Tuneo Yamada

Chapter 9: Cellular and Subcellular Events in Wolffian Lens Regeneration

Publisher Summary Synthetic activities of cells, which are involved in tissue transformation, have been studied utilizing autoradiographic, immunochemical, and electron microscopic techniques. This chapter reviews data obtained so far, and includes related results reported from other laboratories. One of the prominent features of cell behavior observed during Wolffian lens regeneration is ordered, sequential alterations in synthetic activities of regenerate cells. Sonic data suggest that the sequence of synthetic phases observed is comparable to the sequence observed in normal development of the lens. The convergence of regenerative and ontogenetic lens formation implies that Wolffian lens regeneration involves recovery of an embryonic condition by the iris epithelial cells. The data reviewed in this chapter as a whole suggest that the pattern of utilization of the genome characteristic for the iris cannot be changed directly into that characteristic for the lens, but only indirectly by following up the steps with which the lens pattern is acquired during normal development.

DNA synthesis and mitotic activity during early development of chick lens

Summary Various developmental stages of lens rudiment, starting from the early lens placode, were labeled with thymidine −3 H by culturing the blastoderm for 4 hours in a medium containing the labeled precursor. On autoradiograms the frequency of labeled cells was scored for various morphological compartments of the rudiments and for various developmental stages. A comparable study was made on the frequency of mitotic cells. During the stage when the lens placode indicates orientation of nuclei, the cell labeling frequency decreases slightly over the entire area of the placode. From the time of invagination of the placode the central part of the placode starts to decrease in cell-labeling frequency while the peripheral part retains a high frequency. In the lens vesicle stage the internal layer shows a progressive decrease in labeling frequency which leads to complete disappearance of labeling. The external layer of the vesicle has a high level of labeling throughout the stages investigated. The intermediate zone of the lens vesicle shows a progressive decrease and loss of labeling frequency. The data on mitotic frequency indicate a similar decrease, except that the changes start to be evident later than those of cell-labeling frequency. The results imply that DNA replication and mitosis become less frequent and finally cease in the prospective primary and secondary fiber cells as they differentiate into definitive fibers, and that the cells of lens rudiment which are not participating in fiber differentiation retain a high level of cell-labeling and mitotic frequencies.

Immunofluorescence localization of the crystallins in amphibian lens development, with special reference to the γ-crystallins☆

Abstract The ontogeny and localization of the crystallins in the developing lens of R. pipiens has been investigated by means of the immunofluorescence technique. Antibodies to adult R. pipiens total-lens proteins and adult R. pipiens γ-crystallins—whose antigen, tissue, and developmental specificity had been demonstrated—were used in both the direct and indirect (or “sandwich”) immunofluorescence techniques; both methods gave comparable results. These antibody-dye conjugates were applied to sections of embryonic R. pipiens ; a series of lens developmental stages was recognized and defined by us to aid in identification of those stages subjected to immunofluorescence analysis. A positive reaction for the anti-total-lens-protein immunofluorescence reagent was first noted at Lens Developmental Stage VI, in a number of prospective primary lens fiber cells, and a weak, irregular reaction was also noted in the external layer. The intensity of immunofluorescence with this reagent increased in the fiber area and external layer until at Lens Developmental Stage X all cells of the lens were positive; the primary and secondary lens fiber cells and lens epithelium exhibited immunofluorescence in order of decreasing intensity. The first positive reaction with the anti-γ-crystallin immunofluorescence reagent could also be observed at Lens Developmental Stage VI. However, the immunofluorescence was present only in the region of the prospective primary lens fiber cells and was absent in the external layer. The intensity of the immunofluorescence increased in the fiber area until Lens Developmental Stage X, at which time it was greater in the primary fiber cells than that in the secondary fiber cells. No immunofluorescence could be detected in the external layer or in the lens epithelium with this reagent until Stage X. At this time, after the lens is well differentiated and has assumed the basic adult configuration, a weak immunofluorescence reaction was noted in the lens epithelium in a small percentage of cases. At no time, with either immunofluorescence reagent, could an immunofluorescence reaction be observed outside the lens tissue. Such results indicate that the γ-crystallins, previously implicated in bovine and regenerating newt lens fiber differentiation, are also indicative of lens fiber differentiation in the normal, developing lens of the anuran amphibian, R. pipiens . In addition, the relatively late appearance in lens development (i.e., after formation of a lens vesicle) of any immunofluorescence reaction for the crystallins suggest that they are not necessary for lens induction and subsequent lens placode formation.

Dedifferentiation of iris epithelial cells

Abstract Electron and phase-contrast microscopic observations demonstrated that depigmentation of iris epithelial cells in vivo after lentectomy is preceded by alterations of cell shape and increases in microfilaments and microtubules in the periphery of the cell. Extensive cell processes are formed, with tips branching into fine strands that contain melanosomes. The macrophages invading the iris epithelium incorporate pieces of such strands, which are composed of cell membrane, cytoplasmic matrix, and melanosomes. Individual melanosomes are also taken up by macrophages. Some of the strands of iris cell processes seem to degenerate within the intercellular space. Thus, depigmentation of iris epithelial cells is accompanied by loss of a substantial part of the cell surface and cytoplasmic matrix of iris epithelial cells. Measurements of the absolute volume of whole iris epithelial cells, their cytoplasm, and their nuclei were conducted at various stages of depigmentation. These measurements reveal an extensive increase in the volume of the cytoplasm preceding activation of the cell surface and a significant reduction in cytoplasmic volume during the phase of extensive depigmentation. In the case of the nuclear volume, the extensive increase which occurs in parallel with that of the cytoplasmic volume is not followed by a significant change during the depigmentation phase.

Gamma crystallins in Wolffian lens regeneration demonstrated by immunofluorescence

Abstract A fraction containing γ crystallins was isolated from the newt lens, and an antiserum against the fraction was prepared. Tests with immunoelectrophoresis and immunodiffusion indicated that the fraction was not contaminated with other crystallins and that the antiserum reacted with γ crystallins, but not with other crystallins. An immunofluorescent reagent was prepared with this antiserum and used to detect γ crystallins. In the normal lens the reaction was positive in the fiber area but not in the lens epithelium. With this immunofluorescent reagent, appearance and distribution of γ crystallins were studied in Wolffian lens regeneration of adult Triturus viridescens . The γ-crystallin reaction was negative in the regenerates of stages I–IV, as well as in the normal iris epithelium. The reaction became positive in a small number of cells located in the internal wall of lens vesicle at stage V. Subsequently, increase was observed in the number of positive cells and in intensity of immunofluorescence in each cell. All cells which could be recognized as primary lens fiber cells were positive. Later than stage IX, when the secondary lens fiber cells were formed, they all become positive. In all fiber cells, γ crystallins were detected in the nucleus as well as in the cytoplasm. No immunofluorescence was observed in the external layer of lens vesicle or in the lens epithelium of the growing lens. It was concluded that in Wolffian lens regeneration γ crystallins became detectable after the cells entered the terminal cell cycle and were accumulated in the cells during lens-fiber differentiation.

LENS FIBER DIFFERENTIATION AND GAMMA CRYSTALLINS: IMMUNOFLUORESCENT STUDY OF WOLFFIAN REGENERATION.

From the adult lens of Triturus viridescens, a fraction of proteins was isolated which corresponds to γ-crystallins of higher vertebrates. Tests by immuno-electrophoresis indicate that the antiserum against this fraction reacts with γ-crystallins, but not with α- or β-crystallins. With this antiserum, an immunofluorescent reagent has been prepared for detection of γ-crystallins from newts. In the normal lens of the adult newt, these crystallins are detected in fiber cells and fiber material, but not in the epithelial cells. During transformation of the iris into the lens after lens removal, the staining reaction is negative in the regenerating tissue up to the time the prospective primary fiber cells begin to elongate. Subsequently, without exception those cells in fiber differentiation indicate a γ-crystallin reaction. When the secondary fiber cells are produced at the equatorial zone of the regenerating lens, they also begin to show a γ-crystallin reaction. Thus, γ-crystallins characterize fiber differentiation.

Influence of the pituitary on Wolffian lens regeneration

Abstract Removal of the pituitary 3 days before lentectomy retards Wolffian lens regeneration in the adult newt, Notophthalmus viridescens , by two stages over a 21-day period. Hypophysectomy 5 or 10 days after lentectomy does not alter the progress of regeneration during the subsequent 10-day period. Hypophysectomy 3 days before lentectomy also significantly decreases the incorporation of [ 3 H]thymidine by iris epithelial nuclei 5 days after lentectomy but has no statistically significant effect on the incorporation 7 days after lentectomy. Pituitary tissue from newts or frogs enhances the regenerative activity of newt iris epithelial cells in vitro and in many cases promotes lens fiber formation. To a lesser extent, other tissues, such as nerve ganglion, also enhance the production of lens fiber cells from iris epithelium in vitro , whereas muscle tissue does not; and under certain conditions iris epithelial cells were found to depigment and redifferentiate into lens cells in the absence of other tissues in vitro .

Tyrosinase activity in the Wolffian lens regenerating system

Abstract The first part of the paper is devoted to separation and characterization of tyrosinase of Notophthalmus viridescens . After ammonium sulfate fractionation and DEAE-cellulose chromatography, a fraction of extract was obtained which had a 1000-fold increase in enzyme activity and revealed one positive DOPA oxidase band and one negative protein band on acrylamide gel electrophoresis. Some characterization of the enzyme was made using this sample. Specific activity of this fraction was estimated to be 39 × 10 −2 μmole of l -tyrosine/mg in 17 hr. An inhibitor of tyrosinase was demonstrated in the Notophthalmus lens. In the second part of the paper, the level of tyrosinase activity was followed in the iris tissue as it was transformed into the lens after lentectomy in adult N. viridescens . When the activity was based on micrograms of DNA, the dorsal iris increased slightly in activity during the phase of depigmentation. A significant increase in activity was recorded when the lens had been formed by completely depigmented dorsal iris cells. On the other hand, in the ventral iris, where weak depigmentation is followed by repigmentation, the activity level showed a weak increase. Implication of the data for tissue transformation was discussed.

Comparison of γ-crystallins from frog and newt☆

Abstract A comparative study was made on the behavior in various tests of newt and frog γ-crystallins and antibodies against them. The purified γ-crystallins from the two species showed identical patterns in immunodiffusion and immunoelectrophoresis. In agar electrophoresis newt γ-crystallins moved in a single band, while frog γ-crystallins indicated four bands. No difference could be found between antibodies directed to γ-crystallins of the two species in their ability to recognize antigens derived from both species in immunofluorescence, immunodiffusion, and immunoelectrophoresis.

Cell type conversion and galactosyltransferase in lens regeneration

Abstract The level of galactosyltransferase activity was followed during conversion of iris epithelial cells into lens cells in lentectomized adult newts. A moderate increase during the dedifferentiation phase is followed by a remarkable increase when the lens tissue is differentiating. Maturation of the lens is associated with a decline in the enzyme activity.