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Cell Growth and Development
Cell Growth and Development A Model Biologic System OTTO M. LILIEN, M.D., F.A.C.S. With the technical assistance of Harold M. Phillips
MEDICINE is a science of the incomprehensible. When we surgically violate the integrity of the human organism, we assume that the defects we create will be corrected by dividing, differentiating, migrating cells that somehow "know" where to go and what to do. In an effort to elucidate some of the mysteries of cell behavior, the present study was undertaken. The development of a fertilized cell into an insect, a Hower, or a man, involves three cellular processes: division, differentiation, and cell movement. The mechanisms regulating these processes, and their interaction, are not known. In order to study the regulation of cell behavior, we looked for a biologic system in which it would be possible to separate division, differentiation, and cell movement, without disrupting the system. Even in simple metazoans these processes occur simultaneously and with a degree of complexity which makes analysis difficult, if not impossible. We, therefore, turned to one of the most primitive available metazoans. The earliest living forms were either acellular or unicellular. At some critical point in evolution, dividing cells had to remain together, or separate cells had to come together, in order to form a many-celled structure. We will probably never know the precise nature of this historical event, but there exists an order of organisms in which there is a natural separation between cell division, or growth, and morphogenesis. In addition, the step between the single cell and the metazoan is part of its life cycle. The organism is Dictyostelium discoideum, a species of the order Acrasiales, or the cellular slime molds. The first clear identification of a member of the order of Acrasiales was made in 1869 by Brefeld. 1 In 1902 Olive published the earliest compreFrom the Department of Urological Surgery, State University of New York, Upstate Medical Center, and the Veterans Administration Hospital, Syracuse, N. Y. Presented at the 22nd Annual Meeting of the American Fertility Society, Chicago, Ill., Apr. 29-May 1, 1966. The author wishes to express his gratitude to the Medical Illustrations Service of the Syracuse Veterans Administration Hospital.
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hensive study of this presumably primitive order, while in 1935 Raper discovered the species Dictyostelium discoideum. Because of the suitability of this species for the study of a variety of biologic problems, a large body of literature has since appeared concerning the behavior of this interesting organism. Before discussing the specific phases of the life cycle of Dictyostelium discoideum (Fig. 1), it should be emphasized that there is a natural separation between the feeding or vegetative stage, during which cell division and growth occur, and the remainder of the cycle, which is morphogenetic. Feeding stops prior to aggregation, and from that moment on, the energy for the morphogenetic stages comes entirely from reserves stored up during the vegetative stage. It should be further pointed out that the Acrasiales are distinguished from the true myxomycetes, in that the former are made up of uninucleate, ameboid units which never become multinucleate, probably lack sexuality, and do not possess flagella as a means of locomotion. In nature Dictyostelium discoideum grows anywhere there is a supply of gram-negative bacteria, and is usually found in moist humus. In its final, most resistant form, the organism consists of a hairlike stalk, 2-3 mm. tall, with a small sphere or sorus, loaded with spores, at its top. Under the proper conditions of humidity, temperature, and food supply, each spore germinates, giving rise to a single ameba or myxameba (Fig. 2). And so begins the vegetative phase of the life cycle. It is during this phase of the cycle, and only this phase, that cellular division occurs. The sequence of morphogenetic changes which follow the vegetative stage, occurs whether the cells are derived from many spores or from a clone of genetically identical cells derived from a single spore. The amebae, or myxamebae, feed by phagocytosis of predominantly gram-negative bacteria, and reproduce asexually by binary fission. It should be particularly noted that during this phase of multiplication, the cells function as independent units and do not interrelate in any observable way. The vegetative ameba is indistinguishable from other free-living amebae. It has a single nucleus and contractile vacuoles, and moves by means of pseudopods. In addition to lobate pseudopods, we have observed the presence of long, delicate, filiform pseudopods, whose length occasionally exceeds the greatest diameter of the ameba. When the food supply is exhausted, or when a critical population density is reached, cell division stops and morphogenesis begins, with the onset of the preaggregation and aggregation phases. Since food ingestion is limited to the vegetative phase and accumulated anabolites are utilized thereafter, the cells become smaller.4 At the end of an interphase period of some 4-8 hr., the cells elongate (Fig. 3) and interrelate in a remarkable fashion,
Q
VEGETATION
SPORE Spores Stalk
9
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~ Spore,I.9J1
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MIGRATION
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A T I 0 N
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PSEUOOPLASMOOIUM (SLUG) FORMATION
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Fig. 1. Life cycle of Dictyostelium discoideum.
( Acrasin)
VoL. 18, No.3, 1967
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literally seeking each other out, linking u_p head to tail to form long streams of cells which move together toward a common center. Pseudopods at the anterior end of a cell coming into contact with the side of a second elongated cell appear to palpate the side of the cell surface in a retrograde
Fig. 2 (top). Vegetating myxameba and germinating spore. (X 630) Fig. 3 (bottom). Pre-aggregating myxamebae. (X 250)
direction until contact and fixation occur at the most posterior portion of the advancing cell. These linked cells now move as a unit toward the common center. This raises the interesting question as to whether the sides of an advancing ameboid cell move, or whether the anterior portion of the cell is put down with simultaneous absorption of the posterior portion of the cell, while the sides remain stationary. The observed fixation between
322
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the 2 cells occurs when the palpating pseudopods reach the terminal end of the advancing ameba. Our- observation of visible artifacts adherent to the sides of advancing cells tends to support this hypothesis. While streams of cells are moving toward a central point, it can be seen that cells which are not physically connected with these streams are similarly oriented and move in the same direction. Biologists have been intrigued by the problem of how these cells know where to go. While the answer is far from complete, it is a remarkable fact that a steroid-like hormone has been demonstrated to arise from the aggregation center. This substance was given the generic name of "Acrasin," following the experiments demonstrating its existence by Bonner in 1947.3 He demonstrated that when amebae were allowed to aggregate under a layer of water flowing unidirectionally, the amebae upstream of the center showed no orientation, while downstream amebae maintained their orientation toward the center. The presence of a free diffusing agent was thus demonstrated. Substances such as sugar and malic acid, which have previously been shown to orient successfully the spermatozoa of bracken, did not produce orientation with Dictyostelium discoideum. The biochemical nature of Acrasin has been the subject of intensive investigation by a number of workers. In 1960 Heftmann et al. isolated from Dictyostelium disooideum a sterol with biologic activity. They identified this substance as ( a22-Stigmasten-3 {3-ol). Bonner1 and others postulate that Acrasin synthesis predominates at the aggregation center and by diffusion from this central point establishes a gradient along which cells migrate toward its source. It is provocative to consider that even at this primitive level of biologic organization, steroids play a regulatory role in cell behavior. Other steroids that have been tested and found to be effective in producing aggregation include: estradiol, estronesulphate, Stenediol, progesterone, testosterone, 7 and several ergosterols. 17 It is during this stage of preaggregation and aggregation that we have observed regular, periodic, pulsatory cell movement. Using time-lapse motion-picture photography in which movement was speeded up 100 times, we observed that during preaggregation and aggregation, cells and cell streams moving toward an aggregating center (Fig. 4) manifest periodic, simultaneous, synchronous movements in the same direction. In wellorganized streams of cells, an explosive separation of amebae beginning at the aggregation center was seen to propagate as a wave which moved outward from the center along the stream. Identical portions of a cell stream were photographed before, during, and after the passage of this explosive wave of synchronized cell movement (Fig. 5). The movement
t
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of cells away from each other suggests a transient polarization of the membranes tending to separate the cells. Cells not in physical contact with the streams also moved synchronously, apparently receiving some kind of information, the nature of which is completely unknown. In the earlier, less
Fig. 4. Aggregation center with stream of myxamebae. (X 35)
organized preaggregation phase, small groups of cells, alternately moving from one preaggregation center to another, were also seen to move in an organized, pulsatile, synchronous manner (Fig. 3). Analysis of 125 pulses recorded on our film showed a mean pulse interval of 5 min. (Fig. 6). Essentially, the manifestation of pulsatile behavior appears to be the result of a synchronous movement of many cells in the same direction. In several sequences, we have observed that the cells are quiescent after a pulsation, then develop a progressively increasing agitation of the anterior pseudopods which reaches a climax in a positive, forward movement. In these instances, the pulse intervals were prolonged, and are represented by the open circles in Fig. 6. We were able to follow the progression of the explosive waves along the stream, oriented parallel to the long axis of the film frame. A wave could thus be seen to originate at one end of the field and could be followed until it reached the opposite side. By knowing the size of the field and the time interval necessary for the waves to traverse the fields, we were able to determine the rate of transmission of pulsatory movement along a stream of cells. This was of the order of 2 p,jsec. This is the same order of magnitude observed for the saltatory movement of particles in protoplasm. 16
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Toward the end of aggregation, streaming is completed. We have observed several instances in which the orientation gradient is apparently lost when a critical cell mass is reached at the aggregation center. At this point, a previously well-organized stream loses its orientation and individual groups of cells split off to move in random fashion for short distances. This
Fig. 5. Time-lapse sequence photographs of myxamebae stream moving toward aggregation center. A. Before pulsation. B. During pulsation. C. After pulsation. (X 250)
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is associated with the beginning phase of differentiation of the amorphous mass of cells that constitute the aggregation center. This cell mass gradually rises into the air, to form a cigar-shaped slug, which falls on its side forming a pseudoplasmodium. This organism, just visible to the naked eye, Fig. 6. Distribution of 125 pulse intervals of D. discoideum recorded in timelapse motion-picture study. Open circles represent intervals between pulsations characterized by progressive anterior pseudopodal agitation (see text). Transmission rates (microns per second) were 2.5, 2.6, 2.5, 2.3, 2.0, and 1.8, with average of 2.3.
•• • •
•• • • •• ••• •• ••• 2
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• 0 0 0
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• •
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• •
• 0 ••
•• ••
6 7 PULSE INTERVAL IN MINUTES
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9
migrates in a gliding fashion for variable distances over the substrate, depositing a thin slime sheet behind the advancing pseudoplasmodiums. Raper and Fennell suggest that the slime sheet is made up of a combination of cellulose and mucin. It is at this stage that a true metazoan has been formed. Three cell species have appeared and sorted themselves out: the anterior or stalk cells, the middle or spore cells, and the posterior or basal cells. These cells differ morphologically, histochemically, and in their ultimate fate. The cells do not form a syncytium, but retain their uninucleate cellular identity. The pseudoplasmodium is capable of regeneration, and if it is divided, each fragment will regenerate to form a complete organism able to finish its life cycle. After a variable period of migration, which depends on humidity and temperature, the anterior end of the slug becomes stationary, while the posterior end continues to move forward, raising the anterior or stalk cells into the air (Fig. 7). Culmination begins by a reverse fountain movement or infolding of anterior cells. The stalk cells move down through the main mass of amebae to attach to the basal cells in contact with the substrate. When the stalk cells reach the apical region, they become rounded and vacuolated and begin to invaginate through the main mass of cells toward the substrate. 2 Further stalk cells are added to the group from above, pushing their predecessors downward toward the substrate. Raper and Fennell demonstrated that these cells elaborated a stalk sheath, which is a cylinder of cellulose lying outside the cell walls of the stalk cells proper. It is evident
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Fig. 7. Time-lapse sequence photographs of pseudoplasmodium at end of migration and beginning culmination. A. Anterior pole becomes fixed. B and C. Posterior end of slug continues to move forward, elevating anterior cell mass.
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that at this time the mature stalk cells are no longer viable. As this delicate stalk is elaborated, the main mass of amebae is raised away from the substrate, whose inability to provide food for growth triggered the differentiation proce~s. When the main mass of amebae reaches the top of the stalk, the prespore cells show an increasing concentration of polysaccharide unti1 a cellulose spore capsule is formed. The organism is extremely resistant in this state and remains as a fruiting body until a favorable environment once again triggers germination (Fig. 8).
Fig. 8. Terminal culmination with formation of fruiting body, and pseudoplasmodium formation (arrow) .
The entire life cycle has taken about 72 hr. The essential details have been documented with time-lapse studies, in which activity is speeded up 100 times, and are reflected in a 15-min. motion picture. The unique life cycle of this primitive metazoan has permitted a separation for study of the basic problems of cell division, cell differentiation, and cell migration. Despite prolonged and varied analysis from a morphologic, mathematical, biochemical, and physiologic point of view, the essential nature of the phenomena so clearly documented remains a frustrating but tantalizing mystery. APPENDIX I Our time-lapse studies are carried out in the following way: Stock plates of
Dictyostelium discoideum fruiting bodies are stored at 0-3° C. If desiccation is avoided, they remain viable indefinitely. Spores are harvested by touching the sorus or fruiting body with a hypodermic needle. The cells How into the lumen of the needle and are inoculated into a physiologic saline solution containing dead E. coli as nutrient. They are shaken in an incubator at 22° C. for about
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48 hr. An aliquot is removed a:q.d placed on a glass cover slip which is cemented over a hole in the top of a plastic Petri dish, and inverted over the Petri dish bottom containing water. The cells were photographed in this moist chamber at 5-sec. intervals using phase contrast microscopy (Fig. 9).
~W~"~LW~ STOCK PLATES
II TIME LAPSE CAMERA
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PHASE CONTRAST
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PHYS IOLOG I CAL SALINE + DEAD E. COLI.
Fig. 9. Preparation of material for time-lapse studies (see text).
APPENDIX II The significance of the observed rhythmic activity at the cellular level is not known, but may be of fundamental significance. We consider the possibility that biologic cycles and biologic clocks may have their escapement mechanism in an endogenous cell rhythm. In searching the literature, we find a striking similarity in studies of cell behavior carried out with time-lapse photography utilizing tissue-culture explants of brain cells. In 1937 Canti et al. reported the results of a time-lapse cinematography study of the behavior of cells in tissue culture grown from a human oligodendroglioma. They observed an expansion (diastole) and contraction (systole) cycle with a period of approximately 5 min. In 1951, Pomerat12 confirmed the presence of rhythmic, pulsatile activity of cells cultivated from explants of human brain tissue obtained at lobotomy. In 11 of 60 brains studied, pulsatile cells were observed. Cells showing rhythmic activity were found in cultures incubated from 8 to 14 days. In studies of cells derived from adult cat peripheral nervous system tissue, pulsatile behavior was also noted. On analyzing Canti's film, Pomerat calculated the pulsations to be at approximately 5-min. intervals. His analysis revealed that the diastole phase required approximately 3 min., while the systole phase took about 1.5 min. In 1951 Lumsden and Pomerat, in experiments in vitro, observed oligodendrocytes in outgrowths from explants of normal adult rat corpus callosum. These cells
VoL. 18, No.3, 1967
CELL GROWTH
showed rhythmic, pulsatile activity apparently identical with that described in 1935 by Canti et al. The authors described the explant growth as consisting of small aggregates containing 3--8 c~lls each. They noted a tendency of the cells to aggregate into'little clusters or chains, as previously described by Canti. Using the same timing method used by Canti, these workers found that the rate of pulsation of the normal rat oligodendrocyte was also about 5 min. for each contraction cycle. They estimated that the expansion phase took approximately 3 min. and the contraction phase 1.5 min. They concluded that it was probable that pulsatory activity is a general characteristic of normal adult oligodendrocytes, and that it is an intermittent process with active and resting stages. In 1955 Pomerat11 reported his observations on an oligodendroglia! tumor which had contractions occurring at intervals of 7, 6.5, 3.5, and 4.25 min. In 1959 he extended his studies 13 to time-lapse cinematography of Schwann cells and tissue culture of dorsal-root ganglia from newborn rats. These preparations showed a contraction rate of approximately 4-18 min. He studied 12 series from 4 cultures kept in vitro from 5 to 17 days and noted an average pulsation interval of about 8 min. Hild et al. reported that "time lapse cinematographic records of astrocytes revealed slow, rhythmical movement similar to those of amoebae or slime molds" ( Physarum polycephalum). In 1959 Chang et al. extended these studies to electrical stimulation of astrocytes, which Hild, in 1954, had observed to manifest spontaneous contractions similar to the contractions of oligodendrocytes reported by Canti et al. and Pomerat. They observed that following the normal contraction of the cell body in response to an electrical stimulus, cells expanded again to previous size. The duration of the contraction phase ranged from 1.4 to 3.4 min., with an average of 2.8 min., while the relaxation phase ranged from 6 to 16 min., averaging 11 min. A latent period of 1.5-5 min. was occasionally observed between the time of the stimulation and the start of the active contraction of a given cell. State University of New York 750 E. AdamY St. Syracuse, N. Y. 13210 REFERENCES 1. BoNNER, J. T. The Cellular Slime Molds. Princeton Univ. Press, Princeton, 1959. 2. BoNNER, J. T. A descriptive study of the development of the slime mold Dictyostelium discoideum. Amer I Bot 31:175, 1944. 3. BoNNER, J. T. Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium dis.coideum. I Exp Zool106: 1, 1947. 4. BoNNER, J. T., and FRASCELLA, E. B. Variations in cell size during the development of the slime mold, Dictyostelium discoideum. Biol Bull104:291, 1953. 5. CANTI, R. G., BLAND, J. 0. W., and RussELL, D. S. Tissue culture of gliomata. Demonstration. Res Publ Ass Res Nerv Ment Dis 16:1, 1935. 6. CHANG, J. J., and HILD, W. Contractile response to electrical stimulation of glia cells from the mammalian central nervous system cultivated in vitro. I Cell Comp Physiol 53:139, 1959. 7. HEFTMANN, E., WRIGHT, B., and LIDDEL, G. U. The isolation of 6. 22-Stigmasten3 {3-ol from Dictyostelium discoideum. Arch Biochem 91:266, 1960.
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8. HILD, W., CHANG, J. J., and_ TASAKI, I. Electrical responses of astrocytic glia from the mammalian central nervous system cultivated in vitro. Experientia 14:220, 1958. 9. LUMSDEN, C. E., and PoMERAT, C. M. Normal oligodendrocytes in tissue culture; a preliminary report on the pulsatile glial cells in tissue culture from the corpus callosum of the normal adult rat brain. Exp Cell Res 2:103, 1951. 10. OLivE, E. W. :Monograph of the Acrasieae. Proc Boston Soc Nat Hist 30:451, 1902. 11. PoMERAT, C. M. Dynamic neuropathology. I Neuropath Exp Neurol14:28, 1955. 12. PoMERAT, C. M. Pulsatile activity of cells from the human brain in tissue culture. I Nerv Ment Dis 114:430, 1951. 13. PoMERAT, C. M. Rhythmic contraction of schwann cells. Science 130:1759, 1959. 14. RAPER, K. B. Dictyostelium discoideum, a new species of slime molds from decaying leaves. I Agric Res 50:135, 1935. 15. RAPER, K. B., and FENNELL, D. I. Stalk formation in Dictyostelium. Bull Torrey Bot Club 79:25, 1952. 16. REBHUN, L. I. "Saltatory Particle Movements in Cells." In: Primitive Motile System in Cell Biology. Acad. Press, New York, 1964, pp. 503-525. 17. WRIGHT, B. E. Effect of steroids on aggregation in the slime mold Dictyostelium discoideum ( abst.). Presented at the meeting of the American Society of Bacteriologists in Chicago, 1958.
MEDICINE is a science of the incomprehensible. When we surgically violate the integrity of the human organism, we assume that the defects we create will be corrected by dividing, differentiating, migrating cells that somehow "know" where to go and what to do. In an effort to elucidate some of the mysteries of cell behavior, the present study was undertaken. The development of a fertilized cell into an insect, a Hower, or a man, involves three cellular processes: division, differentiation, and cell movement. The mechanisms regulating these processes, and their interaction, are not known. In order to study the regulation of cell behavior, we looked for a biologic system in which it would be possible to separate division, differentiation, and cell movement, without disrupting the system. Even in simple metazoans these processes occur simultaneously and with a degree of complexity which makes analysis difficult, if not impossible. We, therefore, turned to one of the most primitive available metazoans. The earliest living forms were either acellular or unicellular. At some critical point in evolution, dividing cells had to remain together, or separate cells had to come together, in order to form a many-celled structure. We will probably never know the precise nature of this historical event, but there exists an order of organisms in which there is a natural separation between cell division, or growth, and morphogenesis. In addition, the step between the single cell and the metazoan is part of its life cycle. The organism is Dictyostelium discoideum, a species of the order Acrasiales, or the cellular slime molds. The first clear identification of a member of the order of Acrasiales was made in 1869 by Brefeld. 1 In 1902 Olive published the earliest compreFrom the Department of Urological Surgery, State University of New York, Upstate Medical Center, and the Veterans Administration Hospital, Syracuse, N. Y. Presented at the 22nd Annual Meeting of the American Fertility Society, Chicago, Ill., Apr. 29-May 1, 1966. The author wishes to express his gratitude to the Medical Illustrations Service of the Syracuse Veterans Administration Hospital.
318
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hensive study of this presumably primitive order, while in 1935 Raper discovered the species Dictyostelium discoideum. Because of the suitability of this species for the study of a variety of biologic problems, a large body of literature has since appeared concerning the behavior of this interesting organism. Before discussing the specific phases of the life cycle of Dictyostelium discoideum (Fig. 1), it should be emphasized that there is a natural separation between the feeding or vegetative stage, during which cell division and growth occur, and the remainder of the cycle, which is morphogenetic. Feeding stops prior to aggregation, and from that moment on, the energy for the morphogenetic stages comes entirely from reserves stored up during the vegetative stage. It should be further pointed out that the Acrasiales are distinguished from the true myxomycetes, in that the former are made up of uninucleate, ameboid units which never become multinucleate, probably lack sexuality, and do not possess flagella as a means of locomotion. In nature Dictyostelium discoideum grows anywhere there is a supply of gram-negative bacteria, and is usually found in moist humus. In its final, most resistant form, the organism consists of a hairlike stalk, 2-3 mm. tall, with a small sphere or sorus, loaded with spores, at its top. Under the proper conditions of humidity, temperature, and food supply, each spore germinates, giving rise to a single ameba or myxameba (Fig. 2). And so begins the vegetative phase of the life cycle. It is during this phase of the cycle, and only this phase, that cellular division occurs. The sequence of morphogenetic changes which follow the vegetative stage, occurs whether the cells are derived from many spores or from a clone of genetically identical cells derived from a single spore. The amebae, or myxamebae, feed by phagocytosis of predominantly gram-negative bacteria, and reproduce asexually by binary fission. It should be particularly noted that during this phase of multiplication, the cells function as independent units and do not interrelate in any observable way. The vegetative ameba is indistinguishable from other free-living amebae. It has a single nucleus and contractile vacuoles, and moves by means of pseudopods. In addition to lobate pseudopods, we have observed the presence of long, delicate, filiform pseudopods, whose length occasionally exceeds the greatest diameter of the ameba. When the food supply is exhausted, or when a critical population density is reached, cell division stops and morphogenesis begins, with the onset of the preaggregation and aggregation phases. Since food ingestion is limited to the vegetative phase and accumulated anabolites are utilized thereafter, the cells become smaller.4 At the end of an interphase period of some 4-8 hr., the cells elongate (Fig. 3) and interrelate in a remarkable fashion,
Q
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SPORE Spores Stalk
9
•
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~
0
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MIGRATION
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A T I 0 N
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'
''
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Fig. 1. Life cycle of Dictyostelium discoideum.
( Acrasin)
VoL. 18, No.3, 1967
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GROWTH
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literally seeking each other out, linking u_p head to tail to form long streams of cells which move together toward a common center. Pseudopods at the anterior end of a cell coming into contact with the side of a second elongated cell appear to palpate the side of the cell surface in a retrograde
Fig. 2 (top). Vegetating myxameba and germinating spore. (X 630) Fig. 3 (bottom). Pre-aggregating myxamebae. (X 250)
direction until contact and fixation occur at the most posterior portion of the advancing cell. These linked cells now move as a unit toward the common center. This raises the interesting question as to whether the sides of an advancing ameboid cell move, or whether the anterior portion of the cell is put down with simultaneous absorption of the posterior portion of the cell, while the sides remain stationary. The observed fixation between
322
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& STERILITY
the 2 cells occurs when the palpating pseudopods reach the terminal end of the advancing ameba. Our- observation of visible artifacts adherent to the sides of advancing cells tends to support this hypothesis. While streams of cells are moving toward a central point, it can be seen that cells which are not physically connected with these streams are similarly oriented and move in the same direction. Biologists have been intrigued by the problem of how these cells know where to go. While the answer is far from complete, it is a remarkable fact that a steroid-like hormone has been demonstrated to arise from the aggregation center. This substance was given the generic name of "Acrasin," following the experiments demonstrating its existence by Bonner in 1947.3 He demonstrated that when amebae were allowed to aggregate under a layer of water flowing unidirectionally, the amebae upstream of the center showed no orientation, while downstream amebae maintained their orientation toward the center. The presence of a free diffusing agent was thus demonstrated. Substances such as sugar and malic acid, which have previously been shown to orient successfully the spermatozoa of bracken, did not produce orientation with Dictyostelium discoideum. The biochemical nature of Acrasin has been the subject of intensive investigation by a number of workers. In 1960 Heftmann et al. isolated from Dictyostelium disooideum a sterol with biologic activity. They identified this substance as ( a22-Stigmasten-3 {3-ol). Bonner1 and others postulate that Acrasin synthesis predominates at the aggregation center and by diffusion from this central point establishes a gradient along which cells migrate toward its source. It is provocative to consider that even at this primitive level of biologic organization, steroids play a regulatory role in cell behavior. Other steroids that have been tested and found to be effective in producing aggregation include: estradiol, estronesulphate, Stenediol, progesterone, testosterone, 7 and several ergosterols. 17 It is during this stage of preaggregation and aggregation that we have observed regular, periodic, pulsatory cell movement. Using time-lapse motion-picture photography in which movement was speeded up 100 times, we observed that during preaggregation and aggregation, cells and cell streams moving toward an aggregating center (Fig. 4) manifest periodic, simultaneous, synchronous movements in the same direction. In wellorganized streams of cells, an explosive separation of amebae beginning at the aggregation center was seen to propagate as a wave which moved outward from the center along the stream. Identical portions of a cell stream were photographed before, during, and after the passage of this explosive wave of synchronized cell movement (Fig. 5). The movement
t
d
l s t
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18, No. 3, 1967
CELL GROWTH
323
of cells away from each other suggests a transient polarization of the membranes tending to separate the cells. Cells not in physical contact with the streams also moved synchronously, apparently receiving some kind of information, the nature of which is completely unknown. In the earlier, less
Fig. 4. Aggregation center with stream of myxamebae. (X 35)
organized preaggregation phase, small groups of cells, alternately moving from one preaggregation center to another, were also seen to move in an organized, pulsatile, synchronous manner (Fig. 3). Analysis of 125 pulses recorded on our film showed a mean pulse interval of 5 min. (Fig. 6). Essentially, the manifestation of pulsatile behavior appears to be the result of a synchronous movement of many cells in the same direction. In several sequences, we have observed that the cells are quiescent after a pulsation, then develop a progressively increasing agitation of the anterior pseudopods which reaches a climax in a positive, forward movement. In these instances, the pulse intervals were prolonged, and are represented by the open circles in Fig. 6. We were able to follow the progression of the explosive waves along the stream, oriented parallel to the long axis of the film frame. A wave could thus be seen to originate at one end of the field and could be followed until it reached the opposite side. By knowing the size of the field and the time interval necessary for the waves to traverse the fields, we were able to determine the rate of transmission of pulsatory movement along a stream of cells. This was of the order of 2 p,jsec. This is the same order of magnitude observed for the saltatory movement of particles in protoplasm. 16
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& STERILITY
Toward the end of aggregation, streaming is completed. We have observed several instances in which the orientation gradient is apparently lost when a critical cell mass is reached at the aggregation center. At this point, a previously well-organized stream loses its orientation and individual groups of cells split off to move in random fashion for short distances. This
Fig. 5. Time-lapse sequence photographs of myxamebae stream moving toward aggregation center. A. Before pulsation. B. During pulsation. C. After pulsation. (X 250)
e-
0)
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is associated with the beginning phase of differentiation of the amorphous mass of cells that constitute the aggregation center. This cell mass gradually rises into the air, to form a cigar-shaped slug, which falls on its side forming a pseudoplasmodium. This organism, just visible to the naked eye, Fig. 6. Distribution of 125 pulse intervals of D. discoideum recorded in timelapse motion-picture study. Open circles represent intervals between pulsations characterized by progressive anterior pseudopodal agitation (see text). Transmission rates (microns per second) were 2.5, 2.6, 2.5, 2.3, 2.0, and 1.8, with average of 2.3.
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migrates in a gliding fashion for variable distances over the substrate, depositing a thin slime sheet behind the advancing pseudoplasmodiums. Raper and Fennell suggest that the slime sheet is made up of a combination of cellulose and mucin. It is at this stage that a true metazoan has been formed. Three cell species have appeared and sorted themselves out: the anterior or stalk cells, the middle or spore cells, and the posterior or basal cells. These cells differ morphologically, histochemically, and in their ultimate fate. The cells do not form a syncytium, but retain their uninucleate cellular identity. The pseudoplasmodium is capable of regeneration, and if it is divided, each fragment will regenerate to form a complete organism able to finish its life cycle. After a variable period of migration, which depends on humidity and temperature, the anterior end of the slug becomes stationary, while the posterior end continues to move forward, raising the anterior or stalk cells into the air (Fig. 7). Culmination begins by a reverse fountain movement or infolding of anterior cells. The stalk cells move down through the main mass of amebae to attach to the basal cells in contact with the substrate. When the stalk cells reach the apical region, they become rounded and vacuolated and begin to invaginate through the main mass of cells toward the substrate. 2 Further stalk cells are added to the group from above, pushing their predecessors downward toward the substrate. Raper and Fennell demonstrated that these cells elaborated a stalk sheath, which is a cylinder of cellulose lying outside the cell walls of the stalk cells proper. It is evident
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Fig. 7. Time-lapse sequence photographs of pseudoplasmodium at end of migration and beginning culmination. A. Anterior pole becomes fixed. B and C. Posterior end of slug continues to move forward, elevating anterior cell mass.
n d
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that at this time the mature stalk cells are no longer viable. As this delicate stalk is elaborated, the main mass of amebae is raised away from the substrate, whose inability to provide food for growth triggered the differentiation proce~s. When the main mass of amebae reaches the top of the stalk, the prespore cells show an increasing concentration of polysaccharide unti1 a cellulose spore capsule is formed. The organism is extremely resistant in this state and remains as a fruiting body until a favorable environment once again triggers germination (Fig. 8).
Fig. 8. Terminal culmination with formation of fruiting body, and pseudoplasmodium formation (arrow) .
The entire life cycle has taken about 72 hr. The essential details have been documented with time-lapse studies, in which activity is speeded up 100 times, and are reflected in a 15-min. motion picture. The unique life cycle of this primitive metazoan has permitted a separation for study of the basic problems of cell division, cell differentiation, and cell migration. Despite prolonged and varied analysis from a morphologic, mathematical, biochemical, and physiologic point of view, the essential nature of the phenomena so clearly documented remains a frustrating but tantalizing mystery. APPENDIX I Our time-lapse studies are carried out in the following way: Stock plates of
Dictyostelium discoideum fruiting bodies are stored at 0-3° C. If desiccation is avoided, they remain viable indefinitely. Spores are harvested by touching the sorus or fruiting body with a hypodermic needle. The cells How into the lumen of the needle and are inoculated into a physiologic saline solution containing dead E. coli as nutrient. They are shaken in an incubator at 22° C. for about
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48 hr. An aliquot is removed a:q.d placed on a glass cover slip which is cemented over a hole in the top of a plastic Petri dish, and inverted over the Petri dish bottom containing water. The cells were photographed in this moist chamber at 5-sec. intervals using phase contrast microscopy (Fig. 9).
~W~"~LW~ STOCK PLATES
II TIME LAPSE CAMERA
~
r~ .
i~
PHASE CONTRAST
\d
PHYS IOLOG I CAL SALINE + DEAD E. COLI.
Fig. 9. Preparation of material for time-lapse studies (see text).
APPENDIX II The significance of the observed rhythmic activity at the cellular level is not known, but may be of fundamental significance. We consider the possibility that biologic cycles and biologic clocks may have their escapement mechanism in an endogenous cell rhythm. In searching the literature, we find a striking similarity in studies of cell behavior carried out with time-lapse photography utilizing tissue-culture explants of brain cells. In 1937 Canti et al. reported the results of a time-lapse cinematography study of the behavior of cells in tissue culture grown from a human oligodendroglioma. They observed an expansion (diastole) and contraction (systole) cycle with a period of approximately 5 min. In 1951, Pomerat12 confirmed the presence of rhythmic, pulsatile activity of cells cultivated from explants of human brain tissue obtained at lobotomy. In 11 of 60 brains studied, pulsatile cells were observed. Cells showing rhythmic activity were found in cultures incubated from 8 to 14 days. In studies of cells derived from adult cat peripheral nervous system tissue, pulsatile behavior was also noted. On analyzing Canti's film, Pomerat calculated the pulsations to be at approximately 5-min. intervals. His analysis revealed that the diastole phase required approximately 3 min., while the systole phase took about 1.5 min. In 1951 Lumsden and Pomerat, in experiments in vitro, observed oligodendrocytes in outgrowths from explants of normal adult rat corpus callosum. These cells
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CELL GROWTH
showed rhythmic, pulsatile activity apparently identical with that described in 1935 by Canti et al. The authors described the explant growth as consisting of small aggregates containing 3--8 c~lls each. They noted a tendency of the cells to aggregate into'little clusters or chains, as previously described by Canti. Using the same timing method used by Canti, these workers found that the rate of pulsation of the normal rat oligodendrocyte was also about 5 min. for each contraction cycle. They estimated that the expansion phase took approximately 3 min. and the contraction phase 1.5 min. They concluded that it was probable that pulsatory activity is a general characteristic of normal adult oligodendrocytes, and that it is an intermittent process with active and resting stages. In 1955 Pomerat11 reported his observations on an oligodendroglia! tumor which had contractions occurring at intervals of 7, 6.5, 3.5, and 4.25 min. In 1959 he extended his studies 13 to time-lapse cinematography of Schwann cells and tissue culture of dorsal-root ganglia from newborn rats. These preparations showed a contraction rate of approximately 4-18 min. He studied 12 series from 4 cultures kept in vitro from 5 to 17 days and noted an average pulsation interval of about 8 min. Hild et al. reported that "time lapse cinematographic records of astrocytes revealed slow, rhythmical movement similar to those of amoebae or slime molds" ( Physarum polycephalum). In 1959 Chang et al. extended these studies to electrical stimulation of astrocytes, which Hild, in 1954, had observed to manifest spontaneous contractions similar to the contractions of oligodendrocytes reported by Canti et al. and Pomerat. They observed that following the normal contraction of the cell body in response to an electrical stimulus, cells expanded again to previous size. The duration of the contraction phase ranged from 1.4 to 3.4 min., with an average of 2.8 min., while the relaxation phase ranged from 6 to 16 min., averaging 11 min. A latent period of 1.5-5 min. was occasionally observed between the time of the stimulation and the start of the active contraction of a given cell. State University of New York 750 E. AdamY St. Syracuse, N. Y. 13210 REFERENCES 1. BoNNER, J. T. The Cellular Slime Molds. Princeton Univ. Press, Princeton, 1959. 2. BoNNER, J. T. A descriptive study of the development of the slime mold Dictyostelium discoideum. Amer I Bot 31:175, 1944. 3. BoNNER, J. T. Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium dis.coideum. I Exp Zool106: 1, 1947. 4. BoNNER, J. T., and FRASCELLA, E. B. Variations in cell size during the development of the slime mold, Dictyostelium discoideum. Biol Bull104:291, 1953. 5. CANTI, R. G., BLAND, J. 0. W., and RussELL, D. S. Tissue culture of gliomata. Demonstration. Res Publ Ass Res Nerv Ment Dis 16:1, 1935. 6. CHANG, J. J., and HILD, W. Contractile response to electrical stimulation of glia cells from the mammalian central nervous system cultivated in vitro. I Cell Comp Physiol 53:139, 1959. 7. HEFTMANN, E., WRIGHT, B., and LIDDEL, G. U. The isolation of 6. 22-Stigmasten3 {3-ol from Dictyostelium discoideum. Arch Biochem 91:266, 1960.
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8. HILD, W., CHANG, J. J., and_ TASAKI, I. Electrical responses of astrocytic glia from the mammalian central nervous system cultivated in vitro. Experientia 14:220, 1958. 9. LUMSDEN, C. E., and PoMERAT, C. M. Normal oligodendrocytes in tissue culture; a preliminary report on the pulsatile glial cells in tissue culture from the corpus callosum of the normal adult rat brain. Exp Cell Res 2:103, 1951. 10. OLivE, E. W. :Monograph of the Acrasieae. Proc Boston Soc Nat Hist 30:451, 1902. 11. PoMERAT, C. M. Dynamic neuropathology. I Neuropath Exp Neurol14:28, 1955. 12. PoMERAT, C. M. Pulsatile activity of cells from the human brain in tissue culture. I Nerv Ment Dis 114:430, 1951. 13. PoMERAT, C. M. Rhythmic contraction of schwann cells. Science 130:1759, 1959. 14. RAPER, K. B. Dictyostelium discoideum, a new species of slime molds from decaying leaves. I Agric Res 50:135, 1935. 15. RAPER, K. B., and FENNELL, D. I. Stalk formation in Dictyostelium. Bull Torrey Bot Club 79:25, 1952. 16. REBHUN, L. I. "Saltatory Particle Movements in Cells." In: Primitive Motile System in Cell Biology. Acad. Press, New York, 1964, pp. 503-525. 17. WRIGHT, B. E. Effect of steroids on aggregation in the slime mold Dictyostelium discoideum ( abst.). Presented at the meeting of the American Society of Bacteriologists in Chicago, 1958.