The pattern of DNA replication during the initial stages of Wolffian lens regeneration has been examined by an autoradiographic study of tritiated thymidine uptake in zero to five-day regenerates (Triturus viridescens). Scanning of complete sets of serial sections for each iris revealed the absence of labeled cells in normal, one-, and two-day iris epithelia, and a very infrequent occurrence of labeled iris epithelial cells in some three-day regenerates. After this stage, cell labeling is seen in all iris epithelia. Cell labeling frequency increases markedly by four days, and is at a very high level in five-day regenerates.Further data indicate that labeling begins in the dorsal sector of the iris epithelium.By five days after lens removal, however, it reaches siinilar levels in dorsal, lateral, and ventral sectors. Thus, DNA replication is induced throughout the iris epithelium after lens removal, even though the lateral and ventral sectors of the iris epithelium do not participate in actual lens formation.Comparison between the outer and inner laminae of the iris epithelium shows that during all stages studied the great majority of labeled cells are in the inner lamina.Earlier autoradiographic studies of generation and are not sufficient for anWolffian lens regeneration demonstrated swering these specific questions. The that the cells of adult newt iris epithelium autoradiographic study reported and disare activated to replicate DNA soon after cussed here was undertaken in hopes of lens removal, and maintain the activated providing such answers. condition until they start fiber differentiation (unpublished work of R. W. Rever. '64) indicate that another prominent cellular change at the initial stages of regeneration is the activation of ribosomal RNA synthesis. The relationship between induced ribosomal RNA synthesis and induced DNA replication has become an important issue in understanding the control mechanism of this system. For elucidating this issue we need to know whether or not DNA replication is completely repressed in the normal adult iris epithelium, and how long after lens removal the normal condition is maintained before DNA replication begins. We must also know the distribution pattern of the DNA-replicating cells within the iris epithelium in the early stages of regeneration. The autoradiographic studies cited above were focused on the overall pattern of DNA replication during the whole course of re- J. EXP. ZOOL., 171: 425-432Lens removal Lentectomy, either unilateral or bilateral, was performed on 35 adult newts, Triturus viridescens, by the method of Eisenberg and Yamada ('66). In the unilateral lentectomy, the left lens was routinely removed. The right unoperated ins could then serve as a contralateral control. A few entirely normal animals were also included in the experiment.Injection of labeled precursor Regeneration was allowed to proceed for one, two, three, four or five days. At each of these times, some animals were anaesthetized with tricaine (Sigma) and injected intraperiton...
The total numbers of mitotic cells in the iris epithelium, ciliary epithelium, and ora serrata were counted at various time intervals after lentectomy of adult newts, Notophthalmus (Triturus) uiridescens, in bleached serial sections. In some cases colchicine was injected for the purpose of accumulating mitotic figures.In the iris epithelium, which normally shows no mitotic cells, a wave of mitoses is induced by lenfectomy. The wave starts on day 4 and has its peak period during days 7 to 15, when a portion of the iris epithelial cells depigment and form the lens vesicle. In the iris epithelium the wave subsides after formation of the regenerated lens and is gone by day 30. On the other hand, in the regenerated lens the mitotic activity is retained in the lens epithelium. The colchicine treatment has revealed an extremely small number of iris epithelial cells going into mitoses within one day after lentectomy. However, the same treatment failed to demonstrate any mitotic cells on days 0, 2, and 3.Mitotic activity is also induced in the ciliary epithelium, which does not take part in formation of the lens regenerate. This induction is much lower in intensity than that in the iris epithelium.The ora serrata area has a low level of mitotic activity in the normal condition, which is elevated to a high level after day 20. Significance of these observations for the mechanism of lens regeneration is discussed.Estimation of the total cell number of iris and ciliary epithelia during the first 15 days after lentectomy indicated an increase in the iris epithelium, which can be accounted for by the number of mitoses counted during the corresponding period in the same tissue.
After lentectomy of the adult newt eye, non-dividing iris epithelial cells re-enter the cell cycle. Some of the iris epithelial cells become completely depigmented while they are in the cell cycle, and then differentiate into lens cells. The remaining iris epithelial cells become partially depigmented in the induced cell cycle, but resynthesize melanosomes and recover the normal state of iris epithelial cells. The two groups of cells are spatially separated within the iris epithelium. The cell cycle parameters of both groups of iris epithelial cells were estimated by a mathematical procedure on a computerized programme from the percentage of labelled mitotic cells as a function of time after peritoneal injection of [3H]methyl-thymidine on day 6 after lentectomy. The total cell cycle time was found significantly shorter in the cell population with complete depigmentation as compared with that with partial depigmentation. Based on these results the possible role of differential cell cycle time in the control of dedifferentiation was discussed. Grafting of unlabelled iris into the optic cavity of host animals injected 6 h beforehand with [3H]methyl-thymidine, followed by a study of radioactivity of iris epithelial cells of the graft demonstrated incorporation at a low level during the whole period of the experiment in which the cell cycle parameters were estimated. The data used for the estimation were corrected for the delayed incorporation.
Autoradiographic studies of transformation of iris into lens during Wolffian lens regeneration of adult Triturus viridescens indicate an enhancement of RNA synthesis in the nucleus of the iris cells in the early phase of regeneration (Yamada & Karasaki, 1963; unpublished). Activation of protein synthesis and production of lens antigens follow the enhancement of RNA synthesis (Yamada & Takata, 1963; Takata et al., 1964). This sequence of events suggests the possibility that lens removal elicits synthesis of specific RNA's which in their turn induce synthesis of proteins essential for transformation of the tissue. If this is the case, it should be possible to suppress lens regeneration by subjecting the system to an inhibitor of RNA synthesis. In the present present study the possibility was tested, using actinomycin D as the inhibitor.
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