Randall W. Reyer

DNA synthesis and the incorporation of labeled iris cells into the lens during lens regeneration in adult newts

Abstract When the lens was removed from the eye of adultNotophthalmus viridescens and the specific DNA precursor, thymidine-3H, was injected intraperitoneally, the initiation of DNA synthesis was indicated by the appearance of labeled nuclei in the pigmented iris epithelium 4 days after lentectomy. As the cells near the mid-dorsal pupillary margin then undergo depigmentation to form a lens vesicle, DNA replication and mitotic cell division continue in this tissue. Cells which were labeled in the dorsal iris epithelium could be traced into all parts of the young, regenerating lens, including both the lens epithelium and the lens fibers. This radioautographic evidence, together with the histological evidence for continuity of the iris epithelium and the regenerating lens, provides support for the concept that dorsal iris cells transform into lens cells. As soon as the cells in the inner lamina of the lens vesicle begin to elongate to differentiate into primary lens fibers, DNA synthesis ceases in these cells but continues in the cells of the outer part of the lens vesicle and in the stalk connecting the lens vesicle with the rest of the iris epithelium. Cells in the latter areas continue to synthesize DNA and to divide, the daughter cells moving toward the primary lens fibers. In the equatorial zone, these cells begin to elongate, stop dividing and enclose the primary lens fibers with a shell of secondary lens fibers. As the lens grows in size, it detaches from the iris epithelium but the lens epithelium remains as a germinal epithelium providing a continuous supply of new secondary lens fibers. Although DNA synthesis is initiated in the iris epithelium by 7 days after extirpation of both lens and neural retina, the other phases of lens regeneration are delayed until a layer of depigmented cells has been formed by the regenerating neural retina. Unlabeled iris and lens vesicles were implanted into lentectomized eyes at different times after administration of thymidine-3H to the host in order to test for the availability time of the isotope. Conspicuous labeling of the implant nuclei occurred up to two hours after injection and then dropped off so that no label was found from 3.5 hours to 14 days after injection. There was one exception where a light label appeared in a lens vesicle exposed to the host eye environment 2–3 days after isotope injection. It was concluded that the thymidine-3H is available for only 2–3 hours in most instances.

Stimulation of lens regeneration from the newt dorsal iris when implanted into the blastema of the regenerating limb

Dorsal iris from the eyes of adult Notophthalmus viridescens was transplanted into the blastema of regenerating limbs, subcutaneously in the limb or shoulder region, into the dorsal fin of larval newts and into the hindbrain of larval Ambystoma maculatum. The iris implants into the blastema regenerated lens vesicles or lenses with fibers in 40–75% of the cases. Multiple lenses were found in a few instances. No lenses developed from iris implants into the dorsal fin. Twenty percent of subcutaneous implants of iris formed lenses or lens vesicles, but lens regeneration from implants into the brain occurred only rarely. Denervation of the limb at the time of iris transplantation into the blastema greatly reduced the number of lenses regenerated. Studies on nerve fiber distribution in dorsal fin, subcutaneous areas, and denervated and innervated regenerating limbs, using the Bodian method, showed a general correlation between density of nerve fibers in the implant site and the incidence of lens regeneration from iris implants into that site. These results provide some evidence for a trophic action of nerve fibers on lens regeneration from the iris.

The influence of neural retina and lens on lens regeneration from dorsal iris implants in Triturus viridescens larvae.

Abstract In order to study the role of the neural retina and lens on lens regeneration from the iris, sectors of mid-dorsal iris from larval Triturus viridescens donors were implanted into the following sites in larval hosts of the same species: (a) dorsal fin, (b) anterior chamber of the eye, host lens present, (c) vitreous chamber of the eye, host lens absent, and (d) vitreous chamber of the eye, host lens present. In these locations, the implanted iris was subject to the influence of (a) neither neural retina nor lens, (b) probably only lens. (c) neural retina only, or (d) neural retina and lens together. Lens regeneration failed to take place in the anterior chamber or dorsal fin, thus showing the necessity of the neural retinal stimulus. Lens regeneration occurred from implants into the vitreous chamber not only in the absence of the host lens, but also in its presence. However, both the frequency of lens regeneration and the size and growth rate of the regenerates were decreased when the implant was located between the neural retina and host lens, therefore being subject to the influence of both tissues together. It was suggested that the inhibitory action of the host lens on iris implants in this location could be (1) mechanical, by displacing the implant more deeply into the vitreous chamber or (2) chemical, by competing with the lens developing from the implant for the neural retinal factor or through the production of a specific inhibitor for the homologous tissue.

Repolarization of reversed, regenerating lenses in adult newts, Notophthalmus viridescens

Abstract Following removal of the lens from eyes of adult newts, Notophthalmus viridescens, lens regeneration from the dorsal iris was allowed to proceed for 20–30 days. Then a circular disc of iris and the attached regenerating lens was excised, turned inside out, and replaced in the eye so that the lens fiber pole faced the cornea. Eyes were fixed from 1 to 30 days after the second operation. The lens epithelium, facing the vitreous body and neural retina, differentiated into new lens fibers which were laid down on the surface of the fibers present at the time of rotation. The remainder of the lens epithelium grew over the old lens fiber pole and covered the surface facing the cornea. In this way, normal polarity was re-established in the reversed, regenerating lens.

Morphological evidence for lens differentiation from intra-ocular implants of lens epithelium in Ambystoma maculatum

In larvae of Ambystoma maculatum, pieces of lens epithelium and lens capsule were implanted into the pupil of the eye following removal of the host lens. Hosts were fixed from 1 to 31 days after operation. In the first series, some donor lens fibers were included with the implants. These implants developed into lenses in which the old lens fibers were surrounded by new lens fibers which had differentiated from the lens epithelium. In the second series, implants of lens epithelium and lens capsule alone also formed quite normal lenses enclosed by a new lens capsule. These lenses were oriented with the lens epithelium facing the cornea and the lens fiber pole facing the vitreous.

DNA replication in lens vesicles of Ambystoma maculatum embryos implanted into ocular or extra-ocular sites of host larvae.

In Ambystoma maculatum , lens vesicles from donor embryos were transplanted into the dorsal fins or into the lentectomized eyes of larval hosts. From one to 30 days after operation, the hosts were injected with [ 3 H]thymidine and fixed 3 hr later. Autoradiographs were prepared with Kodak NTB-3 liquid emulsion. The lens vesicles developed normally in the larval eyes and differentiated into lenses with primary and secondary lens fibers which grew almost as large as the lenses in the eyes of donor controls. Many cells in the lens epithelium incorporated [ 3 H]thymidine following the three hour exposure to the isotope (mean thymidine index=37·2%). In the fin, lens fibers also differentiated from most of the lens vesicles but the latter remained small and the number of labeled cells in the lens epithelium was greatly reduced (mean thymidine index=7·1%). It was concluded that the larval eye environment, especially the neural retina, supports DNA synthesis and mitotic activity in the cells of the embryonic lens vesicle and the lens epithelium of the young lens.