiation of the iris and retinal pigmented epithelium The iris and retinal pigmented epithelium of larval Xenopus laevis show a high degree of phenotypic stability in situ. This was found in iris tissue even when it had been damaged to various degrees in order to stimulate its latent transdifferentiative competence (see the previous paragraph). However, under some experimental conditions, the iris and pigmented epithelium undergo transdifferentiation processes. In fact, when isolated from its surrounding tissue and implanted in the vitreous chamber (Fig. 21) the dorsal iris undergoes a process of retinal transdifferentiation [lo]. The transdifferentiation process is effected by an initial phase of dedifferentiation of the iris epithelial cells, which gradually become depigmented. The subsequent phase is represented by a cell agglomeration that undertakes new differentiation in a retinal direction (Figs. 22, 23). It is interesting, and should be emphasized, that the degree of transdifferentiation of the iris depends on its localization in the eye environment (Fig. 24). Iris fragments in the vitreous chamber underwent complete retinal transdifferentiation ; implants in the pupil region were transformed into polarized vesicles, in which only the portion facing the vitreous chamber of the host was transdifferentiated. Unlike that of the vitreous chamber, the environment of the anterior chamber cannot provide the stimulus for retinal transdifferentiation of the iris. These data suggest that in the vitreous chamber of larval Xenopus laevis there is some factor(s) which stimulates the retinal-transdifferentiation process of the iris and is distributed in a gradient. Retinal transdifferentiation is also observed in the pigmented epithelium of the tadpole and adult Xenopus laevis
Fig. 22. Autoplastic implant of dorsal iris fragment in the vitreous chamber. Thirty days after implantation, retinal transdifferentiation of the implanted dorsal iris fragment has occurred. x 120. (After Cioni et al. [lo])
[41]. This eye territory, separated from the mesenchyme envelope and implanted into the lens-less eye, transforms into retina under the influence of the retinal factor(s). No retinal transdifferentiation occurs in pigmented epithelium when it is implanted into the enucleated orbit. Unlike the transformation of pigmented epithelium into retina, retinal transdifferentiation of the iris is not prevented by close contact between the iris epithelium and the stroma, a mesenchymal structure corresponding to Bruch's membrane in retinal pigmented epithelium. Retinal transdifferentiation of pigmented epithelium is also seen in larval and adult Rana temporaria [23, 251. In this species, retinal transdifferentiation occurs as in the case of the pigmented epithelium of Xenopus laevis. Moreover, the process can be stimulated by lens epithelium [23]. Fragments of retinal pigmented epithelium cultured in vivo, in contact with the lens epithelium in the tadpole orbit, after a period of intense proliferation, transform into retina. The same tissue, precultivated in vitro with lens epithelium for
13
Conclusion
Fig. 23. Implant of dorsal iris fragmcnt in the vitreous chamber; 30 days after implantation; detail of retinal-transdifferentkted dorsal iris. x 600. (After Cioni et al. [lo])
b
C
Fig. 24. Diagram to illustrate the hypothesis that a gradient of ocular factor(s) exists in the lensectomised eye; a, in the anterior chamber, the implant maintains its original structure, because the level of the inducing factors is below the threshold value; b, in the neighbourhood of the pupil, only the posterior portion of the vesicle receives the inducing factor(s) in quantities greater than the threshold value, thus resulting in transdifferentiation into neural retina (polarized vesicle); c, in the deeper portion of the vitreous chamber, the implanted iris receives the maximum quantity of factor(s), and so it is completely transformed into neural retina
3 days in protein-free medium, and then implanted in the enucleated orbit, transforms into lentoid (see the previous paragraph). As in lens transdifferentiation of the cornea, retinal transdifferentiation of the iris in larval Xenopus lueuis can be induced by nonocular tissues. Fragments of iris, autoplastically implanted into the stump of the amputated hindlimb, transdifferentiate into neural retina [ 1I] ; preliminary data obtained in our laboratory indicate that retinal transdifferentiation of the iris can be induced by the pituitary as well.
At present the available data show that the eye tissues of larval Xenopus laevis have a high degree of reprogramming capacity ; the outer cornea can undergo lens transdifferentiation, while the iris and pigmented epithelium can transdifferentiate into neural retina. The lens-transdifferentiation process of the outer cornea in situ after simple lensectomy is somewhat analogous to the ontogenic process of lens formation from ectoderm. In fact, lens transdifferentiation of the outer cornea is triggered and sustained by a retinal factor(s) that is present in the vitreous chamber. Under normal conditions, the phenotypic stability of the outer cornea is ensured by the integrity of the inner cornea and by the presence of the lens, which prevents spread of the retinal factor(s) towards the outer cornea. In retinal transdifferentiation of the iris, the availability of retinal factor(s) able to stimulate the transdifferentiative process is not sufficient to enable the iris to display its retinal-reprogramming capacity ; the iris must also be isolated from its surrounding tissues. Thus, the iris in normal conditions shows more phenotypic stability than the outer cornea. The situation regarding the retinal pigmented epithelium is quite different, since in a normal eye this tissue does not directly communicate with the vitreous chamber, where the retinal factor(s) is present. As regards the nature and action of the factor(s) that can stimulate transdifferentiative processes of the eye tissues in larval Xenopus laevis, some speculation can now be made. It has been demonstrated that lens transdifferentiation of the outer cornea can be promoted by several tissues or environments [5, 18, 39, 471, although during lens regeneration the stimulus is usually provided by the neural retina, which produces a factor(s) that is probably of a protein nature. Some of these tissues or environments can also induce retinal transdifferentiation of the iris (Table 2). One possible explanation of all, these data could be that the different tissues and environments contain some factor(s) that can obviously replace the retinal factor(s) responsible for stimulating the expression of latent reprogrammative capacities, specific to reacting tissue. In this way, while the outer cornea is always transformed into lens, the dorsal
Table 2. Summary of the tissues or environments stimulating transdifferentiation of the eye tissues in larval Xenopus laevis Reacting eye tissue
Stimulating tissue or environment
Type of transdifferentiation
Outer cornea
Neural retina Limb bud Amputated hindlimb Limb blastema Tentacle blastema Spinal ganglia Spinal cord Pituitary
Lens transdifferentiation
Dorsal iris
Neural retina Amputated hindlimb Pituitary
Retinal transdifferentiation
Retinal pigmented epithelium
Neural retina
14
iris, under the influence of the same tissues, always transdifferentiates into neural retina. On the other hand, it cannot be excluded that lens transdifferentiation of the outer cornea and retinal transdifferentiation of the iris and of retinal pigmented tissue are due to a specific stimulus exerted by different factors that are “instructive” in nature, i.e., by factors able to direct the reprogrammative process in the reacting tissues. If this is so, we have to admit that both factors are present simultaneously, not only in the vitreous chamber, but also in the amputated hindlimb and in the pituitary. The latter hypothesis seems more unlikely than the first. Lopashov [23, 24,261, on the basis of the data regarding the transdifferentiative phenomena of the eye tissues in larval and adult R a m temporaria (see previous paragraphs), formulated another interesting hypothesis, that the direction of the induced transdifferentiation could depend on the type of inducing influences, but this dependence is not unequivocal. The direction of transdifferentiation of frog iris pigmented cells (IPE) and of retinal pigmented cells (RPE) depends to some extent on directive influences from other tissues; however, this does not exclude the possibility that the multipotency of RPE an IPE can depend on the availability in them of some dormant programming determinants. In other words, the type of reprogrammation could depend on the balance between the intrinsic determinants and the influences exerted by type-inducing tissues under different experimental conditions. In Lopashov’s opinion [26] these programming determinants (or factors) are similar to those that are responsible for the induction of eye cells during ontogenesis. References 3 . Bosco L (1988) The problem of lens regeneration in anuran
amphibian tadpoles. Acta Embryol Morphol Exper (in press) 2. Bosco L, Filoni S, Cannata S (1979) Relationships between eye factors and lens-forming transformation in the cornea and pericorneal epidermis of larval Xenopus laevis. J Exp 2001 209~261-282 3. Bosco L, Filoni S, Cioni C (1980) Lens formation from cornea in the presence of the old lens in larval Xenopus laevis. J Exp ZOOI213:9-14 4. Bosco L, Filoni S, Paglioni S (1981) Experimental analysis of the lens-forming competence of the cornea, iris, and retina in Xenopus luevis tadpoles. J Exp Zool 216:267-276 5. Bosco L, Filoni S, Cioni C (1985) Experimental analysis on the capacity of several larval tissues to promote lens-forming transformations in the cornea of Xenopus luevis tadpoles. J Exp Zoo1 233 :221-228 6. Bosco L, Filoni S, Cioni C, Bernardini S (1986) Eye factors and lens-forming transformations of outer cornea in Xenopus laevis larvae. J Exp Zool 240 :401407 7. Campbell JC (1963) Lens regeneration from iris, retina and cornea in lensectomized eyes of Xenopus laevis. Anat Rec 145:214-215 8. Campbell JC, Jones KW (1968) The in vitro development of lens from cornea of larval Xenopus laevis. Dev Biol 17 : 1-1 5 9. Cioni C, Filoni S, Bosco L (1982) Inhibition of lens regeneration in larval Xenopus laevis. J Exp Zool 220: 103-108 10. Cioni C, Filoni S, Aquila C, Bernardini S, Bosco L (1986) Transdifferentiation of eye tissues in anuran amphibians : analysis of the transdifferentiation capacity of the iris of Xenopus laevis larvae. Differentiation 32: 21 5-220 13. Cioni C, Filoni S, Bosco L, Aquila C, Bernardini S (1987) Transdifferentiation of larval Xenopus luevis iris implanted into the amputated hindlimb. Experientia 43 :443-444
12. Connelly TG, Ortiz JR, Yamada T (1973) Influence of the pituitary on Wolffian lens regeneration. Dev Biol 31 : 301-315 13. Eguchi G (1986) Instability in cell commitment of vertebrate pigmented epithelial cells and their transdifferentiation into lens cells. Curr Top Dev Biol 20 :21-37 14. Fekete DA, Brockes JD (1987) A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts. Development 99: 589-602 15. Filoni S, Bosco L, Cioni C (1976) II problema della rigenerazione del cristallino degli Anfibi Anuri negli stadi post-embrionali. Esperienze di asportazione del cristallino in larve di Rana esculentu e Xenopus luevis. Acta Embryol Exper 3 : 319-334 16. Filoni S, Bosco L, Cioni C (1982) The role of neural rctina in lens regeneration from cornea in larval Xenopus luevis. Acta Embryol Morphol Exper ns., 3 : 15-28 17. Filoni S, Bosco L, Cioni C, Venturini G (1983) Lens-forming transformations in larval Xenopus laevis induced by denatured eye-cup or its total whole protein complement. Experientia 39:315-317 18. Filoni S, Bosco L, Cioni C, Burani P (1984) Lens-forming transformations in the outer cornea of larval Xenopus laevis induced by spinal ganglia. J Exp Zoo1 230 :409-416 19. Freeman G (1963) Lens regeneration from the cornea in Xenopus laevis. J Exp Zoo1 154: 39-65 20. Hoptrskaya OA, Zviadadze K G (1981) Transdifferentiation of adult frog iris in retina or lens by exogenous influences. Dev Growth Differ 23 :201-213 21. Kondoh H, Okada TS (1986) Dual regulation of expression of exogenous -crystallin gene in mammalian cells : a search for molecular background of instability in differentiation. Curr Top Dev Biol20: 153-164 22. Kondoh H, Ueda Y, Hayashi S, Okazaki K, Yasuda K, Okada TS (1987) An attempt to assay the state of determination by using transfected genes as probes in transdifferentiation of neural retina into lens. Cell Differ 20 :203-207 23. Lopashov GV (1983) Transdifferentiation of pigmented epithelium induced by the influence of lens epithelium in frogs. Differentiation 24 :27-32 24. Lopashov GV (1983) Induced and spontaneous transdifferentiation and their relation to induction in development. In : Goel SC, Bellairs R (eds) Developmental biology. Afro-Asian perspective. Indian Society of Developmental Biologists, Poona, pp. 87-96 25. Lopashov GV, Sologub AA (1972) Artificial metaplasia of pigmented epithelium into retina in tadpoles and adult frogs. J Embryol Exptl Morphol8 : 521-546 26. Lopashov GV, Golubeva ON, Zviadadze G (1986) Common determinants of embryonic induction and transdifferentiation. In: Progress in developmental biology, pp. 4 3 4 6 27. McAvoy JW (1980) Induction of the lens. Differentiation 17:137-149 28. Moscona AA (1986) Conversion of retina glia cells into lenslike phenotype following disruption of normal cell contact. Curr Top Dev Biol 20: 1-19 29. Nieuwkoop PD, Faber J (1956) Normal table of Xenopus laevis (Daudin). North-Holland, Amsterdam 30. Okada TS (1980) Cellular metaplasia or transdifferentidtion as a model for retinal cell differentiation. Curr Top Dev Biol 26 :360-380 31. Okada TS (1983) Recent progress in studies of transdifferentiation of eye tissues in vitro. Cell Differ 13: 177-1 83 32. Okada TS (1986) Transdifferentiation in animal cells: fact or artefact? Dev Growth Differ 28: 213-221 33. Overton J, Freeman G (1960) Lens regeneration in Xenopus laevis. Anat Rec 137 : 386 34. Piatigorsky J (1981) Lens differentiation in vertebrates. Differentiation 19: 134153 35. Powell JA, Powers C (1973) Effect on lens regeneration of implantation of spinal ganglia into the eyes of the newts, Nothophtalmus. J Exp Zoo1 183:95-114 36. Reyer RW (1966) The influence of neural retina and lens on
15 lens regeneration from dorsal iris implants in Triturus viridescens larvae. Dev Biol 14:214245 37. Reyer RW (1977) The amphibian eye: Development and regeneration. In: Handbook of sensory physiology. Vol. VIIj5. The visual system in vertebrates. Springer-Verlag Berlin Heidelberg, pp. 309-390 38. Reeve JG, Wild A (1978) Lens regeneration from cornea of larval Xenopus laevis in the presence of the old lens. J Embryol Exp Morphol48:205-214 39. Reeve JG, Wild A (1981) Secondary lens formation from the cornea following implantation of larval tissues between the outer and inner corneas of Xenopus laevis tadpoles. J Embryol Exp Morphol64: 121-132 40. Singer M (1965) A theory of the trophic nervous control of amphibian limb regeneration, including a revaluation of quantitative nerve requirements. In: Kiortis W, Trampusch HA1 (eds) Regeneration in Animals and Related Problems. North-Holland, Amsterdam, pp. 2G32 41. Sologub A (1977) Mechanisms of rcpression and derepression of artificial transformation of pigmented epithelium into retina in Xenopus laevis. Roux’s Arch Dev Biol 182:277-291 42. Stone LS (1958) Inhibition of lens regeneration in newt eyes
by isolating the dorsal iris from the neural retina. Anat Rec 120:151-172 43. Stone LS (1958) Lens regeneration in adult newt eyes related to retina pigmented cells and the neural retina factor. J Exp Zoo1 139:69-84 44. Stone LS, Steinitz H (1953) The regeneration of lenses in eyes with intact and regenerating retina in adult Triturus v. viridescens. J Exp Zool 124:435468 45. Yamada T (1977) Control mechanisms in the cell-type conversion in newt lens regeneration. Monogr Dev Biol Basel, Karger 13: 1-124 46. Yamada T (1982) Transdifferentiation of lens cells and its regulation. In: McDevitt DS (ed) Cell biology of the eye. Academic Press, New York, pp. 193-242 47. Waggoner PR (1973) Lens differentiation from the cornea following lens extirpation or cornea transplantation. J Exp Zool 186~97-109 48. Wallace H (1981) Vertebrate limb regeneration. John Wiley, Chichester Accepted in revised form August 27, 1988
Differentiation (1988) 3 9 : 4 1 5
Ontogeny and Neoplasia
0 Springer-Verlag 3 988
Review article Transdifferentiation of ocular tissues in larval Xenopw Iaevis Luigi Bosco Department of Animal and Human Biology, University La Sapienza, Via A. Borelli 50,I-00161 Rome, Italy
Abstract. Transdifferentiation phenomena offer a useful opportunity to study experimentally the mechanisms on which cell phenotypic stability depends. The capacities of vertebrate eye tissues to reprogram cell differentiation are well known in avian and mammalian embryos, and in larval and adult newt. From research into the capacity of anuran eye tissues to reprogram differentiation into a new pathway, considerable data have accumulated concerning the transdifferentiative capacities of eye tissues in larval Xenopus laevis. This work reviews the data concerning the transdifferentiative phenomena of eye tissues in that species and, based on these, aims to establish the extent of our knowledge about the mechanism controlling these processes. In larval Xenopus laevis the outer cornea can regenerate a lens by a lens-transdifferentiation process triggered and substained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye phenotypic stability of the outer cornea is guaranteed by the presence of the inner cornea and lens, which prevent the spread of retinal factor(s). The stimulus for lens transdifferentiation of the outer cornea can be supplied by other tissues as well, but this capacity is not widely distributed. The iris and retinal pigmented epithelium can transdifferentiate into neural retina if isolated from the surrounding tissues and implanted in the vitreous chamber. As for lens transdifferentiation of the outer cornea, retinal transdifferentiation of the iris can be stimulated by certain nonocular tissues as well. Introduction
Among vertebrates, the transdifferentiative phenomena of ocular tissues have been observed in the Amphibia, Aves and Mammalia. Many investigations carried out in avian and mammalian embryos, both in vivo and in vitro, have shown different possibilities of reprogramming in the eye tissues of these animals (see reviews by Eguchi [13], Moscona [28] and Okada [30, 31, 321. As regards the Amphibia, it is known that some completely differentiated ocular tissues can reprogram cell differentiation during regeneration of specific parts of the eye. Larval and adult newt can regenerate lens and neural retina by a process of cellular metaplasia (cell-type conversion), which requires dedifferentiation, proliferation, and new differentiation [37, 45, 461. The conversion of pigmented epithelium into neural retina has also been demonstrated in larval and adult anuran amphibians [lo, 26, 411.
The above mentioned phenomena of cellular metaplasia are distinguished from other examples of reprogramming differentiation, seen in cells that have not completed their differentiation, designated tissue metaplasia. In the latter case the new cell specificity results from changes in the pathway of differentiation in cells that are normally destined to realize a specific cell phenotype, but have not yet achieved it [45]. At present, lens-forming transformation from the outer cornea, which occurs during lens regeneration in larval Xenopus laevis [19], cannot be ascribed to any of the above-mentioned categories of reprogrammative phenomena, since it has not yet been established whether the neoformation of the lens is effected by cells that have already completed this pathway of differentiation or not. Thus, the transformation processes in the ocular tissues of larval Xenopus laevis, which are the scope of this review, can be better grouped under the term transdifferentiation. This term can be applied to a much-wider range of modulation in cell expression than that denoted by cell-type conversion (cellular metaplasia). More precisely, cellular and tissue metaplasia can be considered subcategories of a diversity of reprogrammative phenomena, which as a whole can be correctly indicated by “ transdifferentiation”. Lens transdifferentiation of the outer cornea Among the Anura, lens regeneration has been fully demonstrated only in larval Xenopus laevis. Freeman [19] showed that lensectomised larvae of this species can regenerate a lens from the deep layer of the outer cornea or, if the cornea is removed, from the pericorneal epidermis. The the lensforming capacities of these ocular tissues decrease during the larval period and disappear at metamorphosis. Freeman, besides describing in detail the successive phases of the regenerative process (Figs. 1 4 ) showed, by means of homoplastic grafts of cornea labeled with [3H]-thymidine into the eye cup of unlabeled larvae, that the regenerated lens effectively originates from the outer cornea by lensforming transformation of that epithelial tissue. In the same work, Freeman produced evidence that lens regeneration is contingent upon the presence of the eye cup: the outer cornea of enucleated larvae never regenerates a lens. After the publication of Freeman’s data, lens regeneration in larval Xenopus Zuevis became the object of numerous investigations, and the data now available permit delineation of the morphogenetic processes and tissue interactions that are effected during this phenomenon.
5
st.1
st.2
middle st.3
st. 4
st.5
Fig. 1. Diagram of lens regeneration in larval Xenopus luevis [18]; st.1, 36 hours after lensectomy: the cells of the inner layer of the outer cornea change from squamous to cuboidal; st.2, 3 days after lensectomy: the cells of the inner layer of the outer cornea form an aggregate; middle st.3, 5 days after lensectomy : the aggregate continuous with the inner layer of the outer cornea becomes a pedunclated lens-vesicle; lute st.3, 6 days after lensectomy : the pedunculated lens-vesicle grows in size; st.4, 8 days after lensectomy : the lens fibers start to form in the posterior region of the lensvesicle; st.5,ll days after lensectomy : the primary lens-fiber nucleus has formed, and formation of the secondary lens fibers is underway
Campbell and Jones [S] cultivated in vitro fragments of the outer cornea and of the pericorneal epidermis in different combinations : the outer cornea alone, the outer cornea and surrounding pericorneal epidermis, or pericorneal epidermis alone. When the outer cornea was cultivated in vitro alone or with the surrounding pericorneal epidermis, it generated a new lens, whereas the pericorneal epidermis alone did not show this capacity. From the results obtained, Campbell and Jones concluded that the outer cornea of the normal eye in larval Xenopus laevis has an intrinsic capacity to transform itself into lens and the fact that this does not occur in a normal eye must be attributable to an inhibitory action exerted by the lens itself, while the pericorneal epidermis, to manifest its lens-forming potential, must first transform itself into cornea under the influence of the eye cup. Moreover, they attributed the difference between the in vivo and in vitro results to the difference in availability of nutrients to the outer cornea under the two different experimental conditions. Since enucleation deprives the outer cornea of necessary nutritional factors (supplied by aqueous humor by way of the inner cornea), it should thus inhibit its intrinsic lens-forming capacity. Further experiments showed that this explanation, based on the results obtained from in vitro experiments, does not apply to lens regeneration in vivo. In fact, after removing the lens through an incision in the dorsal eye wall and simultaneously damaging either the outer cornea alone or both corneas (outer and inner cornea; Fig. 5), the outer cornea and pericorneal epidermis undergo lens-forming transformation only when they directly communicate with the vitreous-chamber environment (Figs. 6 , 7) [2]. This means that the factor(s) responsible for lens-forming transformation of the outer cornea cannot reach the
Figs. 2-4. Lens regeneration from the outer cornea in larval Xenopus luevis lensectomized at stage 50 according to Nieuwkoop and Faber [29]. x 280. (after Filoni et al. [15]) Fig. 2 Six days after lensectomy: The formation of primary lens fibers starts in the posterior part of the lens-vesicle (early stage 4) Fig. 3 Eight days after lensectomy: the primary nucleus of the lens fibers is completely formed Fig. 4 Ten days after lensectomy: the secondary fibers are added to the primary fibers (st.5)
6
" 0 "0
Fig. 5. Diagram of various types of experiment. A , simple lensectomy; B, lens removal from the dorsal region of the eye; C. lens removal from the dorsal region of the eye and simultaneous incision of the outer cornea; D,lens removal from the dorsal region of the eye and simultaneous incision of the outer and inner cornea. (After Bosco et al. [2])
outer cornea in this form under normal conditions and thus cannot be considered merely nutritional. The intact inner cornea may be thought of as a barrier preventing the spread of, or metabolically inactivating, the active factor(s). On the other hand, the presence of the lens does not prevent lens-forming transformation of the outer cornea if direct communication between the outer cornea and the vitreouschamber environment is established experimentally. Reeve and Wild [38], after removing the original lens and replacing it in the pupillary space, observed lens transformation of
the outer cornea or of fragments of outer cornea implanted in the vitreous chamber. Although these data indicated that the presence of the lens cannot prevent lens-forming transformation of the outer cornea, some criticism could be made. In most cases the regrafted lenses had a normal histological appearance; in other cases, they were damaged. In the authors' opinion, there was no correlation between lens damage and lens transformation of the cornea. However, as the operated eyes were examined some days after the operation, the possibility that some damage occurred, even in the lenses that appeared normal, could not be excluded. Furthermore, it was not indicated whether lens replacement preserving the original polarity was carried out, and the lens may well have differential activity at each pole. Therefore it was necessary to approach this problem in such a way as to exclude the possibility that the presence of any neoformed lens was due to morphological and functional damage suffered by the original lens and/or its reconstitution from the epithelio-capsular fragments. To this purpose, my coworkers and I carried out three different experiments (Fig. 8) in larval Xenopus lueuis [3] : A. Simple lensectomy B. Incision of outer and inner cornea in the region opposite the dorsal iris, without lens removal C . Incision of outer and inner cornea and perforation of the dorsal iris, without lens removal The results obtained showed that the outer cornea underwent lenstransdifferentiation, even in the presence of the intact old lens, only when direct communication was set up between the outer cornea and the environment of the vitreous chamber (Fig. 9). These data, furthermore, strongly suggested that under normal conditions the inhibitory role played by the lens is mechanical in nature, i.e.,
Figs. 6, 7. Regeneration of the lens after lensectomy from the dorsal region of the eye and simultaneous incision of the outer cornea. Examples show lens regeneration from the pericorneal epidermis. x 220 Fig. 6. Ten days after operation: the regenerated lens is connected to pericorneal epidermis by epithelial clusters (mraws) Fig. 7. Fifteen days after operation the basement membrane of the epidermis is continuous with the lens capsule (urrows)
7
Fig. 8A-C. Diagram of various types of experiment. A Simple lensectomy. B Incision of the outer and inner cornea at dorsal iris without lensectomy. C Incision of the outer and inner cornea and perforation of the iris without lensectomy. (After Bosco et al. [ 3 ] )
Fig. 10. Diagram of various types of experiment. A , simple lensectomy; B, removal of the lens and neural retina; C, removal of the anterior complex (inner cornea, iris and ciliary body) and the lens; D,removal of the anterior complex, the neural retina and lens. (After Filoni et al. [16])
Fig. 9. The neoforming lens-vesicle (arrow) is located in front of the perforation of the iris. D.I., dorsal iris; O.L., old lens. x 320. (After Bosco et al. [3])
the lens occupying the pupillary space prevents spread of the factor(s) from the vitreous chamber, and thus prevents lens transdifferentiation of the outer cornea. This last conclusion is also supported by the results obtained from experiments in which the lens was replaced by a Millipore Filter disk to prevent direct communication between the vitreous and anterior chambers. Under these experimental conditions, lens transdifferentiation of the outer cornea is inhibited [9]. The above-reported data all suggest that in a normal eye of larval Xenopus laevis the phenotypic stability of the outer cornea is ensured by the presence of two barriers, both of which prevent the factor(s) necessary to trigger lens transdifferentiation of the outer cornea from spreading from the vitreous chamber towards the anterior chamber. At present there are some experimental data that indicate the neural retina is the tissue that produces the stimulating factor(s) needed for lens transdifferentiation of the outer cornea. Filoni et al. [16] effected various types of experiment (Fig. 10):
A. Simple lensectomy B. Removal of the lens and neural retina C. Removal of the anterior complex (inner cornea, iris, ciliary body) and lens D. Removal of the anterior complex, neural retina and lens In Experiment A, lens regeneration occurred from the outer cornea. In Experiment B, the removal of the neural retina prevented any lens transdifferentiation of the outer cornea; this result indicates that, in lens regeneration from the outer cornea in vivo, an essential role is played by the neural retina, and also indicates that lens transformation of the outer cornea cannot be sustained by pars ciliaris and pars iridea retinae alone. The removal of the anterior complex and lens effected in Experiment C did not prevent lens regeneration from the outer cornea (Figs. 11, 12). On the contrary, when the neural retina was removed together with the anterior complex and lens (Fig. lOD), lens regeneration from the cornea was completely inhibited. In these experiments a very slight degree of regeneration in the retina, left in situ, was observed. On average, the total volume of the retina ranged from 2.6% (day 3 after the operation) to 5% (day 15 after the operation) of the retinal volume of a normal eye in larvae at stage 50 (according to Nieuwkoop and Faber [29]). This small amount of regenerated retina was not able to promote lens regeneration from the outer cornea. Though the exact nature of the retinal factor(s) has not yet been determined, preliminary data indicate they have a protein nature, as pellets of whole protein complement of the eye cup induce lens-forming transformation of the outer cornea when implanted between the outer and inner cornea [17].
8
Figs. 11, 12. Lens regeneration from the outer cornea after removal of the anterior complex and lens. Fig. 11. Five days after operation: at the end of the epithelial cluster of corneal origin a lens vesicle at stage 4 (arrow) can be seen. C., outer cornea; R.,neural retina. x 320 Fig. 12. Seven days after operation: a lens-vesicle at stage 5 can be seen. x 350
My coworkers and I [6] to ascertain whether lens transdifferentiation of the outer cornea in vivo needed the continuous presence of the stimulus provided by the retinal factor(s) or whether, once triggered by this factor(s), the process could continue in the presence of nutritional factors alone recently carried out experiments based on implantation, between the outer and inner cornea of the host larvae, of the outer cornea taken from lensectomised donors at fixed intervals after lensectomy. The results showed that in vivo lens transdifferentiation of the implanted outer cornea required the continuous presence of retinal factor(s). Furthermore, on its own, the nutritional factor(s) supplied to the outer cornea by the aqueous humour could not sustain continued lens differentiation. Moreover, it was also incapable alone of maintaining the lens-transformation stage, attained by the lens-forming structures, that had previously been induced in the implanted outer cornea by retinal factor(s), whatever that stage was. Absence of the retinal factor(s) determined the arrest of transdifferentiation and then the regression of newly-formed structures. These data strongly suggested that the lens-transdifferentiation process of the outer cornea is not a single-step process, but a morecomplex one, which requires a sequence of interactions, extending over a long period of time, during which the retinal factor(s) must be present until complete lens transdifferentiation of the outer cornea is achieved. There is evidence that crystallin synthesis is regulated at all the generally recognized levels, i.e., transcription of RNA, post-transcriptional changes in RNA, and translation of proteins [27, 341. However, recent studies on the regulation of crystallin expression, carried out by transferring the chicken gene into mouse cells, indicate that the ~
~
level of crystallin synthesis is controlled mainly at the transcriptional level and that there are two steps in the process of transdifferentiation of the lens from neural retina in the chick: acquisition of the capacity to express crystallin genes, and derepression of the endogenous crystallin genes. It is probable that these two steps are involved in other cases of cell differentiation [21, 221. The retinal factor(s) of larval Xenopus luevis might act at any one of these levels. Thus, in vivo the lens-transdifferentiation process of the outer cornea is triggered and sustained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye the phenotypic stability of the outer cornea is guaranteed by the presence of the lens and inner cornea, which represent insurmountable barriers to the retinal factor(s) present in the vitreous chamber. This factor is necessary for the entire lens-transdifferentiation process of the outer cornea, and each of these barriers is able to prevent any direct communication between the outer cornea and the environment of the vitreous chamber. Although during lens regeneration the stimulus for lens transdifferentiation of the outer cornea is provided by the retinal factor(s), there is evidence that it can also be supplied by other tissues. Waggoner [47] grafted fragments of the outer cornea of larval Xenopus luevis into the stump of an amputated hindlimb and observed that in this heterotopic site lens transdifferentiation of the outer cornea is possible. Reeve and Wild [39] implanted pituitary, limb bud, or limb-bud blastema between the outer and inner cornea and demonstrated that these structures, deprived of any nervous connection, are able to trigger and support lens transdifferentiation of the outer cornea. Filoni et al. [18], implanting lumbar ganglia innervating normal and regener-
9
Fig. 14. Implant of tentacle blastema, 10 days after implanting. In the newly formed lens (arrow) the primary lens fiber nucleus has formed and formation of secondary lens fibers is underway (st.5). x 380. (After Bosco et al. [ 5 ] )
Fig. 13. Implant of tentacle blastema; 5 days after implanting. A small cell aggregate at stage 3 (arrow)has formed from the outer cornea. ox., outer cornea; ic., inner cornea. x700 (After Bosco et al. IS])
ating limbs between the outer and inner cornea, observed that these structures can induce lens transdifferentiation of the outer cornea, and that ganglia innervating regenerating limbs are more efficient in this action. These data were interpreted as indicating that the neurotrophic factor(s) that the nerves are thought to emit from their resected terminations during amphibian limb regeneration [14, 40, 481, and which appears to stimulate the division of blastemal cells, can replace the retinal factor(s) during lens transdifferentiation of the outer cornea. Recent investigations of limb regeneration in the newt, carried out with monoclonal antibodies to the cells of a regenerating limb, have provided new evidence about the origin of blastemal cells from Schwann cells and myofibers, and identified a subset of blastemal cells whose division is effectively dependent on the nerve supply [ 141. On the basis of these data, my coworkers and I undertook a series of experiments to establish whether the lensinducing capacity on the outer cornea was present in many other larval tissues or whether it was more restricted, and in the latter case whether one or more common features could be singled out [5]. Fragments of iris ring, spleen, kidney, tail, tail blastema, tentacle, tentacle blastema and spinal cord were implanted between the outer and inner cornea of a normal eye. Of these tissues, only the tail blastema, tentacle blastema and spinal cord showed the capacity to give rise to lens-forming transformation of the cornea (Figs. 13, 14), although to different degrees (Table 1). The other tissues induced no appreciable response in the outer cornea. All these data reinforced the hypothesis previously
advanced, on the basis of results obtained after implanting spinal ganglia between the outer and inner cornea, that lens transdifferentiation of the cornea could be stimulated by the neurotrophic factor produced by the ganglion cells. Since this hypothesis could be applied to lens transdifferentiation induced by spinal-cord neurons and also by dedifferentiated cells of tail blastema, Singer [40] proposed that all undifferentiated embryonic cells produced a neurotrophic factor. A comparison of data obtained in larval Xenopus luevis with those regarding lens transdifferentiation of iris epithelial cells of Urodela shows that tissues able to stimulate the newt iris (retina, spinal ganglia, pituitary) [12, 35, 361 correspond to those which stimulate lens transdifferentiation of the outer cornea in larval Xenopus luevis. In the newt it has been shown that the neurotrophic and pituitary factors promote proliferation, and a correlation has been established between proliferative activity of iris cells and their lens-type conversion [45, 461. Considering the fore-mentioned data, it is possible that, in larval Xenopus laevis also, proliferation of epithelial cells in the outer cornea is the direct target of lens-transdifferentiation-promoting factors. Lens transdifferentiation of the iris and retina Lens regeneration in anuran tadpoles has been the focus of numerous investigations. Some researchers have reported cases of lens regeneration, in larvae of various species, occurring through a process of Wolffian lens regeneration (cellular metaplasia or cell-type conversion), while others have obtained negative results, often in the same species. A series of investigations effected in our laboratory has shown that many cases reported as Wolffian regeneration can be attributed to reorganization of epitheliocapsular fragments of the lens remaining in situ following inadequate lensectomy. At present the available data concerning lens
10 Table 1. Summary of the results obtained following implantation of various larval tissues Experiment
I (Iris)
I1 (Spleen)
I11 (Kidney) IV (Tail) V (Tail blastema) VI (Tcntacle) VII (Tentacle blastema)
No. of cases operated
No. of cases dead/ discarded
No. of cases examined
No. of newly forming lenses
10 10 10
1
5 I 10
10 10 10
-
9 10 9 10 10 9
-
I 10
5
10 10 10
-
Days after operation
5
I 10
1
1
10 10 9 10 10 11
-
-
-
5 I 10
11 10 12
5
10 9 9
9
I 8
4 2 3
10 10 10
10 10 10
4 1 1
7 5
7 10
1 1
regeneration in anuran amphibian tadpoles lead us to consider that this phenomenon is much more restricted than previous data have shown [l]. As I mentioned in the previous paragraph, lens regeneration in anuran tadpoles has been demonstrated only in Xenopus luevis, in which the new lens is originated by a process of lens transdifferentiation. However, until some time ago it was not completely clear, even in this species, whether the lens-forming capacity was present, in situ, in other eye tissues as well. The possibility that lens transdifferentiation of the dorsal iris and retina occurs in Xenopus luevis larvae has been claimed to exist by Campbell [7] and by Overton and Freeman [33]. In particular, Campbell [7] reported that in lensectomised Xenopus luevis larvae the lens can originate from the pupillary edge of the iris (12% of the cases), from the outer cornea (12%), or from the retina near the ciliary body (56%). Nevertheless, the data we obtained in some investigations showed that, when lensectomy was performed without damaging the iris and retina, lens regeneration always occurred via lens transdifferentiation of the outer cornea [2, 151. It was, thus, interesting to re-examine experimentally the possibility of lens transdifferentiation of the iris and retina in situ. To this purpose five experiments were performed (Fig. 15) [4] : I. Simple lensectomy 11. Removal of the lens, outer cornea, and pericorneal integument 111. Removal of the lens, outer cornea, and pericorneal integument plus simultaneous incision of the retina IV. Removal of the lens, outer cornea, and pericorneal integument plus simultaneous incision of the dorsal iris V. Removal of lens and simultaneous lesion of iris and retina
5
-
10 10 15
10 10 14 10 10 11
4
-
5 I 10
-
3
-
10
1
2
Percentage of lens-forming transformations (total)
-
10 10 12
5
I
10
VIII (Spinal cord)
1
Lens regenerating stage
-
-
6 4 4 -
-
II
Ill
IV
Fig. 15. Diagram of various types of experiment; I, simple lensectomy; II, removal of lens, outer cornea, and pericorneal integument plus simultaneous incision of retina; ZII, removal of lens, outer cornea, and pericorneal integument plus simultaneous incision of retina; IV, removal of lens, outer cornea and pericorneal integument plus simultaneous incision of dorsal iris; V , removal of lens and simultaneous lesion of iris and retina. (After Bosco et al. [4])
11
Figs. 16-19. Removal of lens and simultaneous lesion of iris and retina. The series of figures shows a case of apparent lens regeneration from iris. The regenerating lens (st.5) is close up against the iris. Continuity with the cornea is visible in the last two photographs (arrow). c., cornea; di.,dorsal iris. x 600. (After Bosco et al. [6])
The results obtained showed that when lensectomy is performed without damaging eye territories other than the outer and inner cornea, lens regeneration takes place at the expense of the outer cornea only. The absence of the lens transdifferentiation capacity of the iris and retina under these experimental conditions cannot be explained on the basis of inhibition by lens-forming structures of the outer cornea, as there was still no lens transdifferentiation even when the cornea and pericorneal epidermis were removed. The iris and retina did not show in situ lens transdifferentiation capacity, even after they had been deliberately
stimulated, by comparatively extensive damage. The presence of regenerated or regenerating lens was sometimes observed under these experimental conditions, but at the same time it was possible to identify the territory of origin as the pericorneal epidermis or the cornea (Figs. 1 6 1 9 ) . Although these results show that the iris and retina have no lens-forming capacity in situ, the possibility that these tissues of larval Xenopus laevis can demonstrate this capacity under different experimental conditions cannot be excluded. Hoperskaya and Zviadadze [20] showed that pieces of the iris of adult Rana temporaria, wrapped in lens epithe-
12
len.
Fig. 20. Schematic representation of experiment performed. The isolated iris epithelium was combined with lens epithelium; precultivation of sandwiches in vitro for 3 days and implantation into a tadpole orbit. (After Hoperskaya and Zviadadze [20])
lium and cultivated for 3 days in a protein-free medium before implantation into the tadpole orbit, transform into lens in 32% of cases (Fig. 20). Lopashov [20, 211 cultivated sandwiches of lens epithelium of adult Rana temporaria with retinal pigmented epithelium from larvae of the same species in a protein-free medium before implantation into the tadpole orbit. He observed that under these conditions pigmented epithelium transformed into lentoids in 85% of cases. Thus, an event involving lens transdifferentiation of the iris (as in Wolffian regeneration) and of the retinal pigmented epithelium can occur in frogs.
Fig. 21. Diagram showing the two types of implantation of dorsal iris taken from a lensectomised eye. a, autoplastic implant of a dorsal iris fragment in the vitreous chamber of a lensectomised eye; b, autoplastic implant of a dorsal iris fragment in the anterior chamber of a lensectomised eye
Retinal transdifferentiation of the iris and retinal pigmented epithelium The iris and retinal pigmented epithelium of larval Xenopus laevis show a high degree of phenotypic stability in situ. This was found in iris tissue even when it had been damaged to various degrees in order to stimulate its latent transdifferentiative competence (see the previous paragraph). However, under some experimental conditions, the iris and pigmented epithelium undergo transdifferentiation processes. In fact, when isolated from its surrounding tissue and implanted in the vitreous chamber (Fig. 21) the dorsal iris undergoes a process of retinal transdifferentiation [lo]. The transdifferentiation process is effected by an initial phase of dedifferentiation of the iris epithelial cells, which gradually become depigmented. The subsequent phase is represented by a cell agglomeration that undertakes new differentiation in a retinal direction (Figs. 22, 23). It is interesting, and should be emphasized, that the degree of transdifferentiation of the iris depends on its localization in the eye environment (Fig. 24). Iris fragments in the vitreous chamber underwent complete retinal transdifferentiation ; implants in the pupil region were transformed into polarized vesicles, in which only the portion facing the vitreous chamber of the host was transdifferentiated. Unlike that of the vitreous chamber, the environment of the anterior chamber cannot provide the stimulus for retinal transdifferentiation of the iris. These data suggest that in the vitreous chamber of larval Xenopus laevis there is some factor(s) which stimulates the retinal-transdifferentiation process of the iris and is distributed in a gradient. Retinal transdifferentiation is also observed in the pigmented epithelium of the tadpole and adult Xenopus laevis
Fig. 22. Autoplastic implant of dorsal iris fragment in the vitreous chamber. Thirty days after implantation, retinal transdifferentiation of the implanted dorsal iris fragment has occurred. x 120. (After Cioni et al. [lo])
[41]. This eye territory, separated from the mesenchyme envelope and implanted into the lens-less eye, transforms into retina under the influence of the retinal factor(s). No retinal transdifferentiation occurs in pigmented epithelium when it is implanted into the enucleated orbit. Unlike the transformation of pigmented epithelium into retina, retinal transdifferentiation of the iris is not prevented by close contact between the iris epithelium and the stroma, a mesenchymal structure corresponding to Bruch's membrane in retinal pigmented epithelium. Retinal transdifferentiation of pigmented epithelium is also seen in larval and adult Rana temporaria [23, 251. In this species, retinal transdifferentiation occurs as in the case of the pigmented epithelium of Xenopus laevis. Moreover, the process can be stimulated by lens epithelium [23]. Fragments of retinal pigmented epithelium cultured in vivo, in contact with the lens epithelium in the tadpole orbit, after a period of intense proliferation, transform into retina. The same tissue, precultivated in vitro with lens epithelium for
13
Conclusion
Fig. 23. Implant of dorsal iris fragmcnt in the vitreous chamber; 30 days after implantation; detail of retinal-transdifferentkted dorsal iris. x 600. (After Cioni et al. [lo])
b
C
Fig. 24. Diagram to illustrate the hypothesis that a gradient of ocular factor(s) exists in the lensectomised eye; a, in the anterior chamber, the implant maintains its original structure, because the level of the inducing factors is below the threshold value; b, in the neighbourhood of the pupil, only the posterior portion of the vesicle receives the inducing factor(s) in quantities greater than the threshold value, thus resulting in transdifferentiation into neural retina (polarized vesicle); c, in the deeper portion of the vitreous chamber, the implanted iris receives the maximum quantity of factor(s), and so it is completely transformed into neural retina
3 days in protein-free medium, and then implanted in the enucleated orbit, transforms into lentoid (see the previous paragraph). As in lens transdifferentiation of the cornea, retinal transdifferentiation of the iris in larval Xenopus lueuis can be induced by nonocular tissues. Fragments of iris, autoplastically implanted into the stump of the amputated hindlimb, transdifferentiate into neural retina [ 1I] ; preliminary data obtained in our laboratory indicate that retinal transdifferentiation of the iris can be induced by the pituitary as well.
At present the available data show that the eye tissues of larval Xenopus laevis have a high degree of reprogramming capacity ; the outer cornea can undergo lens transdifferentiation, while the iris and pigmented epithelium can transdifferentiate into neural retina. The lens-transdifferentiation process of the outer cornea in situ after simple lensectomy is somewhat analogous to the ontogenic process of lens formation from ectoderm. In fact, lens transdifferentiation of the outer cornea is triggered and sustained by a retinal factor(s) that is present in the vitreous chamber. Under normal conditions, the phenotypic stability of the outer cornea is ensured by the integrity of the inner cornea and by the presence of the lens, which prevents spread of the retinal factor(s) towards the outer cornea. In retinal transdifferentiation of the iris, the availability of retinal factor(s) able to stimulate the transdifferentiative process is not sufficient to enable the iris to display its retinal-reprogramming capacity ; the iris must also be isolated from its surrounding tissues. Thus, the iris in normal conditions shows more phenotypic stability than the outer cornea. The situation regarding the retinal pigmented epithelium is quite different, since in a normal eye this tissue does not directly communicate with the vitreous chamber, where the retinal factor(s) is present. As regards the nature and action of the factor(s) that can stimulate transdifferentiative processes of the eye tissues in larval Xenopus laevis, some speculation can now be made. It has been demonstrated that lens transdifferentiation of the outer cornea can be promoted by several tissues or environments [5, 18, 39, 471, although during lens regeneration the stimulus is usually provided by the neural retina, which produces a factor(s) that is probably of a protein nature. Some of these tissues or environments can also induce retinal transdifferentiation of the iris (Table 2). One possible explanation of all, these data could be that the different tissues and environments contain some factor(s) that can obviously replace the retinal factor(s) responsible for stimulating the expression of latent reprogrammative capacities, specific to reacting tissue. In this way, while the outer cornea is always transformed into lens, the dorsal
Table 2. Summary of the tissues or environments stimulating transdifferentiation of the eye tissues in larval Xenopus laevis Reacting eye tissue
Stimulating tissue or environment
Type of transdifferentiation
Outer cornea
Neural retina Limb bud Amputated hindlimb Limb blastema Tentacle blastema Spinal ganglia Spinal cord Pituitary
Lens transdifferentiation
Dorsal iris
Neural retina Amputated hindlimb Pituitary
Retinal transdifferentiation
Retinal pigmented epithelium
Neural retina
14
iris, under the influence of the same tissues, always transdifferentiates into neural retina. On the other hand, it cannot be excluded that lens transdifferentiation of the outer cornea and retinal transdifferentiation of the iris and of retinal pigmented tissue are due to a specific stimulus exerted by different factors that are “instructive” in nature, i.e., by factors able to direct the reprogrammative process in the reacting tissues. If this is so, we have to admit that both factors are present simultaneously, not only in the vitreous chamber, but also in the amputated hindlimb and in the pituitary. The latter hypothesis seems more unlikely than the first. Lopashov [23, 24,261, on the basis of the data regarding the transdifferentiative phenomena of the eye tissues in larval and adult R a m temporaria (see previous paragraphs), formulated another interesting hypothesis, that the direction of the induced transdifferentiation could depend on the type of inducing influences, but this dependence is not unequivocal. The direction of transdifferentiation of frog iris pigmented cells (IPE) and of retinal pigmented cells (RPE) depends to some extent on directive influences from other tissues; however, this does not exclude the possibility that the multipotency of RPE an IPE can depend on the availability in them of some dormant programming determinants. In other words, the type of reprogrammation could depend on the balance between the intrinsic determinants and the influences exerted by type-inducing tissues under different experimental conditions. In Lopashov’s opinion [26] these programming determinants (or factors) are similar to those that are responsible for the induction of eye cells during ontogenesis. References 3 . Bosco L (1988) The problem of lens regeneration in anuran
amphibian tadpoles. Acta Embryol Morphol Exper (in press) 2. Bosco L, Filoni S, Cannata S (1979) Relationships between eye factors and lens-forming transformation in the cornea and pericorneal epidermis of larval Xenopus laevis. J Exp 2001 209~261-282 3. Bosco L, Filoni S, Cioni C (1980) Lens formation from cornea in the presence of the old lens in larval Xenopus laevis. J Exp ZOOI213:9-14 4. Bosco L, Filoni S, Paglioni S (1981) Experimental analysis of the lens-forming competence of the cornea, iris, and retina in Xenopus luevis tadpoles. J Exp Zool 216:267-276 5. Bosco L, Filoni S, Cioni C (1985) Experimental analysis on the capacity of several larval tissues to promote lens-forming transformations in the cornea of Xenopus luevis tadpoles. J Exp Zoo1 233 :221-228 6. Bosco L, Filoni S, Cioni C, Bernardini S (1986) Eye factors and lens-forming transformations of outer cornea in Xenopus laevis larvae. J Exp Zool 240 :401407 7. Campbell JC (1963) Lens regeneration from iris, retina and cornea in lensectomized eyes of Xenopus laevis. Anat Rec 145:214-215 8. Campbell JC, Jones KW (1968) The in vitro development of lens from cornea of larval Xenopus laevis. Dev Biol 17 : 1-1 5 9. Cioni C, Filoni S, Bosco L (1982) Inhibition of lens regeneration in larval Xenopus laevis. J Exp Zool 220: 103-108 10. Cioni C, Filoni S, Aquila C, Bernardini S, Bosco L (1986) Transdifferentiation of eye tissues in anuran amphibians : analysis of the transdifferentiation capacity of the iris of Xenopus laevis larvae. Differentiation 32: 21 5-220 13. Cioni C, Filoni S, Bosco L, Aquila C, Bernardini S (1987) Transdifferentiation of larval Xenopus luevis iris implanted into the amputated hindlimb. Experientia 43 :443-444
12. Connelly TG, Ortiz JR, Yamada T (1973) Influence of the pituitary on Wolffian lens regeneration. Dev Biol 31 : 301-315 13. Eguchi G (1986) Instability in cell commitment of vertebrate pigmented epithelial cells and their transdifferentiation into lens cells. Curr Top Dev Biol 20 :21-37 14. Fekete DA, Brockes JD (1987) A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts. Development 99: 589-602 15. Filoni S, Bosco L, Cioni C (1976) II problema della rigenerazione del cristallino degli Anfibi Anuri negli stadi post-embrionali. Esperienze di asportazione del cristallino in larve di Rana esculentu e Xenopus luevis. Acta Embryol Exper 3 : 319-334 16. Filoni S, Bosco L, Cioni C (1982) The role of neural rctina in lens regeneration from cornea in larval Xenopus luevis. Acta Embryol Morphol Exper ns., 3 : 15-28 17. Filoni S, Bosco L, Cioni C, Venturini G (1983) Lens-forming transformations in larval Xenopus laevis induced by denatured eye-cup or its total whole protein complement. Experientia 39:315-317 18. Filoni S, Bosco L, Cioni C, Burani P (1984) Lens-forming transformations in the outer cornea of larval Xenopus laevis induced by spinal ganglia. J Exp Zoo1 230 :409-416 19. Freeman G (1963) Lens regeneration from the cornea in Xenopus laevis. J Exp Zoo1 154: 39-65 20. Hoptrskaya OA, Zviadadze K G (1981) Transdifferentiation of adult frog iris in retina or lens by exogenous influences. Dev Growth Differ 23 :201-213 21. Kondoh H, Okada TS (1986) Dual regulation of expression of exogenous -crystallin gene in mammalian cells : a search for molecular background of instability in differentiation. Curr Top Dev Biol20: 153-164 22. Kondoh H, Ueda Y, Hayashi S, Okazaki K, Yasuda K, Okada TS (1987) An attempt to assay the state of determination by using transfected genes as probes in transdifferentiation of neural retina into lens. Cell Differ 20 :203-207 23. Lopashov GV (1983) Transdifferentiation of pigmented epithelium induced by the influence of lens epithelium in frogs. Differentiation 24 :27-32 24. Lopashov GV (1983) Induced and spontaneous transdifferentiation and their relation to induction in development. In : Goel SC, Bellairs R (eds) Developmental biology. Afro-Asian perspective. Indian Society of Developmental Biologists, Poona, pp. 87-96 25. Lopashov GV, Sologub AA (1972) Artificial metaplasia of pigmented epithelium into retina in tadpoles and adult frogs. J Embryol Exptl Morphol8 : 521-546 26. Lopashov GV, Golubeva ON, Zviadadze G (1986) Common determinants of embryonic induction and transdifferentiation. In: Progress in developmental biology, pp. 4 3 4 6 27. McAvoy JW (1980) Induction of the lens. Differentiation 17:137-149 28. Moscona AA (1986) Conversion of retina glia cells into lenslike phenotype following disruption of normal cell contact. Curr Top Dev Biol 20: 1-19 29. Nieuwkoop PD, Faber J (1956) Normal table of Xenopus laevis (Daudin). North-Holland, Amsterdam 30. Okada TS (1980) Cellular metaplasia or transdifferentidtion as a model for retinal cell differentiation. Curr Top Dev Biol 26 :360-380 31. Okada TS (1983) Recent progress in studies of transdifferentiation of eye tissues in vitro. Cell Differ 13: 177-1 83 32. Okada TS (1986) Transdifferentiation in animal cells: fact or artefact? Dev Growth Differ 28: 213-221 33. Overton J, Freeman G (1960) Lens regeneration in Xenopus laevis. Anat Rec 137 : 386 34. Piatigorsky J (1981) Lens differentiation in vertebrates. Differentiation 19: 134153 35. Powell JA, Powers C (1973) Effect on lens regeneration of implantation of spinal ganglia into the eyes of the newts, Nothophtalmus. J Exp Zoo1 183:95-114 36. Reyer RW (1966) The influence of neural retina and lens on
15 lens regeneration from dorsal iris implants in Triturus viridescens larvae. Dev Biol 14:214245 37. Reyer RW (1977) The amphibian eye: Development and regeneration. In: Handbook of sensory physiology. Vol. VIIj5. The visual system in vertebrates. Springer-Verlag Berlin Heidelberg, pp. 309-390 38. Reeve JG, Wild A (1978) Lens regeneration from cornea of larval Xenopus laevis in the presence of the old lens. J Embryol Exp Morphol48:205-214 39. Reeve JG, Wild A (1981) Secondary lens formation from the cornea following implantation of larval tissues between the outer and inner corneas of Xenopus laevis tadpoles. J Embryol Exp Morphol64: 121-132 40. Singer M (1965) A theory of the trophic nervous control of amphibian limb regeneration, including a revaluation of quantitative nerve requirements. In: Kiortis W, Trampusch HA1 (eds) Regeneration in Animals and Related Problems. North-Holland, Amsterdam, pp. 2G32 41. Sologub A (1977) Mechanisms of rcpression and derepression of artificial transformation of pigmented epithelium into retina in Xenopus laevis. Roux’s Arch Dev Biol 182:277-291 42. Stone LS (1958) Inhibition of lens regeneration in newt eyes
by isolating the dorsal iris from the neural retina. Anat Rec 120:151-172 43. Stone LS (1958) Lens regeneration in adult newt eyes related to retina pigmented cells and the neural retina factor. J Exp Zoo1 139:69-84 44. Stone LS, Steinitz H (1953) The regeneration of lenses in eyes with intact and regenerating retina in adult Triturus v. viridescens. J Exp Zool 124:435468 45. Yamada T (1977) Control mechanisms in the cell-type conversion in newt lens regeneration. Monogr Dev Biol Basel, Karger 13: 1-124 46. Yamada T (1982) Transdifferentiation of lens cells and its regulation. In: McDevitt DS (ed) Cell biology of the eye. Academic Press, New York, pp. 193-242 47. Waggoner PR (1973) Lens differentiation from the cornea following lens extirpation or cornea transplantation. J Exp Zool 186~97-109 48. Wallace H (1981) Vertebrate limb regeneration. John Wiley, Chichester Accepted in revised form August 27, 1988