Preparation of Material for Light and Electron Microscopy

The fine structural changes occurring in the columnar absorbing cells of the intestinal epithelium during metamorphosis of the bullfrog, Rana catesbeiana, have been examined by phase contrast and electron microscopy. Tissue samples taken just posterior to the entrance of the hepatopancreatic duct were fixed in vcronal acetate-buffered osmium tetroxide and embedded in methacrylate. Under the action of the metamorphic stimulus (thyroid hormone), specific and characteristic responses were given by differentiated larval cells and undifferentiated basal cells within the same epithelium. The functional larval cells underwent degenerative changes and were retained for a time within the metamorphosing epithelium. Dense bodies appeared and increased in number in association with the loss of normal cell structure. Because of their morphology and time of formation, these bodies have been tentatively identified as lysosomes. Early in metamorphosis the basal cells did not change, but they subsequently proliferated to form a new cell layer beneath the remaining degenerating cells that lined the lumen. After the dying cells were sloughed into the gut, the new epithelium differentiated to form the adult tissue. The columnar epithelial cells of the mature animal differed in their fine structural organization from their larval precursors. Therefore, their adult configuration was molded by the action of the metamorphic stimulus. I N T R O D U C T I O N Amphibian metamorphosis is a phenomenon that is especially attractive to those interested in understanding morphogenetic processes. It is obvious that the sudden onset of metamorphic changes that progress rapidly and dramatically allows easy collection of developmental stages. But more than this, the period of final maturation that transforms larva into froglet occurs under the influence of the thyroid secretion. The first indication of this hormonal control of differentiation came in the work of Gudernatsch (1). His observations were extended and confirmed in a variety of experiments by many different investigators. Etkin (2) has provided a review of the body of this work, which led to widespread recognition of the thyroid hormone as a causative agent--probably the unique one--in amphibian metamorphosis. As the structure of several physiologically active components of the thyroid gland hormone became known, investigators produced structural changes through the use of known chemical agents. Exploitation of this method has led to more detailed 579 on A uust 8, 2017 jcb.rress. org D ow nladed fom understanding of the interaction between the hormone and responding tissue, and it is now well established that each tissue and even individual cell types within tissues respond specifically to the metamorphic stimulus. This point was well illustrated on the anatomical level by the experiment of Schwind (3), on the histological level by the work of Weiss and Rossetti (4), and more recently on a biochemical level in investigations such as that of Finamore and Frieden (5). Therefore it seems reasonable to expect that equally specific cytological changes occur both as a basis for the grosser, well known events of metamorphosis and as a reflection of the subtle, well ordered biochemical changes more recently described. As yet, however, fine structural changes have not been observed. Although formerly it was, of course, impossible to record the effects of the metamorphic stimulus upon the fine structure of cells or to follow with certainty the fate of individual cells, now study by electron microscopy can provide unequivocal identification of cell types and detection of cytological changes within them. Indeed, this developmental problem seems especially suited to this particular method of investigation. The short, dramatic nature of events allows the complete process to be observed easily and tissue samples to be taken at frequent intervals. Furthermore, the changes already revealed by other means hold promise that cytological changes seen under the electron microscope will be clear-cut and easy to define. With these considerations in mind, an investigation of fine structural changes in the intestinal epithelium of the bullfrog during metamorphosis was undertaken. The intestinal epithelium promised to be a suitable object for such an investigation primarily because previous studies had established that striking metamorphic changes occur in this organ. The general shortening of the tract, changes in size and position of the stomach, and rearrangement of the coils of the intestine were described many years ago (6-10). In addition, histological and cytological observations of the epithelial changes in the anuran intestine (7, 8, l l -13 ) demonstrated that cell degeneration and regeneration were important in reshaping the tissue during metamorphosis. Furthermore, in spite of complicated tissue changes, there seemed little doubt that comparable samples of epithelium could easily be located throughout metamorphosis by removing the area just posterior to the entrance of the hepatopancreatic duct into the intestine. The observations made here will be concerned with the specific responses of the. functional larval columnar epithelial cells, which are destroyed and removed from the epithelium. Their behavior will be contrasted with that of the basal or germinative cells, which are stimulated to divide and to undergo cytodifferentiation preparatory to the formation of the adult epithelium. The differential response of these two cell types, which is to be examined here in detail, is an early event in the metamorphic process and prepares the way for development of the adult intestinal epithelium. Finally, it will be shown that the differentiation of basal cells follows a new course under the influence of the metamorphic stimulus, producing cells which differ in fine structure from their larval precursors. M A T E R I A L S A N D M E T H O D S