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Tentacular and oral-disc regeneration in the sea anemone, Aiptasia diaphana
III. Autoradiographic analysis of patterns of tritiated thymidine uptake
By IRWIN I. SINGER 1
From the Department of Biology, New York University
SUMMARY Autoradiography with [ H]thymidine and electron microscopy were used todetermine (a) the patterns of cellular division exhibited by intact anemones, (b) if measurable increases in cellular proliferation accompany oral-disc regeneration, (c) whether interstitial cells are present in Aiptasia, and (d) if these cells could be responsible for the latter proliferative patterns. An oral-aboral gradient in cellular proliferation was exhibited by the epidermis of uncutanemones, with the highest levels in the tentacles. Wound healing did not require cell proliferation and did not immediately stimulate cellular division which was associated with subsequent morphogenetic events. Indices of presumptive oral-disc [3H]thymidine uptake into nuclei increased tenfold with the outgrowth of the new tentacles. This increase occurred in the epidermis, while only small amounts ofgastrodermal proliferation were detected. It is hypothesized that the epidermis contributes new cells to the expanding gastrodermis during tentacle budding. Most of the [3H]thymidine-labeled nuclei were localized in the basal portions of the epidermis of intact anemones and 1- to 2-day-old regenerates; very few gastrodermal nuclei accumulated the label. Nests of interstitial cells and transforminginterstitial cells were localized in the exact epidermal regions where nuclear labeling took place, suggesting that the proliferative patterns of intact and regenerating Aiptasia are a function of their interstitial cell distribution.
INTRODUCTION The successive structural events culminating in the formation of a new oraldisc and tentacle crown in the sea anemone, Aiptasia diaphana, includetwo important steps: (1) the fusion of the cut pharyngeal and columnar tissues, referred to in this paper as wound healing, and (2) the appearance of the primordia of the tentacles, 3 days after amputation, which is designated as tentacle budding (Singer & Palmer, 1969). The purpose of the study reported here is to determine if measurable increases in cellular division accompany theseregenerative events. The solution of this problem would shed considerable light on the cellular
1 Author's address: Department of Biology, Saint Louis University, 1504 South Grand Boulevard, Saint Louis, Missouri, 63104, U.S.A.
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basis of the regeneration in Aiptasia. For example, if no increases in the rates of cellular division occur before or during tentacle budding,then one would expect that the cells which are responsible for the building of the tentacles are distally migrating columnar cells which have transformed into tentacular cells. Similar mechanisms have been used to explain the reconstitution of complete dwarfs from isolated gastrodermal fragments (Davis, Burnett, Haynes & Mumaw, 1966) and hypostomal mucous cell regeneration (Rose & Burnett, 1968) inHydra viridis. It is also noteworthy that neither increased mitotic activity nor heightened rates of nuclear [3H]thymidine uptake have been detected in budding or regenerating Hydra (Li & Lenhoff, 1961; Spagenberg, 1961; Breslin-Spagenberg & Eakin, 1962; Clarkson & Wolpert, 1967; Park, Ortmeyer & Blankenbaker, 1967; Clarkson, 1969). Alternatively, tentacle buds could be formed by the rapid andrepeated division of all columnar cell types (or a single totipotent cell) thus resulting in tissue outgrowth from the spaces between the gastric septae on the presumptive oral-disc of Aiptasia. Comparable increases in mitotic rates have been demonstrated during the reconstitution of polyps from fragments of Amelia aurita scyphistomae larvae (Steinberg, 1963). Quantitative autoradiography with...