The morphology of tadpole (Rana catesbeiana) liver under varying conditions of organ culture.код для вставкиСкачать
The Morphology of Tadpole (Rancr ccrfesbeiana) Liver under Varying Conditions of Organ Culture THOMAS PETER BENNETT AND HENRY XREIGSTEIN Biological Science, Florida State University, Tallahassee, Florida 32306 and Stanford University Medical School, Stanford, California 94305 ABSTRACT Primary explants from Rana catesbeiana tadpole liver have been maintained in organ culture for six days using methods of cultivation which are described in detail. The organization and morphology of the cells in culture have been defined by light and electron microscopy. By adjustment of the experimental conditions of culture, after six days, the cells are morphologically identical to those freshly excised from tadpole liver. Soon after being put into culture, however, the chromatin of the nuclei becomes condensed, and only after about 30 hours in culture do t h e original euchromatic characteristics of the nucleus reappear. The effects of changes in the culture conditions on these morphological characteristics are presented. The system that has been developed is discussed with a view toward elucidating the basis of the cytological changes which occur during anuran metamorphosis. Recent studies in this laboratory (Bennett et al., '70; Bennett and Glenn, '70) have provided morphological criteria for differentiation of tadpole ( R a m catesbeiana) liver cells during thyroxine-induced and natural metamorphosis. Cytological alterations include change of the nuclei from a euchromatic to heterochromatic condition, an increase in the size of mitochondria and changes in their cristae from broad lamellar to a smaller more tubular appearance, a proliferation of the rough endoplasmic reticulum through the cytoplasm, and changes in the Golgi complex. In preliminary reports, Cohen ('70) and Spiegle and Spiegle ('70) have made some of these observations on respectively R. catesbeiana and R . pipiens. Because of individual differences among tadpoles in their time of response to thyroxine (Weber, '67) and tissue variability, the development of organ or cell cultures of their tissues is of considerable importance for elucidating the mechanisms of thyroxine-induced changes in biochemical and cytological patterns during metamorphosis. ANAT. kc., 176: 461-474. The extensive literature (Garvey, '61; Hillis and Bang, '62; Majno, '64; Blatt et al., '69; Simnet and Balls, '70; Balls, Simnett and Arthur, '69; Monnickendam, Miller and Balls, '70; Shambaugh et al., '69; Rafferty, '69) about culturing parenchymal cells suggest that i t is necessary that very careful morphological studies on cells in culture must precede or be closely correlated with biochemical studies. It is clear that if liver cells or liver tissue are placed in sterile culture medium, stable enzyme activities may be measured for days and weeks even though cells may be dying, lysing and releasing their contents into the sterile medium (Majno, '64). Although both biochemical and cytological studies have been published about organ culture of tadpole tail (Tata, '66; Gross and Lapiere, '62; Weber, '69), frog skin (Vanable, '65) and frog lens (Rothstein et al., '65), and other anuran cells (Freed and Mezger-Freed, '69; Freed, Mezger-Freed and Schatz, '69), only biochemical data on tadpole liver in culture (Bennett et al., '69; Shambaugh et al., '69; Balinsky et al., '70) along with preliminary Received Sept. 21, '72. Accepted Mar. 6 , '73. 461 462 THOMAS PETER BENNETT AND HENRY KREIGSTEIN statements (Bennett, '71) about the cy- dium, the tissue was sliced (0.5 mm, thicktology of the tissues have been reported. ness) with a Stadie-Riggsmicrotome which In at least one study the cells were stated had been washed in 95% ethanol followed to be in a degenerating state (Blatt et al., by air drying during exposure to UV. The slices were gently rinsed from the micro'69; Cohen, '70). The purpose of this study was to define tome with fresh culture medium into a the techniques for preparing and condi- Petri dish. When all the liver had been tions for maintaining R . catesbeiana tad- sliced with a microtome, the pieces which pole liver cells in vitro over a period of had been transferred to the medium in six days in a close approximation to their the Petri dish were then transferred to native morphological state. This period is culture medium on bite wax. These larger sufficient to observe in vivo effects of thy- slices of tissue were diced into pieces about roxine (Bennett et al., '70; Frieden and 1 mm square with razor blades. Thus the Just, '70) and should be sufficient time for final tissue fragments were approximately in vitro study of the mechanism of thy- 1 mm X 1mm X 0.5 mm. In early studies the size of tissue was monitored with a roxine action. plastic centimeter ruler or ocular micromMATERIALS AND METHODS eter. The final tissue size was judged to All animals used were Class 1 (Ben- be 1 mm 0.5. Subsequently the size was nett and Glenn, '70), that is Stage IX-XI11 judged by eye with occasional standardiof Taylor and Kollros ('46) R. catesbeiana zations as described above. One dimension, tadpoles (Connecticut Valley Biological determined by the Stadie-Riggs microtome, Supply Co., Southampton, Mass.). These was fixed. The slices were gently removed tadpoles have short hind limbs but no fore- with a stainless steel micro-spatula to a limbs and are said to be in late pre- container of medium. From here two fragments were transferred with a spatula into metamorphosis. The culture medium used in these ex- a culture tube (12 mm X 75 mm, 2003, periments was based on the formulation Falcon Plastics, Los Angeles, Calif.) conof Wolf and Quimby ('64) and was pur- taining 0.4 ml of culture medium. The chased from the Grand Island Biological tube was capped and when all tubes had Supply Company, Grant Island, N.Y. This been so prepared (about 50-75 tubes per medium was modified to contain 100 mg 30 minutes per tadpole) they were placed glucose per liter except as indicated in in a Dubnoff metabolic shaking incubator (Precision Scientific Co., Chicago, Ill.). individual experiments. Organ cultures of liver explants (ap- All operations were conducted at 22"proximately 1 mm X 1 mm X 0.5 mm), 24°C. Incubation was at 26"C, at a slow were prepared from tadpole liver under the shake setting (50 shakes per minute), with following conditions. All instruments were continuous gas flow (95% air-5% CQI) autoclaved and sterile disposable pipettes, for the time indicated in the experiments. Petri dishes, etc., (Falcon Plastics, Los Contaminated culture tubes, as determined Angeles, Calif.) were used. The equip- by cloudiness or pH change of the medium, ment was further sterilized by ultra-violet were discarded. (UV) radiation in a hood where all operaFig. 1 Phase contrast micrographs of liver tions were performed. The tadpole was cells during a six day culture experiment. X 400. killed by decapitation, the abdomen was Scale shows 25 $I. Fig. 1A Liver immediately after being put swabbed with 95% ethanol and the liver was removed by dissecting it free of the into culture. Note the cord-like arrangement of cell*$(Co) and the large sinusoids (Si). Arrows gallbladder and blood vessels. It was put point out nuclei. into a s m d pool (5-6 ml) of sterile culFig. 1B Appearance of cells after 48 hours ture medium on a sterile bite wax plate in culture. Fig. 1C After 96 hours of culture. ("Byte-Ryte," Mizzy, Incorp., Clifton Forge, Fig. 1D After 144 hours of culture. Note that Va.). After being momentarily placed on cord-like arrangement of hepatocytes has been sterile filter paper to remove excess me- preserved. * MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE Figure I 463 464 THOMAS PETER BENNETT AND HENRY KREIGSTEIN Fig. 2 Electron micrographs of cells a t varying periods in culture during a six day culture experiment such as shown in figure 1. x 13,300. Scale shows 1p. Fig. 2A Tadpole liver recently excised and #containing rough endoplasmic reticulum (RER), Golgi ( G ) , bile canaliculi (bc), mitochondria ( M ) , a euchromatic nucleus ( N ) , glycogen (gl), and lysosomes (ly). Fig. 2B A similar view after 48 hours in culture. MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE 465 Fig. 2C After 96 hours in culture. Fig. 2 D After 144 hours of culture. Note the preservation at increasing times of cytoplasmic features and characteristics. 466 THOMAS PETER BENNETT AND HENRY KREIGSTEIN Tissue for microscopy was fixed in general morphology is not changed noticeglutaraldehyde, post-fixed in osmium te- ably. The examination of large areas of troxide, dehydrated and embedded in randomly selected cultured tissue showed Epon; sections were stained with lead cit- cells that were more closely packed than rate and uranyl acetate (Glenn and Ben- those in freshly excised tissue and slightly nett, '68; Bennett and Glenn, '70). Electron more elongate cells with typical nuclei. microscopy was carried out with a Siemens Only seldom were necrotic areas observed. Elmiskop or a Philips 300 at primary magElectron microscopy of cultured cells nifications of 2000 to 30,000 X. For light microscopy, sections 0.5 to 3 p in thickness The representative electron micrographs were cut and stained with Toluidine Blue shown in figure 2 summarize the features for transmitted light optics, or mounted of cells from a six day time course exunstained for phase contrast optics. periment similar to that described above. Explants were cultured for a period of In figure 2A, the ultrastructural character72 hours in Wolf and Quimby medium istics of pre-metamorphic tadpole liver are containing varying amounts of glucose : clearly displayed and correspond to those 800 mg/liter, 100 mg/liter and 10 mg/ earlier reported (Bennett and Glenn, '70). liter. For biochemical analysis, tissues from Small, rod-shaped mitochondria occur four culture tubes were pooled for each scattered throughout the cytoplasm and assay and processed as described above. they have prominent, tubulo-lamellar Glycogen was determined by the method cristae. The occurrence of lysosomes, glyof Dubois et al. ('56) after homogeniza- cogen rosettes, and occasional stacks of tion of weighed pieces of cultured tissue. rough endoplasmic reticulum (RER) are features characteristic of R. catesbeiana RESULTS liver. A bile canaliculus is also shown, Light microscopy of organ cultured cells as are several multi-vesicular Golgi com(fig. 1 ) shows tadpole liver tissue as it plexes. The chromatin of the nucleus, alappears after 0, 48, 96, and 144 hours of though not prominently shown here, is culture. The cells of liver tissue at the bewell dispersed and the nucleus can be ginning of the culture period (fig. 1A) characterized as euchromatic. occur in clusters with two or three cells Micrographs of cells in culture for 48, in contact with each other. The sinusoids 96, and 144 hours, respectively, are preare prominent and leucocytes and red blood cells are clearly visible. Individual sented in figures 2B-D for comparison with the freshly excised tissue. As can be cells are trapezoidal or polygonal in shape seen, the appearance of the organelles and and have a single prominent nucleus. The their arrangements are preserved essennuclei are asymetrically located and have tially as in the fresh sample. For example, several densely staining areas which represent nucleolar regions and clusters of Fig. 3 Electron micrographs of the major orheterochromatin, as revealed by electron ganelles in liver cells after three days in culture. Fig. 3A Nucleus ( N ) with nucleolus (Nu). microscopy (see below), hucleoplasm is predominantly euchromatic (Eu), After two days in culture (fig. l B ) , one with occasional areas of diffuse heterochromatin striking change is a more close-packing of (He). AROWS emphasize fine structure of the the cells and reduction of the well defined nuclear envelope. x 9,380. Scale shows 1 p . Fig. 3B Field showing mitochondria (M), sinusoidal spaces. Some cell division has surrounded by glycogen (91) and rough endooccurred as evidenced by clusters of plasmic reticulum (RER). x 27,600. Scale show6 smaller, more tightly packed cells. De- 0.5 p. Fig. 3.2 Rough endoplasmic reticulum in struction of the blood cells in the sinus- close juxtaposition to mitochondria (M), glycogen oids has occurred to a large extent and (91 and arrow) L, lipid. Other arrows point to there is considerable debris surrounding ribosomes. x 38,600. Scale shows 0 . 2 5 , ~ . Fig, 3D Golgi complex with cisternae (GC) the parenchymal cells. After longer periods and vesicles distinctly resolved. Also shown are in culture, 96 and 144 hours (fig. l C , D ) , glycogen (91) granules. x 29,300. Scale shows cells remain closely packed, but their 0.5 p. ~~ MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE Figure 3 467 468 THOMAS PETER BENNETT AND HENRY KREIGSTEIN mitochondria1 cristae remain distended and lamellar; the chromatin of the nucleus remains dispersed. Electron microscopy of organelles Figure 3 summarizes the results of higher magnification studies of selected organelles; i.e., the nucleus, mitochondria, RER, and Golgi in three day culture experiments. The nucleus, as shown in figure 3A, is predominantly euchromatic, with some condensed chromatin associated with the nucleolar region. Figure 3B reveals that the morphology of pre-metamorphic mitochondria has been preserved during this culture period. Profiles of the cristae remain broad, flat and irregular; dramatic changes in the sizes and shapes of mitochondria do not occur as happens during metamorphosis (Bennett and Glenn, ’70). A higher magnification view of RER is shown in figure 3C. In addition, mitochondria and clusters of glycogen may be discerned between the stacks of cisternae of the RER. Two well developed Golgi complexes are shown in figure 3D. They consist of stacks of cisternae, in close opposition to glycogen, smooth endoplasmic reticulum, etc. Glycogen content In vivo, liver glycogen levels are under complex controls. In organ culture, however, this balance may be disrupted because of different levels of hormones in serum contained in the culture medium or because of the level of glucose in the medium. It is therefore necessary to adjust the culture conditions of the cells in order to obtain normal levels of glycogen coiitent. Our approach was to try to regulate glycogen content by regulating the glucose concentration in the medium. The following experiments established glycogen levels in freshly excised liver tissue and in organ cultured tissue under varying conditions of glucose concentration in the culture medium. Figure 4 compares low magnification electron micrographs of liver cells before and after 72 hours of culture in media with various concentrations of glucose. Glycogen rosettes are prominent in the cytoplasm of parenchymal cells at zero time., After 72 hours in culture medium with the “standard concentration of glucose, i.e., the concentration in Wolf and Quimby medium (800 mg/l), the cells become m e d with glycogen and sections thus appear abnormal by comparison with control tissue (fig. 4A,B). At the lower concentrations of 100 mg/liter or 10 mg/ liter, the liver maintains a more normal appearance with regard to glycogen content and glycogen rosettes occur with a frequency comparable to that seen in tadpole liver cells. Quantitative results are presented in table 1. The various glycogen contents, expressed on the basis of pg protein or milligram wet weight of tissue, confirms the qualitative microscopic impression that lower glucose concentrations result in a situation more like that in vivo. TABLE 1 Effect of glucose concentration on glycogen content of orgaia cultured liver1 ~ Glucose concentration pg glycogen per pg protein gg glycogen per m g hssue ~ ms/l 0.059 ‘-c 0.0030 16.5 k 0.10 100 0.072 k0.018 17.622.4 800 2 0.12 i- 0.018 32.8 f6.6 10 1 Freshly excised tissue gave values of 0.084 +- 0.014 pg per pg protein. ZConcentration present in Wolf and Quimby Medium. Fig. 4 Electron microscore survey pictures of liver from the same tadsole at zero time and after three days culture in media with various concentrations of glucose. x 5,930. Scale shows 1 P. Fig. 4A Zero time. Freshly excised and prepared for culture. Fig. 4B Cultured with 800 m % / l glucose, the “standard’ content of glucose in Wolf and Quimby medium for three days. Note the superabundance of glycogen rosettes (91). Fig. 4 C Cells in tissue cultured with 100 m g / l glucose. Fig. 4D Cells as they appear after three days with 10 m g / l glucose. The heterochromatic nucleus in ( A ) is due to pre-chilling of the tadpole, as described by Kriegstein and Bennett (’73). N, nucleus; gl, glycogen; L, lipid. MORPHOLOGY OF TADPOLE LIVER I N ORGAN CULTURE Figure 4 469 470 THOMAS PETER BENNETT AND HENRY KREIGSTEIN Reversible chromatin condensation of liver cell nuclei during short-term organ culture Since the rates of amino acid, uridine and thymidine incorporation by tadpole liver showed slight decreases during the first 24-40 hours in culture, after which incorporation rates recovered (Bennett and Kriegstein, ’73), it was of interest to investigate morphological correlates of these responses. Cultures were prepared according to METHODS and the early time periods were monitored by microscopy. Figure 5 summarizes the results of a time course experiment during 36 hours in culture with the tissue being monitored with phase contrast optics. Figure 5A illustrates fresh tissue with its characteristic parenchymal cell nuclei which are euchromatic. After 12 hours in culture (fig. 5 B ) , the nuclei are altered in appearance by having a greater distinction between euchromatic and heterochromatic regions. At 24 hours (fig. 5C) the even, grey euchromaticity of the nuclei has been restored, and by 36 hours (fig. 5D) the nuclei look like those observed in the starting tissue (fig. 5A). The electron micrographs in figure 6 compare a cell nucleus from recently excised liver tissue with a nucleus from a cultured tissue cell after 12 hours in culture. The former nucleus is predominantly euchromatic, with a homogeneous distribution of chromatin, gradually becoming more heterochromatic near the nuclear membrane and about the nucleolus. In contrast, the nucleus from short-term cultured tissue, has distinct condensed chromatin as well as euchromatic regions. The areas of condensed chromatin along the inner surface of the nuclear membrane and that associated with the nucleolus are both more extensive and more denseiy staining than the corresponding chromatin in freshly excised tissue. DISCUSSION A standard amphibian cell culture medium (Wolf and Quimby, ’64) which has been modified to contain a lower concentration of glucose, has been used under defined experimental conditions to main- tain liver fragments (1 mm X 1 mm X 0.5 mm) from a single tadpole for an experimental period of six days. Approximately 50 to 75 experimental tubes from a single tadpole, each containing two pieces of liver tissue, are incubated under an air-COz atmosphere in a Dubnoff Shaker. Our culture conditions, in contrast to those of Shambaugh et al. (’69) in which “viability of the liver cubes was not altered significantly by incubation in oxygen, type of medium used, or use of smaller cube size,” had to be precisely regulated, as described in METHODS, with regard to these parameters in order to achieve the quality of morphological and biochemical integrity described in the present publication. Under many other different conditions, we observed anomolous cell morphology, large areas of necrotic tissue and biochemical features comparable to those reported by other investigators for a dying liver cell population (see review by Majno, ’64). These other conditions that were tested included modifications of the culture medium (e.g., pH, ionic strength, serum content) of the ratio of amounts of medium to tissue, size of tissue fragments, incubation atmosphere and the conditions for preliminary handling of tissue. In concurrence with Shambaugh et al. (’69) we observed that “viability was adversely affected by higher temperatures of incubation, inadequate shaking of the flasks during incubation, and prolonged preparation time after liver removal ( 2 to 4 hours).” On the basis of light microscopy studies, medium containing 10% fetal calf serum proved superior to that containing frog or iadpole serum; the latter resulted in large Fig, 5 Light micrographs showing nuclear changes in liver cells durins their first 36 hours in culture, All tissues are from one animal. rbc, red blood cell; hp, hepatocytes. x 1,000. Scale shows l o p . Fig. 5A Appearance of freshly excised parenchymal cells. Arrows point to nuclei, which are euchromatic. Fig. 5B Tissue after 12 hours in culture. The nuclei (see arrows) are now heterochromatic. Fig. 5C After 24 hours the cell have nuclei that are becoming euchromatic again. Fig. 5D Thirty-six hour cultured tissue has cells with euchromatic nuclei as in A. MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE Figure 5 471 472 THOMAS PETER BENNETT AND HENRY KREIGSTEIN Figure 6 MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE accumulations of lipid (Kriegstein, ’71). Pieces of tissue that exceeded 2 mm in size generally showed large areas of necrosis. Two pieces of tissue per 0.4 ml of medium could be satisfactorily maintained for six days. One piece of tissue could be used in this volume of medium, however, three or four pieces generally showed necrosis. The results reported here demonstrate that the normal premetamorphic fine structure features of parenchymal cells and their organelles are well preserved for a period of six days in culture. However, on being put into culture medium and during the early period (up to about 30 hours) in culture, parenchymal cell nuclei become, according to the terminology of Fawcett (’66) and Porter and Bonneville (’70), heterochromatic; later they return to their so called euchromatic state. O u r results suggest that in our case the “trauma,” often mentioned in connection with cell culture work, that occurs when tissue adapts to in vitro conditions is reflected in defined fine structural changes (i.e., chromatin condensation) in the cultured cells. After an adjustment period these changes are reversed completely or in part. The most obvious suggestion, following current writings, is that chromatin condensation occurs as a result of perhaps changes in ionic strength, of cations, or other ingredients in the in vitro milieu and that this condensed chromatin is genetically inactive. Thus the biochemical, morphological and culture effects become interrelated. One is seemingly left with a broad spectrum of possible causes for which perhaps the best term is “trauma” and the cell morphology and biochemistry (Bennett and Kriegstein, ’73) reflect recovery of the cells from the “shock” of ‘lag” phase which occurs when fresh tissue “adapts” to in vitro conditions. Fig. 6 Comparison of control and incubated (12 houre) liver cell nuclei. x 6,370. Scale shows 1 fi. Fig. 6A Control. Eucliromatin ( E u ) , heterochromatin (He), and the nucleolus ( N u ) are clearly shown in this cross-section of a typical nucleus. Fig. 6B Incubated nucleus. Heterochromatic ( H e ) regions, together with densely stlaining spots of heterochromatin (Ch), occur i n formerly euchromatic areas. The euchromatic regions are now more distinct. 473 In the present work, emphasis has been placed upon determining the viability and also the maintenance of differentiated features of premetarnorphic tadpole liver in culture. The assessment of these states for tissues rests on indirect biochemical criteria and more direct morphological criteria. As reported elsewhere (Bennett and Kriegstein, ’73) the patterns for biochemical constituents (e.g., dry weight, wet weight, DNA, RNA and protein content) during periods in culture as well as the gross metabolic patterns reflected by incorporation of radioactive amino acids, uridine, and thymidine, corroborate our morphological observations about the tissue. The tadpole liver organ culture system as here reported is convenient to set up, uses a slightly modified medium which is readily available, and allows as many as 50 to 75 individual tubes of liver explants to be prepared from a single tadpole liver. This insures genetic uniformity and a reduction in so-called “biological variability.” These features make the organ culture system suitable for further development directed toward the in vitro study of metamorphic responses of tadpole liver and the mode of action of thyroxine. LITERATURE CITED Balinsky, J. B., G. E. Shambaugh and P. P. Cohen 1970 Glutamate dehydrogenase biosynthesis in amphibian liver preparations. J. Biol. Chem., 245: 128-137. Balls, M., J. D. Simnet and E. Arthur 1969 Organ cultures of normal and neoplastic amphibian tissues. Biology of Amphibian Tumors. M. Mizeu, ed. Springer-Verlag, New York, pp. 385-398. Benxett, T. P. 1971 Moruhological and biochemical studies on tadpole liver in organ culture. Abstract 44. The American Society for Cell Biology. 11th Annual Meeting. Bennett, T. P., and J. S. Glenn 1970 Fine structural changes i n liver cells of Rana catesbeiana during natural metamorphosis. Develop. Biol., 22: 535-560. Bennett, T. P., J. S. Glenn and H. Sheldon 1970 Changes in the fine structure of tadpole ( R a m catesbeiana) liver during thyroxine-induced metamorphosis. Develop. Biol., 22: 232-248. Bennett, T. P., H. Kriegstein and J. S. Glenn 1969 Thyroxine stimulation of ornithine transcarbamylase activity and protein synthesis in tadpole (Rana catesbeiana) liver in organ culture. Biochem. Biophys. Res. Commun., 34: 412-417. 474 THOMAS PETER BENNETT AND HENRY KREIGSTEIN Bennett, T. P., and H. Kriegstein 1973 Biochemical Studies on tadpole (Rana catesbeiana) liver in organ culture, submitted to Biochem. Biophys. Res. Comm. Blatt, L. M., K. H. Kim and P. P. Cohen 1969 The effect of thyroxine on ribonucleic acid synthesis by premetamorphic tadpole liver cell suspensions. J. Biol. Chem., 244: 48014807. Cohen, P. P. 1970 Biochemical differentiation during amphibian metamorphosis. Science, 168: 533-543. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Eebers and F. Smith 1956 Colorimetric method for determination of sugar and related substances. Analy. Chem., 28: 350-354. Fawcett, D. W. 1966 An atlas of fine structure. The cell, its organelles and inclusions. W. B. Saunders Company, Philadelphia, Pennsylvania, pp. 2-28. Freed, J. J., and L. Mmzger-Freed 1969 Culture methods for anuran cells. In: Methods in Cell Physiology. D. M. Preseott, ed. Academic Press, New York, 4: 19-47. Freed, J. J., L. Menzger-Freed and S. A. Schatz 1969 Characteristics of cell lines from haploid and diploid anuran embryos. Biology of Amphibian Tumors. M. Mizell, ed. SpringerVerlag, New York, pp. 101-111. Frieden, E., and J. Just 1970 Hormonal responses in amphibian metamorphosis. In: Mechanisms of Hormone Action. G. Litwack, ed. Appleton-Century-Crofts, New York, New York, pp. 1-52. Garvey, J. S. 1961 Separation and in vitro culture of cells from liver tissue. Nature, 191: 972-974. Glenn, J. S., and T. P. Bennett 1968 A rapid method for preparing tissue for light and electron microscopy. J. Histochem. Cytochem., 16: 815-819. Gross, J., and C. M. Lapiere 1962 Collagenolytic activity i n amphibian tissues: a tissue culture assay. Proc. Natl. Acad. Sci. U. S., 48: 1014-1 018. Hillis, W. D., and F. B. Bang 1962 The cultivation of human embryonic liver cells. Exper. Cell Res., 26: 9-36. Kriegstein, H., and T. P. Bennett 1973 Chromatin condensation and nucleolar segregation induced in nuclei of parenchymal cells of Rana catesbeiana tadpoles. Expt. Cell Res., in press. Majno, G. 1964 Death or' liver tissue. In the Liver, Morphology, Biochemistry, and Physi- ology, Ch. Roullier, ed. Academic Press, New York, 2: 267-313. Monnickendam, M. A., J. L. Maller and M. Balls 1970 Cell proliferation in vitro and in vivo in visceral organs of the adult newt. J. Morph., 132: 359-453. Porter, K. R., and M. Bonneville 1970 Fine structure of cells and tissues. Third ed. Lea and Febiger, Philadelphia, Pennsylvania, pp. 20-25. Rafferty, K. A. 1969 Mass culture of amphibian cells : methods and observations concerning stability of cell type. In: Biology of Amphibian Tumors. M. Mizell, ed. Springer-Verlag, New York, pp. 52-81. Rothstein, H., J. M. Lander and A. Weinsieder 1965 In vitro culture of amFhibian lenses. Nature, 206: 1267-1269. Shambaugh, G.E.,J. B. Balinsky and P. P. Cohen 1969 Synthesis of carbamvl phosphate synthetase i n amphibian liver in vitro. J. Biol. Chem., 244: 52955308. Simnet, J. D., and M. Balls 1969 Cell proliferation in Xenopus tissues: a comparison of mitotic incidence in vivo and in organ culture. J. Morph., 127: 363-372. Spregle, E. S., and M. Spiegle 1970 Some observations on the ultrastructure of the hepatocyte in the metamorphosing tadpole. Exp. Cell Res., 61: 103-1 12. Tata, J. R. 1966 Requirement for RNA and protein synthesis for induced regression of the tadpole tail in organ culture. Develop. Biol., 13: 77-94. Taylor, A. C., and J. J. Kollros 1946 Stages i n the normal development of Rana pipiens larvae. Anat. Rec., 94: 7-23. Vanable, J. 1965 Organ culture of Xenopus laevis larval skin. Am. Zool., 5: 663. Weber, R. 1967 Biochemistry of amphibian metamorphosis. In: The Biochemistry of Animal Development. R. Weber, ed. Academic Press, New York, 2: 227-301. 1969 The isolated tadpole tail as a model system for studies on the mechanism of hormone-dependent tissue involution. Gen. Comp. Endocrind. Suppl., 2: 408-416. Wolf, K., and M. C. Quimby 1964 Amphibian cell culture: permanent cell line from the bullfrog ( R a n a catesbeiana). Science, 144: 1578-1580.