Ultrastructure and glycoconjugate histochemistry of the lens capsule during lens regeneration from the iris in the Newt.

The lens capsule of the regenerating lens develops from the basal lamina of the iris epithelium. As the lens differentiates and grows in size, the lens capsule increases in thickness by the formation of successive layers of basal laminar material resulting in a structure composed of increasing numbers of parallel lamellae. This initial arrangement may be lost and a more homogeneous composition of fine granules and filaments may characterize some parts of the capsule in older lenses. The developing lens capsule stains with the PAS and PAPD methods but not with the PAPS, AB and HID methods. Therefore, it appears to contain only neutral, non-acidic glycoconjugates (probably glycoproteins) which may contain fucose, galactose or mannose. This conclusion is supported by the incorporation of 3H-fucose into the developing lens capsule of regenerating lenses beginning at a stage with initial lens fiber formation. Except for the apparent absence of acidic glycoconjugates in the regenerating lens capsule, these events are similar to those described for developing lenses in chick and mammalian embryos. The differentiation of lens fibers by elongation, loss of organelles and accumulation of fine, electron-dense particles in the cytoplasm also parallels embryonic, lens fiber differentiation.

Morphology and glycoconjugate histochemistry of the palpebral glands of the adult newt, Notophthalmus viridescens.

The eyelids of the newt were studied in 10 microns serial paraffin and 1-2 microns plastic sections using standard histological stains and special stains for glycoconjugates. The eyelids contain four different glands. Simple acinar serous and simple acinar mucous glands occur in the skin; unicellular mucous glands occur in the conjunctiva; and convoluted tubular seromucous glands are present in connective tissue beneath the conjunctiva. The first two are identical to cutaneous glands found elsewhere on the head and body. The simple acinar serous glands are surrounded by myoepithelial cells and release their secretion, which is composed largely of proteins with minimal glycoconjugate content, by a holocrine mechanism. The secretory product of the simple acinar mucous glands is composed of neutral glycoconjugates with a minor content of acidic glycoconjugates; the mucin exhibits strong PAS and PAPD staining and weak staining by AB and PAPS methods. The unicellular conjunctival mucous glands secrete both neutral and acidic glycoconjugates as shown by positive reactions with PAS, PAPD, PAPS, and AB methods. Convoluted tubular seromucous glands in the ventral eyelid synthesize both proteins and neutral glycoconjugates. The mucous secretions of the conjunctival glands probably provide lubrication and protection for the cornea.

Macrophage invasion and phagocytic activity during lens regeneration from the iris epithelium in newts.

Following removal of the lens through the cornea, early stages of lens regeneration from the dorsal iris of the adult newt, Notophthalmus viridescens, were studied using light and electron microscopic observations on sectioned, plastic-embedded irises. Specimens were fixed in Karnovsky's fixative every 2 days from 0 to 12 and 15 days after lentectomy. Infiltration of the iris epithelium by macrophages and their phagocytosis of melanosomes and small fragments of iris epithelial cells were observed. These macrophages were characterized by coarse nuclear chromatin, numerous mitochondria, free ribosomes, granular endoplasmic reticulum, Golgi complexes, vesicles, lysosomes, and phagosomes containing ingested melanosomes. Lamellipodia of varying length projected from their surface. Most of the cells lying on or close to the posterior surface of the iris could be identified as macrophages by these criteria. During this period, there was enlargement of the intercellular spaces within the iris epithelium. The iris epithelial cells near the margin of the pupil elongated, lost their melanin pigment and some associated cytoplasm, and acquired abundant free polyribosomes to form a lens vesicle of depigmented cells.

Macrophage mobilization and morphology during lens regeneration from the iris epithelium in newts: studies with correlated scanning and transmission electron microscopy.

The lens was removed from both eyes of adult newts (Notophthalmus viridescens), and the eyes were fixed in Karnovsky's fixative every 2 days 0-20 days after operation. Anterior half-eyes were prepared by standard procedures for scanning electron microscopy of the surface. Before fixation, the posterior iris surface was cleaned of adhering vitreous mechanically with forceps or by treatment with bovine testicular hyaluronidase or with hyaluronidase and collagenase. Some specimens were cryofractured in buffer or ethanol transverse to the mid-dorsal iris, and the fractured surface viewed with scanning electron microscopy (SEM). Cells with various combinations of ridges, blebs, filopodia, and lamellipodia were observed adhering to the posterior surface of the iris by 6 days after lentectomy. These cells, which exhibited the surface characteristics of macrophages, became more numerous in specimens fixed after longer intervals. Invasion of the iris epithelium was observed in a cryofractured specimen. After observations with SEM, selected specimens were embedded in plastic and sectioned for study with transmission electron microscopy (TEM). The cells on the iris surface had the cytological characteristics of macrophages, and other macrophages were located within the iris epithelium. In specimens fixed 16 or more days after lentectomy, a bulging lens vesicle was regenerating from the dorsal pupillary margin of the iris. Macrophages were absent or few on the surface of this developing lens but remained scattered over the adjoining iris. Roles that might be played by these macrophages during the transdifferentiation of iris epithelium into lens are discussed.

Availability time of tritium-labeled DNA precursors in newt eyes following intraperitoneal injection of 3H-thymidine.

Following intraperitoneal injection of 3H-thymidine into host newts, iris together with a regenerating lens was transplanted from a donor eye into a lentectomized host eye at frequent intervals for 20 hours and then every 1 or 2 days for 14 days. The eyes were fixed 2 hours and 1 or 2 days after implantation and autoradiographs prepared. Following fixation 2 hours after operation, incorporation of 3H-thymidine into DNA, as evidenced by grain counts over nuclei, fell rapidly for 3.5 hours after injection and was no longer apparent after 4.5 hours. However, almost one-half of the implants were lightly labeled when they remained in the host eyes for 1 or 2 days beginning from 1 to 14 days after isotope injection. When these implanted, regenerating lenses were left in the host eyes for longer periods of time, then a light label was found over nuclei in most of the implants remaining in the eye for 3 to 24 days. When 3H-thymidine was injected from 1 to 3 days after extirpation of both lens and neural retina, before DNA synthesis had been initiated in the pigmented retinal epithelium or iris, there were numerous cases of labeled nuclei among depigmenting cells of the pigmented retinal epithelium which was regenerating a new neural retina from 2 to 25 days after isotope injection. Depigmenting cells of the dorsal iris and regenerating lens were similarly labeled. These results provide evidence for the continued availability of small amounts of tritiated DNA-precursor molecules which can be incorporated in DNA of proliferating cells long after the initial injection of 3H-thymidine.

Dedifferentiation of iris epithelium during lens regeneration in newt larvae.

Early stages in lens regeneration from the pigmented epithelium of the dorsal iris were studied in larval Notophthalmus viridescens by means of transmission electron microscopy. Normal iris epithelium is composed of two layers of low cuboidal cells. packed with melanosomes and surrounded by a basal lamina. Scattered desmosomes attach adjacent cells. Following lens removal, the intercellular spaces enlarge and the epithelial cells increase in size. Some irregular microvilli from these cells extent into the intercellular spaces. Macrophages invade the iris epithelium and phagocytize melanosomes discharged from the pigmented cells. These invading macrophages have numerous microprojections and are often separated from the surface by a very thin layer of iris epithelial cell cytoplasm. In the iris cells, nucleoli become more prominent and granular, polyribosomes increase greatly in number, melanosomes gradually disappear, mitochondria become more numerous, and mitotic activity is greatly augmented. Fine cell processes of adjacent interdigitate near the external surface, where numerous micropinocytotic vesicles can be seen. Over the external surface, the basal lamina may be disrupted or duplicated in places where pseudopodia project from iris cells or a macrophage has entered an intercellular space. It is lacking on the lumenal surface. Sloughed membranes are often found in these intercellular spaces.

Lens regeneration from the pupillary margin of the eyecup in newt embryos.

Early stages in lens regeneration from the dorsal margin of the pupil, following removal of the young lens from the embryonic eyecup, were studied with transmission electron microscopy in Notophthalmus viridescens. At the stage of operation, the eyecup cells have an undifferentiated, embryonic appearance with numerous free ribosomes and scattered mitochondria. In normal embryonic eyes containing a lens, the iris epithelium differentiates from the edge of the optic cup by growth and flattening of the cells to form a two-layered cuboidal epithelium. There is extensive melanogenesis in both inner and outer layers. In lentectomized eyes, this flattening does not occur and melanogenesis takes place mainly in the outer layer of epithelium. Mitoses are abundant and a vesicle of unpigmented columnar cells, enclosed by a basal lamina, develops from the dorsal pupillary margin of the eyecup. Macrophages are present and phagocytize some of the melanin but less than during dedifferentiation of pigmented iris epithelium. During this regeneration of a lens vesicle, there is an increase in the number of polyribosomes, granular endoplasmic reticulum, and mitochondria in preparation for synthesis of the lens proteins. Micropinocytosis occurs at the cells surfaces.

Soluble protein synthesis in the neural retina during lens regeneration.

Lens regeneration in newts involves transformation of pigmented dorsal iris epithelial cells into lens cells. This process is somehow stimulated by the neural retina. In the present study, soluble proteins and their synthesis were analyzed in normal neural retina and in neural retina 12 days after lentectomy by means of disc and slab gel electrophoresis. Electrophoretic analysis revealed 16 prominent polypeptides (M.W. 18,500 to 185,000 daltons). The electrophoretic banding pattern of the neural retina was similar in normal eyes and in eyes 12 days after lentectomy. To determine which of these bands were being synthesized. 3H-leucine or 35S-methionine was injected prior to tissue preparation for electrophoresis. About 12 bands were found to be radioactive, again no major difference was seen in the radioactivity pattern of soluble proteins obtained from normal and lentectomized eyes. Since no major differences, were observed, the present results suggest that the neural retinal factor, if a soluble protein, is not a new protein but is present in normal retina.

Role of neural retina and vitreous body during lens regeneration: transplantation and autoradiography.

The trophic influence of neural retina in regeneration of the lens from dorsal iris is well known. The first part of this study involved transplantation of dorsal iris pieces into various intra-ocular positions in order to determine the field of action of the neural retina. The most favorable location for lens regeneration was the pupillary space, closely followed by the vitreous chamber. Lens regeneration was retarded from implants lying in the anterior chamber; this was most evident when grafts were placed in front of the iris and not in contact with the swollen vitreous body. Incorporation of 3H-leucine in the neural retina and vitreous body was also investigated using autoradiography. No significant difference in labeling was seen between the control and lentectomized neural retina at all time intervals studied. However, increased labeling above that in the controls was observed over the vitreous body between 8 to 20 days after lentectomy. There was also an increase in the number of silver grains over the vitreous body with increasing time between injection of isotope and sacrifice of the animal. These results provide evidence that vitreous body plays an important role in the process of lens regeneration, perhaps serving as a preferential pathway for transfer of the neural retinal factor to the dorsal iris.

DNA synthesis during lens regeneration in larval Xenopus laevis.

Larvae of Xenopus laevis at stages 50-53 were lentectomized and then injected with tritiated thymidine at various times after lentectomy. In series I, the animals were injected 1,2,3,4,6,8,10,12,15, or 17 days after lens removal and fixed three hours after injection. Autoradiograpns of serial cross sections through the eyes were prepared. Increased incorporation of thymidine in the cells of the regenerating lens was first observed two days (stage 2) after lentectomy. All of the cells of the lens vesicle incorporated H3 thymidine until stage 4 at which time the cells in the inner wall of the vesicle began to differentiate into lens fibers. Labelling then became restricted to the peripheral cells (prospective lens epithelium and prospective lens fibers). At stage 5 of regeneration, only cells of the lens epithelium incorporated H3 thymidine. In Series II, animals injected three or four days after lentectomy were fixed daily from one to seven days after injection. Many stage 3 and stage 4 regenerates were recovered with label throughout and stage 5 regenerates were found, seven days after injection, with label over the lens fibers as well as over the lens epithelium.