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Keywords:

  • cornea;
  • lens;
  • optic vesicle;
  • Xenopus laevis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Many studies on lens development have demonstrated that in Xenopus laevis the early signals from the anterior neural plate perform an essential role by establishing a lens-forming bias in head ectoderm. During gastrula stages, all non-neural ectoderm has some competence to respond to lens-inductive interactions, but this potential becomes restricted to the presumptive lens ectoderm (PLE) and the surrounding regions of the head during neurulation (Henry & Grainger, 1987, 1990; Servetnick & Grainger, 1991; Grainger et al. 1997). At the end of neurulation, enough lens induction has occurred to commit the PLE to a lens-forming fate. In fact, this tissue differentiates lens cells when cultured in vitro and forms ‘free lenses’ in embryos submitted to the removal of the optic vesicle just prior to the time the developing optic vesicle comes into contact with the PLE (Henry & Grainger, 1990; Grainger, 1992). Instead, the optic vesicle, once thought to be sufficient for lens induction, appears to be a very weak lens inductor. In fact, the transplantation of the optic vesicle under the competent non-biased ventral ectoderm does not promote lens formation. The optic vesicle promotes lens differentiation only when is transplanted under the biased head ectoderm (Grainger et al. 1988, 1997; Saha et al. 1989).

After lentectomy, larval X. laevis can regenerate a new lens through the process of cornea–lens transdifferentiation (Freeman, 1963). This process is triggered by factors present in the vitreous chamber and produced by the neural retina (Filoni et al. 1982, 2006; Bosco et al. 1993). Fragments of outer cornea or pericorneal epidermis implanted into the vitreous chamber transdifferentiate into lens (Reeve & Wild, 1978; Filoni et al. 1980; Bosco & Filoni, 1992), whereas fragments of epidermis outside the lentogenic area (the area extending for twice the diameter of the eye cup, according to Freeman, 1963) maintain the epidermal phenotype (Bosco & Filoni, 1992; Cannata et al. 2003).

Embryological manipulations on X. laevis embryos and larvae have demonstrated that the maintaining of the lens-regenerating competence (that is, the capacity to regenerate a lens under the influence of the retinal factor) in the lentogenic area of the larva is the result of tissue interactions linked to the bias of head ectoderm during the early phase of lens induction and to corneal induction during the last phase of eye development (Cannata et al. 2003). In fact, the removal of the optic vesicle at neurula stages does not prevent the maintenance of the lens-regenerating competence in the lentogenic area of the larva. Moreover, when an eye is transplanted under the head epidermis outside the lentogenic area of an early larva, this promotes the re-acquisition of the lens-regenerating competence in this epidermis (Arresta et al. 2005). Recently (Gargioli et al. 2008), by using Real-time-PCR, we observed that a relationship exists between lens-regenerating competence in the cornea and epidermis of larval X. laevis and pax6 expression. In fact, pax6 is expressed only in the lentogenic area and is re-expressed in the head epidermis covering a transplanted eye. Moreover, following pax6 RNA injection in the embryo at two-cell stage, pax6 misexpression promoted lens-regenerating competence in the ventral epidermis. These data indicated that pax6 was a factor of lens-regenerating competence. Because during eye development the bias of the PLE is characterized by pax6 expression, which follows the decrease of otx2 expression (Zygar et al. 1998; Ogino & Yasuda, 2000; Khosrowshahian et al. 2005), we hypothesized that in the larval cornea and epidermis with lens-regenerating competence a condition similar to that of the embryonic biased ectoderm is maintained or acquired (Gargioli et al. 2008). In this paper we verify this hypothesis by testing the capacity of the outer cornea and epidermis with lens-regenerating competence to respond to the optic vesicle, the very weak inductor which promotes lens formation only in the biased ectoderm. The rationale underlying this study is that if the lens-regenerating competence in the outer cornea and epidermis is similar to the lens-forming competence of the biased ectoderm, then the optic vesicle should promote lens formation also in the lentogenic areas of the larva. We show that competent larval cornea and epidermis synthesize lens proteins under the influence of the embryonic optic vesicle such as the biased ectoderm, confirming our previous hypothesis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The X. laevis embryos and larvae were obtained by hormone-induced mating, kept at a temperature of 20 °C and staged according to the normal table of Nieuwkoop & Faber (1956). In preparation for surgery, tadpoles were anaesthetized in 1 : 3000 MS 222 (Sigma, St Louis, USA).

Surgical procedures

Operations on embryos

Before the operations, the jelly coat and the vitelline envelopes were removed from the embryos with fine tweezers. To retain the embryo on its side (right side) during surgery, it was placed within a tiny depression made in plastic modeling clay which lined the base of a Petri dish.

Optic vesicle and eye cup removal in embryos at stages 22/23 and 35/36. In embryos at stage 22/23, the optic vesicles have achieved contact with PLE (Henry & Grainger, 1987). In embryos at stage 35/36 the retinal layers of the eye cup begin to differentiate (Nieuwkoop & Faber, 1956). The PLE of embryos at stage 22/23 and the presumptive outer cornea of embryos at stage 35/36 were lifted using hair loops and a tungsten needle. The exposed optic vesicle of embryos at stage 22/23 and the eye cup of embryos at stage 35/36 were then excised with a tungsten needle. The operations were conducted in 100% Steimberg's solution containing 100 ug/mL−1 streptomycin, 100 units mL−1 penicillin (Gibco-Invitrogen Paisley, UK).

Operations on larvae

During the operation and for 24 h afterwards, the larvae were kept in full-strength Holtfreter's solution. Thereafter, they were gradually transferred to dechlorinated tap water and fed on powder nettles.

Five different kinds of experiments were carried out (Fig. 1).

image

Figure 1. Diagram of experiments I–V. I. Experiment I: Implant of an optic vesicle taken from an embryo at stage 22/23 plus an outer cornea fragment of a larva at stage 53. II. Experiment II: Implant of an outer cornea fragment of a larva at stage 53. III. Experiment III: Implant of an eye cup taken from an embryo at stage 35/36 plus an outer cornea fragment of a larva at stage 53. IV. Experiment IV: Implant of an optic vesicle taken from an embryo at stage 22/23 plus a fragment of head epidermis covering a transplanted eye of a larva at stage 53. V. Experiment V: Implant of an optic vesicle taken from an embryo at stage 22/23 plus a fragment of head epidermis of a larva at stage 53. In all experiments, the different tissues were implanted into the enucleated orbit of stage 56 host larvae together with a Millipore filter disk. e.c., eye cup; e.o., enucleated orbit; f, filter disk; h.e., head epidermis; h.e.c.t.e., head epidermis covering a transplanted eye; o.c., outer cornea; o.v., optic vesicle.

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Experiment I: Implant into the enucleated orbit of a sandwich of outer cornea plus optic vesicle. Each sandwich, consisting of an optic vesicle between two sheets of the outer cornea, was made in the following manner. The optic vesicle taken from an embryo at stage 22/23 was placed in a hole made in the centre of a Millipore filter disc with a diameter of 0.6 mm. Both sides of the filter were covered with a sheet of outer cornea and pericorneal epidermis dissected from stage 53 larvae. The inner surfaces of the two corneal sheets faced the optic vesicle. Thanks to the filter, the inner layer of the cornea remained in tight proximity to the optic vesicle. The sandwich was then implanted into the enucleated orbit of a host larva at stage 56. Twenty sandwiches were implanted. The host larvae were sacrificed 72 h after implanting.

Experiment II: Implant into the enucleated orbit of outer cornea. Both sides of a Millipore filter disc similar to that used in experiment I were covered with a sheet of outer cornea and pericorneal epidermis dissected from a stage 53 larva. Twenty sandwiches were implanted into the enucleated orbit of stage 56 host larvae. These larvae were sacrificed 72 h after implanting.

Experiment III: Implant into the enucleated orbit of a sandwich of outer cornea plus eye cup. Each sandwich, prepared as described in experiment I, consisted of an eye cup taken from an embryo at stage 35/36 placed between two sheets of outer cornea and pericorneal epidermis dissected from stage 53 larvae. Twenty sandwiches were implanted into the enucleated orbit of stage 56 host larvae. These larvae were sacrificed 48 h after implanting.

Experiment IV: Implant into the enucleated orbit of a sandwich of head epidermis covering a transplanted eye plus optic vesicle. After detaching the very thin adhesion between the outer and inner corneas of the right eye of stage 44 larvae, the outer cornea was removed. The eye was enucleated and auto-transplanted beneath the epidermis covering the endbrain (this epidermal region is outside the lentogenic area, Freeman, 1963). The transplant bed was obtained after removing the dorsal part of the endbrain. To ensure that the enucleated eye did not carry some outer cornea with it, each eye was carefully examined under the stereomicroscope before transplantation. Moreover, 10 eyes were fixed in Bouin's liquid immediately after enucleation (operative control), dehydrated and embedded in paraffin. The 7-µm-thick serial sections were stained with haematoxylin & eosin. In no case was any outer cornea adhering to the inner cornea observed. After the operation, the larvae were transferred to Holtfreter's solution and allowed to reach stage 53.

Each sandwich, prepared as described in experiment I, consisted of an optic vesicle taken from an embryo at stage 22/23 between two sheets of head epidermis covering a transplanted eye, dissected from stage 53 larvae. Twenty sandwiches were implanted into the enucleated orbit of stage 56 host larvae. These larvae were sacrificed 72 h after implanting.

Experiment V: Implant into the enucleated orbit of a sandwich of head epidermis plus optic vesicle. Each sandwich, prepared as described in experiment I, consisted of an optic vesicle from an embryo at stage 22/23 placed between two sheets of head epidermis covering the endbrain, dissected from stage 53 larvae. Twenty sandwiches were implanted into the enucleated orbit of stage 56 host larvae. These larvae were sacrificed 72 h after implanting.

To identify the implanted tissue, all experiments were performed using, as nuclear marker, 5-bromodeoxyuridine (5-BrdU, Amersham Int., Little Chalfont, UK), which is incorporated into replicating DNA. Labeled cornea or epidermis fragments were excised from stage 53 donor larvae repeatedly injected with the 5-BrdU (one intraperitoneal injection of 3 µL every 24 h, for 3 days). The host larvae were sacrificed in 1 : 1000 MS 222 and fixed in Carnoy's liquid. Their right orbits were then dehydrated, embedded in paraffin, cut into 7-µm-thick serial sections and tested for detection of both lens proteins and incorporated BrdU in the implanted tissue, as follows. Dewaxed sections were hydrated and incubated with an anti-lens polyclonal antibody (pAbL) (Filoni et al. 1995). Secondary antibody HRP-conjugated goat anti-rabbit (Biorad, Hercules, USA) was then applied and the immunoreactions were developed using 3-amino-9-ethylcarbazole as substrate (AEC, Sigma). After washing in phosphate-buffered saline (PBS), sections were incubated with an anti-BrdU monoclonal antibody (Amersham Int.). The bound antibody was detected using peroxidase-conjugated antibody against mouse immunoglobulin and polymerizing diaminobenzidine in the presence of nickel and cobalt (Amersham Int.). Implants were scored positive for lens transdifferentiation only when they reacted positively to pAbL.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Experiment I: implant into the enucleated orbit of stage 53 outer cornea plus stage 22/23 optic vesicle

In 12 of 20 (60%) cornea implants examined 3 days after implanting, some cells, isolated or reunited in a small aggregate, underwent pAbL positive lens transformations (Table 1, Fig. 2A).

Table 1.  Results of implantation of outer cornea or epidermis from stage 53 larvae into the enucleated orbit of stage 56 host larvae. In experiments I, IV and V, fragments of outer cornea or epidermis were implanted together with optic vesicles taken from stage 22/23 embryos. In experiment III, fragments of outer cornea were implanted together with eye cups taken from stage 35/36 embryos. The implants were examined 2 or 3 days after implantation
Experiment number (implanted tissue)No. of implants examinedNo. of TS implants (pAbL+)TS%
  • a

    Implants were fixed 3 days after implantation.

  • b

    Implants were fixed 2 days after implantation.

  • TS, transdifferentiated implants, positive to pAbL (anti-lens polyclonal antibody).

I (optic vesicle plus outer cornea)a201260
II (outer cornea)a20 0 0
III (eye cup plus outer cornea)b20 0 0
IV (optic vesicle plus head epidermis covering a transplanted eye)a201155
V (optic vesicle plus head epidermis)a20 0 0
image

Figure 2. Results of experiments I–V. Immunohistochemistry with anti-lens polyclonal antibody (pAbL) and anti-Brdu monoclonal antibody on fragments of outer cornea or epidermis dissected from BrdU-injected stage 53 larvae. The tissues were implanted into the enucleated orbit of normal non-injected stage 56 host larvae together with a Millipore filter disk. In all experiments, several cells of the implants were BrdU-labeled (black nuclei, white arrowheads) indicating the donor origin of the tissue. A. Experiment I. Implant of a larval cornea fragment plus an optic vesicle taken from an embryo at stage 22/23 3 days after implantation. Red pAbL positive cell aggregate is shown (arrow), revealing lens transdifferentiation in part of the cornea implant labeled by black BrdU positive nuclei. Inner box, detail of the implant showing black nuclei in the transdifferentiated (red) part of the implant. B. Experiment II. Implant of a larval cornea fragment 3 days after implantation. No transdifferentiation was observed in the implanted cornea, as in the BrdU-labeled tissue (black nuclei), no red pAbL positive signal was detected. C. Experiment III: Implant of a larval cornea fragment plus an eye cup taken from an embryo at stage 35/36 2 days after implantation. No transdifferentiation was observed in the implanted cornea, as in the BrdU-labeled tissue (black nuclei), no red pAbL positive signal was detected. D. Experiment IV. Implant of a fragment of larval head epidermis covering a transplanted eye plus an optic vesicle taken from an embryo at stage 22/23 3 days after implantation. Red pAbL positive cell aggregate is shown (arrow), revealing lens transdifferentiation in part of the head epidermis implant labeled by black BrdU-positive nuclei. Inner box, detail of the implant showing black nuclei in the transdifferentiated (red) part of the implant. E. Experiment V. Implant of a fragment of larval head epidermis plus an optic vesicle taken from an embryo at stage 22/23 3 days after implantation. No transdifferentiation was observed in the implanted head epidermis, as in the BrdU labeled tissue (black nuclei), no red pAbL-positive signal was detected. The arrow marks the retinal pigmented epithelium. ec, eye cup; f, filter disk; ov, optic vesicle. Scale bar: 50 µm.

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Experiment II: implant into the enucleated orbit of stage 53 outer cornea

None of the 20 cornea implants examined 3 days after implanting showed signs of lentogenic transdifferentiation as all the implant cells were pAbL negative (Table 1, Fig. 2B).

Experiment III: implant into the enucleated orbit of stage 53 outer cornea plus stage 35/36 eye cup

None of the 20 cornea implants examined 2 days after implanting showed signs of lentogenic transdifferentiation as all the implant cells were pAbL negative (Table 1, Fig. 2C).

Experiment IV: implant into the enucleated orbit of stage 53 head epidermis covering a transplanted eye plus stage 22/23 optic vesicle

In 11 of 20 cases (55%) examined 3 days after implantation, some epidermal cells, isolated or reunited in a small aggregate, underwent pAbL positive lens transformations (Table 1, Fig. 2D).

Experiment V: Implant into the enucleated orbit of stage 53 head epidermis plus stage 22/23 optic vesicle

None of the 20 cases examined 3 days after implanting showed signs of lentogenic transdifferentiation as all the transplant cells were pAbL negative (Table 1, Fig. 2E).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The results of experiments I and II show that, in corneal fragments placed in contact with stage 22/23 optic vesicle and implanted into the enucleated orbit of host larvae, the synthesis of crystallins was revealed by specific anti-lens antibodies as early as the third day after operation. On the other hand, cornea fragments implanted into the enucleated orbit without the optic vesicle maintained the corneal phenotype. This indicates that the cornea of X. laevis larvae responds not only to the inductive action of the factor produced by the larval neural retina (Filoni et al. 1982, 2006; Bosco et al. 1993), but also to signals coming from the embryonic optic vesicle. However, after little more than a day (26 h, at a temperature of 20 °C) the optic vesicle at stage 22/23 becomes, at stage 35/36, an eye-cup in which the retinal layers start to form (Nieuwkoop & Faber, 1956). Thus, a possible objection arises that the transdifferentiation of the cornea implants of experiment I was triggered not by the optic vesicle but by the retinal tissue which it derives from. This is unlikely, as in such a hypothesis the retinal factor would have promoted the synthesis of crystallins in the cornea in less than 2 days, whereas it is known that, after lentectomy or corneal implantation into the vitreous chamber, crystallin synthesis in corneal cells is detectable by immunoreactions only from the third day after operation (Filoni et al. 2006). In fact, the results of experiment III demonstrate that there is no detectable synthesis of crystallins in corneal fragments transplanted in the enucleated orbit together with a stage 35/36 eye cup and fixed after 2 days. Therefore, in experiment I the optic vesicle has exercised its inductive action on corneal cells from the moment of implant.

Similarly to what happens for the cornea, fragments of head epidermis covering a transplanted eye start the synthesis of crystallins as early as the 3rd day (experiment IV) when they are implanted into the enucleated orbit together with the optic vesicle at stage 22/23. However, fragments of head epidermis implanted without the optic vesicle maintain the epidermal phenotype (experiment V). Therefore, the transplant of an eye below the head epidermis restores the capacity to respond not only to the inductive action of the retina, as previously observed (Arresta et al. 2005), but also to signals coming from the optic vesicle just as the cornea does.

Taking the results obtained in the present work as a whole, it is clear that the optic vesicle is able to promote synthesis of crystallins in the competent larval areas. As the optic vesicle is considered a very weak inductor, capable of promoting crystallin synthesis only on an ectoderm that has undergone bias (Henry & Grainger, 1987; Grainger et al. 1988; Saha et al. 1989), our results here indicate that in X. laevis larvae the persistence and re-acquisition of the lens-regenerating competence corresponds to the persistence and re-acquisition of a condition similar to that of the biased ectoderm. Thus, the data obtained in this work using techniques of experimental embryology confirm the data previously obtained by Real-time RT-PCR on gene expression in the lentogenic areas of larval X. laevis (Gargioli et al. 2008). In fact, the Real-time RT-PCR analysis showed that the pattern of gene expression in these areas was similar to that observed in the early embryo during the PLE bias. Nevertheless, there is a fundamental difference between the biased PLE and the larval cornea and epidermis with lens-regenerating competence. Whereas the biased ectoderm can auto-differentiate, forming free lenses in the absence of the optic vesicle (Henry & Grainger, 1990), larval cornea and epidermis with lens-regenerating competence always need an inducer to transdifferentiate into lens tissue.

Henry & Mittleman (1995) observed that embryonic ectoderm implanted into the vitreous chamber of host tadpoles differentiated lens cells in a greater percentage than embryonic ectoderm transplanted over the embryonic optic vesicle. The authors concluded that the stronger inductive action of the tadpole eye cavity might be related to the large size of the larval eye and to a greater concentration of lens inductive factors. If this is true, given that the outer cornea is capable of responding to the optic vesicle as well, the retinal inductor factor level in the larval eye would be superior to that required by the cornea for lens transdifferentiation. As lens regeneration is a multi-step process requiring an adequate retinal level right up to the final stage (Reeve & Wild, 1978; Bosco et al. 1979, 1986, 1993), it is likely that the presence of a high concentration level of inductive factor in the larval eye increases the probability that after lentectomy the cornea to lens transdifferentiation process is not only triggered, but is also completed.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Grant sponsor: MIUR (Ministero dell’Università e della Ricerca Scientifica); Grant number 2004057145-006.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References