Rend. Fis. Acc. La)~cei s. 9, v. 5:63-69 (1994)

Embriologia e morfogenesi. --Differentiation of transplanted lens epithelium of larval Xenopus laevis. Nota(*)di LUlGI Bosco e DANm~ WILLEMS, presentata dal Corrisp. E. Capanna.

ABSTaACr. - - In the present research the lens epithelium of larval Xenopus laevis was isolated and autoplastically implanted into the enudeated orbit to ascertain the autonomous differentiative capacity of this tissue. The results obtained show that the eye cup is not necessary for the differentiation of the lens epithelium into lens fiber ceils and suggest an intrinsic lens differentiation capacity of this tissue.

KEy WOROS: Lens epithelium; Differentiation; Xenopus laevis.

RIASSUNTO. - - Differenziamento di trapianti di epitelio della lente di lame di Xenopus laevis. Nella presente ricerca l'epitelio delia lente di larve di Xenopus laevis ~ stato isolato ed impiantato autoplasticamente a[ fine di accertare la possibile capacit~i di~erenziativa autonoma di questo tessuto. I risultati ottenuti mostrano che il clifferenziamento dell'epitelio della lente ~ indipendente dalla presenza dell'occhio e suggeriscono quindi I'esistenza di capacit~ differenziative autonome di questo tessuto oculare.

INTRODUCTION

During development of Vertebrates the lens formation is the result of inductive interactions by which the presumptive lens ectoderm becomes able to form a lens. A critical role in formation of the lens has been attributed to the optic vesicle (Spemann, 1901, 1938; Herbst, 1901). There are, however, reports of formation of a free lens in the absence of the optic vesicle (for a review see Jacobson and Sater, 1988). This finding has been ascribed to an inductive action of dorsolateral endomesodermal tissues on the presumptive lens ectoderm before the formation of the optic vesicle. Experimental data, recently obtained in Xenopus laevis (Henry and Grainger, 1987, 1990; Grainger et al., 1988) show that in lens morphogenesis the presumptive anterior neural plate is an essential early lens inductor and the dorsolateral endomesodermal tissues and optic vesicle are not individually sufficient inductors of the lens. The presumptive lens ectoderm isolated and cultured at early stages of development with the adjacent neural plate tissues is able to form a lens vesicle showing a lens-cell specifity as revealed by immunofluorescence test with anti-lens antibodies.

After the formation of the lens vesicle, the cells in the posterior part of the developing lens elongate and fill the cavity of the lens vesicle forming the primary fiber cells.

There is evidence that the optic cup environment induces cell elongation and primary fiber formation (for a review see McAvoy, 1980). The prolonged dependence of the elongation and differentiation of lens fiber cells upon retinal influences have been demonstrated experimenta]]y in chick (Coulombre and Cou]ombre, 1963) and mouse

(*) Pervenuta ali'Accademia i122 settembre 1993.

6 4 L. BOSCO - D. WILLEMS

(Muthukkaruppan, 1965) embroys. A factor called lentropin that promotes cell elonga- tion and cristallins synthesis in primary explants of embryonic chick lens epithelia has been partially purified from the vitreous body (Beebe et al., 1980).

After the completion of morphogenesis, the lens continues to grow throughout life by the proliferating activity of lens epithelium. Analysis of the pattern of DNA synthesis (Harding et al., 1959) and mitosis (Von Sallman, 1952) on lens epithelium indicates that cell proliferation in adult lens is confined to a narrow band of cells in the preequatorial region of the lens called the germinative zone, whereas fiber cells do not divide (for a review see McAvoy, 1980; Reddan, 1982).

In order to determine whether the lens epithelial cells of Vertebrates are able or not to grow and differentiate autonomally, the lens epithelium has been isolatecl in vivo and in vitro.

Takeichi (1970) observed the differentiation of fiber cells from 6-day-old chick embryo lens epithelium when it was transplanted onto the chorio-allantoic membrane. The lens epithelium explanted from a 6-day-old chick embryo maintained in tissue culture is able to differentiate into lens fiber cells (Piatigorsky et al., 1973).

Although primary explants of lens epithelia lose in tissue culture the ability to differentiate into lens fiber cells near the time of hatching (Piatigorsky and Rothschild, 1972), long-term cultures of dissociated cells from hatched chicken will develop fiber- like cells in the form of lentoid bodies (Okada et al., 1971).

As regards Amphibia, Mc Devitt and Yamada (1969) in a short communication reported that the lens epithelium of larval Rana pipiens will differentiate or <<modulate>) under specific tissue culture conditions to form cells that possess the morphological and biochemical/immunological characteristics of lens fiber cells. By contrast mature lens epithelial cells of amphibians proliferate rather than elongate when grown in organ culture (Reddan and Rothstein, 1966; Rothstein et al., 1965). However, a systematic research of isolated lens epithelial cells of a single species of Anura at different stages of development has not been conducted. In the present research we have tested the ability of lens epithelial cells from larval Xenopus laevis at stage 50 (according to Nieuwkoop and Faber, 1956) isolated from the eye and implanted into the enucleated orbit.

M A T E R I A L S A N D M E T H O D S

Implantation experiments were performed using Xenopus laevis tadpoles obtained from a single pair after ovulation and amplexus induced by gonadotrophic hormones (Profasi, Serono). During operations, animals were anaesthetized with MS 222 (Sandoz) 1:3000 and kept in full-strength Holtfreter's solution for about 24 h. After operation, the medium was changed progressively to tap water (24-25 ~ Larvae were fed on boiled nettle powder; they were fixed in Bouin's solution, embedded in paraffin and cut into 7 um thick serial sections. Sections were stained with Azan's method according to Heidenhain (1915).

Experimental design: Autoplastic implantation of the lens epithelium of right eye into the enucleated left orbit (60 operations).

D I I ~ F E R E N T I A T I O N O F T R A N S P L A N T E D . . . 65

The lens was removed from the right eye ofXenopus laevis at stage 50 using the same operative technique described in a previous paper (Bosco, 1988), placed into a plastic tissue culture dish containing full-strength Holtffeter's solution and the fiber mass was surgically separated from the epithelium. The epithelium was placed capsule-down on the surface of the culture dish and the peripheral regions were trimmed away with a micro scalpel.

The left eye cup, lens and inner cornea were removed through a dorsal incision using the operative technique described elsewhere (Bosco et al., 1993), and the lens epithelium previously isolated was implanted into the enucleated orbit. Fifteen larvae were fixed immediately afterward to serve as controls of operation. The other operated animals were fixed at 2, 4, 7 days after implantation.

Preparation of anti-total-lens protein antibody: Xenopus laevis adults were sacrificed and the lenses were removed and freed from surrounding tissue. The lenses were washed three times in ice-cold 5 mM phosphate buffer, pH 7, and stored at - 20 ~ Soluble lens proteins were obtained by homogenizing the lenses in the same buffer and removing insoluble materials by centrifugation following the procedure of McDevitt and Brahma (1973). Female rabbits were injected subcutaneously with a 1:1 mixture of complete Freund's adjuvant and 250 mg of soluble lens proteins, three or more times at intervals of three weeks. The animals were bled one week after a booster injection of the soluble proteins without adjuvant. The serum obtained was tested against the homologous antigen by immunoelectrophoresis in 1% agarose with 0.05 mM veronal buffer, pH 8.4, on microscope slides at 5 volts/cm. Staining was performed with Coomassie Brillant Blue R-250.

Retrospeclive immunofluorescence: Retrospective immunofluorescence of the antigen was carried out according to Mikailov and Gorgolyuk (1979). The preparation was placed into chilled xylene to remove canada balsam (10 ~ 2-7 h), and washed first in 2-3 changes of 95 ~ ethanol (1 h each) and then in 3 changes of buffered saline (pH 7.1, 30 min. each). The above anti-total-lens protein antibody was utilized as the bottom layer in the <~sandwich~ immunofluorescence technique of Weller and Coons (1954), in combination with commercial (Nordic, Pharmaceuticals and Diagnostic, The Netherlands) goat anti- rabbit gamma globulin antibody conjugated with fluorescein isothiocyanate.

R E S U L T S

The histological examination of the lens epithelia effected immediately after the isolation and implantation into the enucleated orbit showed that they consisted of a single layer of cuboidal cells (fig. 1, I) with the adhering capsule that normally delimits the outer surface of the lens (fig. 1, 2).

In the cases examined two days after the operation, the implanted lens epithelium fragments had originated hollow or solid vesicles (fig. 1, 3) that showed an active cellular proliferation. Four days after operation in 13 out of 15 cases, the newly formed

66 L . B O S C O - D . W I L L E M S

Fig. 1. - Autoplastic implantation of lens epithelium of larval Xenopus laevis at stage 50, into the enudeated orbit. 1: lens epithelium Rxed immediately after the operation, x 1360; 2: normal lens of larval Xenopus laevis at stage 50. x 680; 3: two clays after operation: the implanted lens epithelium originates hollow or solid vesicles, x 1360; 4: four days after operation: in the inner part of the newly-formed lens-like structure a

primary lens fiber nucleus (arrow) is observed, x 850.

aggregates were formed of elongated acidophilic cells with granular cytoplasm (fig. 1, 4), all histological distinctive features of the lens fiber cells (Papaconstantinou, 1967). The

lens specificity of the newly-formed structures was confirmed by high positive reaction to

the indirect immunofluorescence method with anti-total-lens proteins antibody (fig. 2). The newly-formed lens-like structure did not show the ordinate organization

DIFFERENTIATION OF TRANSPLANTED ... 67

typical of a normal lens. In most cases the lens fiber cells were present also at the boundary of the newly-formed structure and a lens epithelium was not observed. In some case in the inner part of the lens-like formation a primary lens fiber nucleus could be observed (fig. 2).

Fig. 2. - Autoplastic implantation of lens epithelium of larval Xenopus laevis at stage 50 into the enucleated orbit. Four days after operation: the newly-formed structure gives a positive reaction by the immunofluore-

scent staining method with anti-total-lens proteins, x 1200.

The cases examined seven days after operation did not reveal any substantial difference if compared with those examined four days after operation.

DISCUSSION

The results obtained in this research show that lens epithelium of larval Xenopus laevis at stage 50 is able to grow and differentiate into lens fiber cells when isolated from the eye and transplanted into the enucleated orbit.

The differentiation of lens fiber cells is realized after a proliferative activity of the implanted fragment. The lens-like structures formed in the enucleated orbit do not show an ordinate organization corresponding to that of a normal lens. In most cases, in the inner region of the transplanted fragment the formation of prinaary lens fiber nucleus occurred, but neither secondary lens fibers forming a secondary nucleus nor external well differentiated lens epithelium were observed. The newly-formed lens-like structures tested by indirect immunofluorescence method with anti-total-lens antibodies showed the highest positive reactivity in correspondance of the primary lens fiber nucleus and a diffuse positive reaction through all the newly-formed structure.

Prolonged dependence of lens growth and differentiation upon retinal influences is demonstrated by the classic lens inversion experiment of Coulombre and Coulombre (1963) where they turned the lens of a 5 day-old chick through 180 ~ so that the epithelium which normally faced away from the eye cup now faced the eye cup. Under the influence of the new environment the epithelial cells of the central zone elongated

68 L. BOSCO - D. WILLEMS

and differentiated into lens fiber cells. Subsequently, data obtained in mouse and chick under different experimental conditions confirmed this result (Muthukkaruppan, 1965; Philpott and Coulombre, 1965; McAvoy, 1980; Beebe et al., 1980).

On the contrary, the present results show that the Xenopus lens epithelium can grow and differentiate without any influence of the eye cup. Different explanations are possible. The eye of the larval Xenopus could be a <~system>> substantially different from that of chick and mouse; the influence of the retina on the differentiation of the lens epithelium pointed out in chick and mouse could be absent in Xenopus. Another possibility, which has not been rigorously excluded by the present experiment is that the retinal factor(s) influencing lens fiber differentiation or its equivalent was present in body circulation of the enucleated larval Xenopus. Preliminary results obtained, in our laboratory, from in vitro experiments seem to exclude this latter hypothesis. We are now carring on further experiments in order to clarify this process.

This work was supported by the National Council of Researches (C.N.R.), grant n. 90.03224. CT04, and by the Ministry of Education.

I wish to thank Prof. G, Chieffi for the critical review of the manuscript.

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