Rend. Fis. Ace. la)wei s. 9, v. 5:261-268 (1994)

Embriologia e morfogenesi . - - First evidence o f lens-transdifferentiation o f larval

Xenopus laevis induced by brain-detCved acidic FGF. Nota di LUIGI B o s c o , GIORGIO VENTtrVaNI e D a N m ~ WmLEMS, presentata (*) dal Corrisp. E. Capanna.

ABSTRACT. - - It has been shown that lens regeneration from outer cornea in larval Xenopus laevis is de- pendent on the influence of neural retina both in situ and in tissue culture. In the present research the outer cornea of larval Xenopus laevis was cultured for 4-10 days in serum supplemented diluted Leibovitz L 15 added with 500 ng/rul of brain derived acidic FGF. The obtained results show that aFGF can stimulate lens-forming transformation of the outer cornea and suggest that this growth factor may be one of the pep- tidic factors normally produced by the retina during lens regeneration.

KEY WORDS: FGF; Lens; Transdifferentiation; Anura.

RtaSSUNTO. - - Primi risultati di transdifferenziamento lentogeno della cornea di larve di Xenopus laevis in- dotto da aFGE Precedenti ricerche hanno dimostrato chela rigenerazione della lente dalla cornea esterna di larve di Xenopus laevis dipende dall'influenza esercitata dalla retina neurale sia in situ sia in coltura di tes- suto. Nella presente ricerca la cornea esterna dl larve di Xenopus laevis ~ stata coltivata in vitro in Leibovitz L 15 diluito, in presenza di siero fetale di vitello e di 500 ng/ml di FGF acido. I risultati ottenuti evidenzian- do che il transdifferenziamento lentogeno della cornea pub essere stimolato da aFGF e suggeriscono che tale fattore di crescita potrebbe essere prodotto dalla retina neurale durante la rigenerazione della lente.

INTRODUCTION

Among the Anura lens regeneration appears to be a very limited phenomenon (for a

review see Bosco, 1988a; Bosco et al., 1993a); only Xenopus laevis larvae are able to re- generate a lens after lentectomy through a transdifferenziative process of the outer cornea (Freeman, 1963). Epithelial cells of the deep layer of the outer cornea are able

to reprogram differentiation into a new pathway during lens regeneration under the in- fluence of a factor(s) produced by the neural retina, both in situ and in cornea tissue culture (Bosco et al., 1993b). Results obtained in grafting experiments have demon- strated that lens regeneration from cornea in Xenopus laevis larvae can be triggered and sustained by the neural retina isolated from larvae and adults of different species which

are not able to realize lens regeneration (Bosco and Wlllems, 1992; Bosco et al.,

1993c). Moreover, although during tens regeneration the stimulus for lens-transdiffer- entiation of the outer cornea is provided by neural retina there is evidence that, under different experimental conditions, it can be supplied by other tissues, i.e. pituitary,

spinal ganglia, limb bud, amputated hind limb, limb blastema, tentacle blastema and

spinal cord (for a review see Bosco, 1988b). It has been hypothesized that all the above mentioned tissues can secrete lens re-

generation stimulating growth factors (Bosco, 1988b). As far as the Urodela are concerned, newts and other Salamandridae (Stone, 1967)

(*) Nella seduta del 12 marzo 1994.

262 L. BOSCO ET AL.

can regenerate a new lens from the dorsal iris margin (Wachs, 1914). The lens regener- ation is stimulated by the neural retina both in the eye (Wachs, 1914) and in iris organ culture (Connelly et al., 1973; Yamada et al., 1973). In iris organ culture lens regenera- tion did not occur when no stimulating agents were present (Eguchi, 1967; Stone and Gallagher, 1958). The lens regeneration from dorsal iris can be sustained also by the pi- tuitary in situ and in organ culture (Connelly et al., 1973; Cuny et al., 1984; Zalokar, 1944).

A comparison of the lens regeneration process between Anura and Urodela clearly reveals some affinities.

Recently Cuny et al. (1986) cultured newt dorsal iris in a medium added with vari- ous concentrations of Fibrobast Growth Factor (FGF), a mitogen for many mesenchy- mal cells; bovine retina-derived basic Fibroblast Growth Factor (Eye Derived Growth Factor I), acidic FGF (EDGF II), non retained retinal factor after heparin-affinity cro- matography (EDGF HI) or brain-derived basic FGF. These authors observed that EDGF II and EDGF III can stimulate lens regeneration from cultured newts irises.

In the present paper we report the first results showing, the lens-transdifferentiation of larval Xenopus laevis outer cornea stimulated in vitro by bovine brain derived acidic FGF.

M A T E R I A L S A N D ME'VIIODS

Three experiments were carried out. In all the experiments larvae of Xenopus laevis at stage 49-50 (according to Nieuwkoop and Faber, 1956) were used. All larvae were obtained from a single pair after amplexus and ovulation induced by gonadotropic hor- mones (Profasi, Serono). In all the experiments, larvae were anaesthetized with MS 222 (Sandoz) 1:3000 before operation and fixation.

Evperiment I (control experiment): simple lentectomy of larval Xenopus laevis at stage 49-50 (30 operations). This experiment was carried out to determine the percentage of regeneration, as this percentage varies considerably (Waggoner and Reyer, 1975).

The lenses were removed by the operative technique previously described (Bosco, 1988a). Operations were carried out in Holtfreter's solution and after operation the larvae were maintained in Holtfreter's solution for three days before transfer to spring water. Entire larvae were fixed in Bouin's solution 3-5-7-10 and 15 days after operation and embedded in paraffin. Serial sections were cut at 7 ~m thickness and stained by the Mallory-Azan's method according to Heidenhain (1915).

Experiment II (control experiment): explant and in vitro culture of outer cornea of larval Xenopus laevis at stage 49-50 (60 cases). In this experiment cultures were ter- mined after four (30 cases) and ten days (30 cases).

- isolation of outer cornea: the larvae were immersed for 30 sec in 1% Euclorine solution (Zambelletti), then rinsed three times in sterile Holtfreter's solution. Opera-

F I R S T E V I D E N C E O F L E N S - T R A N S D I F F E R E N T I A T I O N . . . 263

tions were carried out in sterile Holtfreter's solution containing 100 U/ml penicillin, 100 ~g/ml streptomycin and 0.25 l*g/ml Fungizone (GIBCO). After a small incision in the dorsal pericorneal epidermis, the outer cornea was gently separated from the inner cornea with a thin hook-shape tungsten needle. The cornea was removed through a cir- cular incision made with iridectomy scissors. The tissue removed were rinsed four times in Leibovitz L 15 (Flow) diluted with distilled water (2 : 1) and containing 100 U/ml penicillin, 100 t,g/ml streptomycin (GIBCO), and 1.5 glutamine (200raM);

- culture medium: Leibovitz L 15 (Flow) diluted with distilled water (2:1) and containing 100 U/ml penicillin, 100 t-tg/ml streptomycin (GIBCO), 1.5% glutamine (200 mMol) and 10% inactivated foetal calf serum (GIBCO) was used;

- culture methods: explanted cornea was placed in a plastic organ-culture dish (35 • 10 mm Falcon Plastics) with culture medium. The cultivated fragmens floated in the medium and did not adhere to the bottom of the dishes. Approximately 2 ml of me- dium was used per dish containing ten cultures. The culture medium was renewed every day. Cultures were termined after 4 and 10 days.

Histological methods: cultures were fixed in 95% ethanol at 4 ~ embedded in paraffin, cut into 5 l*m serial sections, and stained by the Mallory-Azan's method ac- cording to Heidenhain (1915).

Expen'ment III: explant and in vitro culture of outer cornea of larval Xenopus laevis at stage 49-50, in medium added with brain-derived acidic FGF (60 cases).

The culture methods were the same as experiment II, but in this experiment 500 ng/ml of bovine brain-derived acidic FGF (Sigma: F 3635), were added to the culture medium after dilution with distilled water. The culture medium was renewed every day. The histological procedures were the same as in experiment II.

Preparation of anti-total-lens protein antibody: Xenopus laevis adults were sacrificed, lenses were removed, freed of other tissues. Then they were washed three times with ice-cold 5 mMol phosphate buffer pH 7 and stored at - 20~ Soluble lens proteins were obtained by homogeneization in the same buffer; insoluble materials were re- moved by centrifugation following the procedure of McDevitt and Brahma (1973). A mixture of complete Freund's adjuvant and 25 mg of lens proteins was injected 3 times into rabbits over a period of 3 weeks. The animals were bled one week after a booster injection without adjuvant. The serum obtained was tested against the omologous anti- gen by immunoelectrophoresis in 1% agarose gel 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.

Retrospective immunofluorescence: retrospective immunofluorescence was effected by the method of Mikailov and Gorgolyuk (1979). For the immunochemical analysis, the preparations were immersed in chilled xylene to remove canada balsam (10~ 2-7 hr) and then washed with 95% ethanol (2-3 changes 1 hr each) and three changes of

264 L. BOSCO ET AL.

buffered saline (pH 7.1 30 min each). Anti-total-lens protein antibody was used as the first antibody in the ~<sandwich)~ immunofluorescence technique of Weller and Coons (1954) after its cross absortion with adult and larval lentectomized Xenopus laevis tissue powder. Then the preparations were treated with commercial (Nordis Pharmaceutical and Diagnostic, the Nederlands) goat anti-rabbit gamma globulin antibody conjugated with fluorescein isothiocyanate.

As a preliminary control before the retrospective immunofluorescence test, normal Xenopus laevis eyes were incubated with rabbit non-immune serum and then with goat anti-rabbit gamma globulin antibody conjugated with fluorescein isothiocyanate. Nega- tive results were obtained.

RESULTS

Experiment I: simple lentectomy in larval Xenopus laevis at stage 49-50. In 26 out of the 30 cases (86%) examined the outer cornea underwent lens-forming

transformation. Results on the regeneration process were consistent with those report- ed by Freeman (1963).

Experiment II: explant and in vitro culture of outer cornea of larval Xenopus laevis at stage 49-50.

None of the 60 cases examined after four days (30 cases) and ten days (30 cases) outer corneal lens-forming transformation. The most conspicuos histological modifica- tion observed in some cases was a general volumetric increase of the double epithelial sheet. The indirect immunofluorescence test gave negative results in 15 cases exam- ined. The results obtained in this experiment were consistent with those we previously reported (Bosco et al., 1993b).

Experiment Ill: explant and in vitro culture of outer cornea of larval Xenopus laevis at stage 49-50 in medium added with brain-derived acidic FGF.

Under the experimental conditions we adopted the outer cornea showed lens-form- ing transformation in 45 of the 60 cases (70%) examined. It was not possible to stage the lens-forming transformations .according to the method adopted by Freeman (1963) for staging the lens-forming transformations during the lens regeneration in the eye as in our cultures most of the observed lens-forming transformations were atipical. In the positive cases the cultured cornea underwent lens-forming transformation producing lens-structures with fibers oriented in the antero-posterior plane (fig. 1,1) or irregularly arranged rather than concentrically. Moreover no lens epithelium was present. Never- theless, careful histological examination of the lens-forming structures revealed that the cells underwent the characteristic changes observed in fiber cell differentiation (Papa- constantinou, 1967; Piatigorsky, 1981), i.e. cell elongation, enlargement of nucleus and nucleoli, strong citoplasmatic acidophilia. In some cases the lens-forming transforma- tions showed a primary-like lens fiber nucleus (fig. 1,2).

The lens-forming transformations gave positive reaction by the indirect immunoflu- orescence method with anti-total lens proteins (fig. 2).

F I R S T E V I D E N C E O F L E N S - T R A N S D I F F E R E N T I A T I O N . . . 265

Fig. 1. - Experiment III: explant and in vitro culture of outer cornea of larval Xenopus laevl} at stage 49-50, in medium added with brain-derived acidic FGF; 1: four-day cultured cornea undergoes lens-forming tran- sformation producing lens structure with fibers oriented in antero-posterior plane, x 1750; 2: ten-day cul-

tured cornea shows a primary-like lens fiber nucleus, x 875.

DIscussioN

The results reported in the present paper provide the first evidence of lens-forming transformation of larval Xenopus laevis outer cornea stimulated by aFGF.

Various tissues have been tested for their lens-regeneration-stimulating influence on the

outer cornea. Neuronal tissues such as neural retina, spinal ganglia, regenerating spinal ganglia, spinal cord (Reeve and Wild, 1981; Filoni et al., 1984; Bosco et al., 1985, 1993b);

266 L. BOSCO ETAL,

Fig. 2. - Experiment III: explant and in vitro culture of outer cornea of larval Xenopus laevis at stage 49-50 in medium added with brain-derived acidic FGF. Ten-day cultured cornea undergoes lens-forming tran-

sformation showing immunofluorescence with anti-total lens proteins, x 1200.

regenerative blastema such as limb blastema, tail blastema, tentacle blastema (Reeve and Wild, 1981; Bosco et al., 1985); limb bud and pituitary gland, are able to stimulate lens- forming transformation of the outer cornea. Well differentiated lens structures will form from larval Xenopus laevis outer cornea when it is implanted into the stump of an amputat- ed hind limb of larval Xenopus (Waggoner, 1973) or into the stump of an amputated and no innervated hind limb of precocious larvae of the same species (Filoni et al., 1991).

The lens-forming influence of the above mentioned tissues could be attributed to the presence of the same or closely related factors. FGFs could be implied in this phe- nomenon. Acidic FGF has been detected in brain (B6hlen et al., 1985; Gimenez-Gal- lego et al., 1985; Pettman et al., 1986), retina (Baird et aL, 1985; Courty et al., 1985) and pituitary gland (B6hlen et al., 1985; D'Amore and Klagsbrun, 1984). Basic FGF has been detected in kidney (Esch et al., 1985; Mormede et al., 1985), in acqueous (Schulz et al., 1993), corpus luteum (Esch et al., 1985), liver (Mormede et al., 1985), macrophages (Gospodarowicz et al., 1985) and placenta (Gospodarowicz et al., 1985), as well as in neural tissues. Experiments are currently under way in our laboratory to understand the role of aFGF in lens-forming transformation of the outer cornea and to test other growth factors for their possible lens-formation stimulating influence.

I wish to thank Prof. Alberto Stefanelli for the critical review of the manuscript.

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