CELL DIFFERENTIATION AND FINE STRUCTURES IN THE DEVELOPMENT OF THE CELLULAR SLIME MOLDS' ABSTRACT

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1 Development, Growth and Differentiation, Vol.1 f, No.3 (Dec., 1969) CELL DIFFERENTIATION AND FINE STRUCTURES IN THE DEVELOPMENT OF THE CELLULAR SLIME MOLDS' YASUO MAEDA and IKUO TAKEUCHI Department of Botany, Faculty of Science, Kyoto University, Kyoto, Japan ABSTRACT Changes in fine structures during the development of the cellular slime molds D. discoideum and D. mucovoides were studied, with emphasis on the regional differentiation between the prestalk and prespore cells of the slug. Cells in the prestalk region were in closer contact than those in the prespore region. Some differences were also noticed in the structure of plasma membrane between the two types of cells. An endoplasmic reticulum, vesicle, autophagic vacuole, and cytoplasmic fibril were found more abundantly in the prestalk cell than in the prespore cell. In the prespore cells there were observed a number of prespore specific vacuoles of ca. 0.61~ diameter which consist of membraneous and fibrous structures. The vacuole was never found in the prestalk cells, and was a sole structure that existed only in one of the two types of cells. A possible function of such a vacuole was discussed in relation to spore differentiation. No differences in structure and distribution of mitochondria and crystal bodies were noticed between the prestalk and prespore cells, although these structures underwent considerable changes during the development. The nucleolus underwent considerable structual differentiation between the prestalk and prespore cells as well as during the course of development. INTRODUCTION In the vegetative growth phase of the cellular slime molds an amoeba grows and multiplies independently, feeding on a suitable species of bacteria. Some time after amoebae finish feeding, they stream together to a collecting point (aggregation). The cell aggregate thus formed initially erects itself, but later lies down on the substratum in the shape of a slug. The slug migrates for a period of time (migration), until it rises into the air to form a fruiting body which consists of a mass of spores and a supporting cellular stalk (culmination). An essential part of the work was reported at the annual meeting of the Experimental Morphology and Embryology Association (MAEDA and TAKEUCHI, 1967). 232

2 FINE STRUCTURES IN SLIME MOLD DEVELOPMENT 233 During this process, the anterior part of the slug differentiates into stalk cells, while the posterior part differentiates into spores. It has been demonstrated by a variety of histochemical and immunohistochemical studies that the anterior prestalk cells of the slug have different staining properties from the posterior prespore cells. Several electronmicroscopic studies of the cellular slime molds have been conducted with special emphasis on cellulose fibers present in the wall of stalk and spore cells (M~HLETHALER, 1956; GEZELIUS and RANBY, 1957), on cell to cell contact in cell aggregates (MERCER and SHAFFER, 1960), and on some of the fine structures found in the cells at the various stages of development (GEZELIUS, 1959, 196 I). During the preparation of this manuscript Hohl et al. published two papers which dealt with the culmination of several species of this organism, and they observed the behavior of a unique structure named prespore vacuole (PV) during the process of spore differentiation (HOHL et al., 1968; HOHL and HAMAMOTO, 1969). In spite of these investigations, few reports have been available concerning the ultrastructural changes specifically related to cell differentiation in the slug. It is the purpose of the present work to investigate intra- and inter-cellular changes in fine structures during the development of this organism, with special emphasis on the regional differentiation between the anterior prestalk and the posterior prespore cells of the slug. MATERIALS AND METHODS Dictyostelium discoideum, strain NC-4 was the organism mainly used in the present study, and D. mucoroides, No. 11 was used in comparison. The latter differs from the former in that stalk formation starts at the end of aggregation and continues while the slug migrates. In consequence, differences in staining between the prestalk and prespore cells appear earlier in the latter than in the former (BONNER et al., 1955; TAKEUCHI, 1960). Amoebae were grown with Escherichiu coli on a standard nutrient agar medium (BONNER, 1947). They were collected at the stationary growth phase, and freed from bacteria by repeated (four times) centrifugation with chilled standard salt solution. In order to obtain electronmicroscopic specimens of vegetative and interphase (between the vegetative and aggregation stages) amoebae, suspension of washed amoebae was placed on Epoxy resin in the presence and the absence of bacteria respectively. After a certain period of incubation, the amoebae were fixed and dehydrated on the resin. In order to obtain cell masses at the various stages of development, washed amoebae were incubated on non-nutrient agar and the cell masses were selected at their proper stages. The slug was taken after it had migrated about 1.5 cm. Cells and tissues were fixed at room temperature in 1% solution of osmium tetraoxide dissolved in standard salt solution. The former was fixed for 5 minutes and the latter for

3 234 Y. MAEDA AND I. TAKEUCHI 20 minutes. After dehydration in a series of ethanol, specimens were embedded in Epoxy resin. Ultrathin sections ( A thick) were cut with a LKB ultrotome. Sections were double-stained with uranyl acetate and lead citrate and were observed with a Japanese Electronics JEM-7 or a Hitachi HU-11D. RESULTS I. Cell differentiation and fine structures in D. discoideum A. Cell contuct Cells in the aggregation center of D. discoideum were found to be in close contact with an intercellular space of 200 A wide, as is the case of animal tissue cells. This confirms the observation previously made by MERCER and SHAFFER (1960). It was further shown in the present study that considerable differences in cell contact were observed between the prestalk and prespore regions of the slug. In the former, cells were in contact similar to or closer than those in the aggregate (Fig. 1). In contrast, cells in the prespore region were more loosely packed, leaving large intercellular spaces scattered around the regular spaces of 200 A (Fig.2). This indicates that cell adhesion in the prespore region is much weaker than that in the aggregation center as well as in the prestalk region. Some differences were also noticed in the structure of the plasma membrane between the prestalk and prespore cells. In the former, the plasma membranc of ca. 100 A thick consisted of an outer and inner layers of 30 A each which sandwiched a middle layer of 40 A (Fig.3A). In the latter case, a similar structure was also observed in some parts of the cells, but in other parts the membrane was extended to a width of 200 A owing to the increase in thickness of thc outer and middle layers (Fig.3B). In such region, the electron density of the outer layer was also increased. B. fn tr~c~~lul~ir structures Substantial differences in intracellular structures were appreciated between the prestalk and prespore cells. Some structures were observed in either type of cells, but differed in the extent of development. Other structures, however, were specific for one type of cells and entirely absent from another type. Only Fig.l Prestalk cells from a slug of D. discoideum. The cells are tightly packed. Many vesicles (V) are observed around the nucleus (N). A considerable number of autophagic vacuoles (AV) and rough surfaced endoplasmic reticulum (ER) are scattered in the cytoplasm. Arrows indicate the bundles of cytoplasmic fibrils which run in the direction of pseudopods. Cb; crystal body. M; mitochondria. x 11,100.

4 FINE STRUCTURES IN SLIME MOLD DEVELOPMENT 23 5 Fig. 1

5 236 Y. MAEDA AND 1. TAKEUCHI Fig. 2

6 FINE STKUCTURES IN SLIME MOLD DEVELOPMENT 23 7 one of the latter kind of structures was identified thus far, that is a prespore specific vacuole described presently. Besides such regional differences, structural changes during the development will be described. 1. Vacuolar System (11 Endoplusmic reticulum A rough surfaced endoplasmic reticulum (ER) was not well developed in the vegetative and interphase amoebae. In contrast, a considerable development of ER was remarked in the prestalk cells of the slug, where the cavity of a part of ER was fairly extended and was occasionally found to contain fibrils of 50 A diameter (Fig.4). In the prespore cells, however, only a few ER was observed, as was the case of the vegetative Fig.3 A portion of the plasma membranes in prestalk cells (A) and prespore cells (B) of D. discoideum. In B, the membrane is thickened in the electron dense outer (0) and transparent middle (m) layers. x 80,000. Fig.2 Prespore cells from a slug of D. discoideum. The cells have sporadic large intercellular spaces, in contrast to the prestalk cells as shown in Fig.1. In the cytoplasm, a number of prespore-specific vacuoles (PSV) are observed, whereas autophagic vacuoles, vesicles, and rough surfaced endoplasmic reticula are not well developed. X 13,300.

7 238 Y. MAEDA AND 1. TAKEUCHI Fig.4 A rough surfaced endoplasmic reticulum (ER) well developed in the prestalk cell of D. discoideum. The cavity of ER is fairly extended and contains fibrous structure. X 32,000. and interphase amoebae. 6) Vesicle Only a small number of vesicles were found in the vegetative and interphase amoebae. The prestalk cells of the slug, however, contained numerous vesicles, most of which were clustered around the nuclei (Fig.5 and Fig. 1). Some electronmicrographs indicated that some of these vesicles fuse each other to form a large vacuole. On the other hand, the prespore cells had much less number of vesicles, giving an appearance similar to the vegetative amoebae. c) Autophugic vacuole The vegetative and interphase amoebae contained a large number of food and contractile vacuoles. In contrast, a different kind of large vacuoles of 0.6 to 1.2,~ diameter developed in the prestalk cells of the slug (Fig.5). The vacuole enclosed fragments of the cytoplasm including various kinds of organelles such as mitochondrion which appeared to be in the process of digestion. Some electronmicrographs also demonstrated the process in which the cytoplasm was engulfed by the vacuole. From these observations, it was inferred that autophagocytosis of the cytoplasm is in progress in the prestalk cells. Since cells at the morphogenetic stages are devoid of a food

8 FINE STRUCTURES IN SLIME MOLD DEVELOPMENT 239 Fig5 The vesicles (V) and autophagic vacuoles (AV) in a prestalk cell of D. discoideum. The autophagic vacuole encloses a fragment of the cytoplasm (FC) which appears in the process of digestion. X 26,700. source, the energy and material needed for morphogenesis must be supplied by digestion of their own cytoplasm. The autophagic vacuoles in the prestalk cells increased in size and number, as they differentiated into stalk cells. As a result, an extremely large vacuole occupied the bulk of a mature stalk cell. On the contrary, the vacuoles were not well developed in the prespore cells of the slug, indicating that autophagocytosis is somehow prevented in those cells. This is probably related to the fact that the prespore cells must preserve their own material for the next generation since they differentiate into spores. d) Prespore-specific vacuole (PSV) In the prespore cells of the slug there were observed a considerable number of specific vacuoles of 0.5 to 0.7~ diameter, which will be referred to as PSV hereafter (Fig.2). Under a high magnification, the unit membrane of the vacuole was lined with an electrondense membraneous structure of ca. 200 A thick which enclosed an aggregate of filaments of 50 A diameter (Fig.6). The vacuole was characteristic of the prespore cell and was never found in the prestalk cell. This is a sole structure thus far found that shows discrete localization between the two types of cells.

9 240 Y. MAEDA AND 1. TAKEUCHI Fig6 The prespore-specific vacuoles (PSV) in a prespore cell of D. discoideum. The unit membrane of the vacuole is lined with membraneous structure (arrows) which encloses an aggregate of filaments (f). X In later development, the PSV was found in the prespore cells of the culminating cell mass, but was never found in mature spores. 2. Cytoplasmic fibril After the formation of a slug, bundles of fibrils of ca. 50 A diameter developed in the cytoplasm. Its development was conspicuous in the prestalk cells, where bundles of ca. 0. ILL diameter run either parallel to the cell surface or in the direction of pseudopods (Fig.1). In contrast, such fibrils were infrequently observed in the prespore cells. 3. Crystal body A considerable number of crystalline structures with ca. 50 A interlattice space were observed in the cells which had ceased to feed (Fig.1). A similar crystal was described by GEZELIUS (1959). The crystal was retained during the entire period of morphogenesis and was present in mature spores. It disappeared during the germination of spores and was completely absent from the vegetative amoebae. A part of ER was frequently observed to enclose a small crystal, suggesting

10 FINE STRUCTURES IN SLIME MOLD DEVELOPMENT 24 1 that the crystal is synthesized inside EK. The crystal was a sole structure that clearly discriminated the interphase amoebae from the vegetative amoebae. However, no differential distribution was found between the prestalk and prespore cells of the slug. 4. Mitochondria The mitochondria in the vegetative amoebae were generally round in shape and held tubular cristae. In contrast, cells in and after the interphase contained longer mitochondria, whose cristae were reticular. In such cells a part of the mitochondrion was frequently observed to be vacuolized and enclose electrondense material. No difference in the number and structure of mitochondria was noticed between the prestalk and prespore cells of the slug. During the process of spore differentiation, however, a mitochondrion became a rosette-like body with obscure internal structure, and many ribosomes were attached to its outer membrane. 5. Nucleus No structural changes in nuclear membrane and nucleoplasm were ap- Nucleolus prestalk cell stalk cell prespore cell spore cell Vegetative )amoeba I nterphase Aggregate slug (Precuhination ) Fruiting body Fig.7 A diagram showing ultrastructural changes in the nucleolus during the development of D. disocideum.

11 242 Y. MAEDA AND I. TAKEUCHI preciated during the process of cell differentiation. On the contrary, the nucleolus underwent considerable structural changes during the development, as diagrammed in Fig.7. The nucleolus in the vegetative and interphase amoebae consisted of fibrous matrix and uniformly distributed, ribosome-like granules of ca. 200 A diameter. During the aggregation stage, the nucleolus temporarily lost some granules. In the slug, the fibrous component of the nucleolus was separated from the granular component (Fig. 8). Furthermore, it showed a considerable structural differentiation between the prestalk and prespore cells. In the former, it contained large (500 A diameter) and highly electron-dense granules (Fig.8A). On the other hand, the size and number of granules were considerably smaller in the prespore cells (Fig.8B). When the prestalk cells differentiated to stalk cells, the nucleolus gradually shrinked and lost granules of high electron density. During the process of spore differentiation, however, the granules Fig.8 The nucleoli in a prestalk cell (A) and a prespore cell (B) of D. discoideum. The fibrous component (f) is separated from the granular component (9). Large and highly electron-dense granules (arrow) are present in the nucleolus of the prestalk cell, while the size and electron density of the granules are considerably smaller in the prespore cell. X 58,700.

12 FINE STRUCTURES IN SLlME MOLD DEVELOPMENT 243 increased their size, number, and electron density. Soon afterwards, the granular and fibrous components were mixed, and the granules were uniformly distributed. 11. Cell differentiation and fine structures in D. mucoroides. The information described above with D. discoideurn was essentially the same as that with D. mucoroides. Structural differences of cell contact and autophagic vacuoles between the prestalk and prespore cells were even accentuated in D. mucoroides. The development of PSV in the prespore cells was also more prominent in this species than in D. discoideum. DISCUSSION Substantial differences in various fine structures were demonstrated between the prestalk and prespore cells of the slug. Among those, the PSV was a sole structure that existed only in one of the two types of cells. According to the immunohistochemical studies with D. mucoroides, the antispore serum absorbed with interphase amoebae specifically stains cytoplasmic granules in the prespore cells (TAKEUCHI, 1963). These granules seem to be identical with the PSV, considering their size and distribution. On the other hand, the absorbed antiserum stains only the surface of mature spores (TAKEUCHI, 1963). This is in good agreement with the present finding that spores which had completed coat formation were devoid of any PSV. These facts suggest that during culmination the PSV is released from the cells and its content constitutes the spore coat. In fact, another electronmicroscopic study on spore differentiation has revealed that the unit membrane of a PSV fuses with the plasma membrane, and that the interior membraneous structure was excreted and covered the ou tside of the plasma membrane (MAEDA, ADACHI and TAKEUCHI, unpublished). The ibove idea is also supported by the fact that the outer membrane of the spore coat had an appearance similar to the lining membrane of the PSV. At any rate, elucidation of the regulatory mechanism of the appearance and disappearance of the PSV seems important to understand differentiation of the prespore and spore cells. Investigation along this line is under way. A structure similar to the PSV has been independently reported and named PV by HOHL and HAMAMOTO (1969) in their recent paper. They also observed the behavior of the structure during spore differentiation and obtained essentially the same results as mentioned above. Differentiation between the prestalk and prespore cells was also manifested by a difference in cell contact; the former were tightly packed as animal tissue cells, while the latter had larger intercellular spaces. In fact, when the slug was

13 244 Y. MAEDA AND I. TAKEUCHI disaggregated either in a 0.15 M solution of NaCl containing versene or in a solution of proteolytic enzymes containing dimercaptopropanol, the anterior part of the slug showed stronger resistance to disaggregation than the posterior part (TAKEUCHI and YAHUNO, unpublished). In the two mutant strains of D. discoideum which are incapable of constructing the fruiting body, all the cells in the slug were found to show large intercellular spaces like the prespore cells of a normal slug. This suggests that normal differentiation of the prestalk cells was somehow inhibited in those strains. The function of cytoplasmic fibrils which were predominantly observed in the prestalk cells is little known at present. Generally, cytoplasmic fibrils of ca. 50 A were found in the cells which were active in cytoplasmic streaming (KOMNICK and WOHLFARTH-BOTTERMANN, 1965; NACAI and REBHUN, 1966), and were considered to be composed of contractile proteins (HATANO et al., 1967). The cytoplasmic fibrils in slime mold amoebae may also be of the same nature and be related to amoeboid movement. This idea was supported by the fact that bundles of the fibrils mostly run in the direction of pseudopods. On the other hand, those cytoplasmic fibrils are similar in appearance to the fibrils which during culmination were secreted out of prestalk cells to form the stalk sheath or the wall of stalk cells (MAEDA, 1967). Accordingly, it is also likely that the cytoplasmic fibrils constitute the stalk sheath. Which possibility is right will be answered by a chemical analysis of the fibrils. At any rate, throughout the development a considerable correlation was found between the development of cytoplasmic fibrils and that of ER. Furthermore, the fibrous structures contained in the cavity of ER were similar in appearance to the cytoplasmic fibrils. These suggest that the cytoplasmic fibrils are synthesized or stored in the ER. During the development of this organism, the nucleolus underwent regional as well as temporal changes to a considerable extent. The fact that the nucleolus plays an important role in the differentiation of stalk and spore cells was demonstrated by the development of an anucleolus mutant of D. discoideum isolated by S. ISHIDA. The mutant carries out completely normal develop ment (vegetative growth, cell aggregation, slug formation, and its migration) up to the beginning of culmination, where the development stops and no differentiation of stalk or spore cells occurs. The mutant cells were found to hold normal nucleoli at thc vegetative stage, but the nucleoli gradually disappeared after the formation of a slug. Even after their disappearance, the slug still continued its normal morphogenetic movement, but no stalk or spore cells were differentiated. These indicate the importance of the nucleolus for the final cell

14 FINE STRUCTURES IN SLIME MOLD DEVELOPMENT 245 differentiation in this organism. This work was in part supported by a Research Grant from the Ministry of Education. REFERENCES BONNER, J. T., Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium discoideum. J. Exp. Zool., 106, BoNNER, J. T., A. D. CHIQUOINE, and M. Q. KOLDERIE, A histochemical study of differentiation in the cellular slime molds. J. Exp. Zool., 130, GEZELIUS, K., The ultrastructure of cells and cellulose membranes in Acrasiae. Exp. Cell Res., 18, GEZELIUS, K., Further studies in the ultrastructure of Acrasiae. Exp. Cell Res., 23, GEZELIUS, K. and B. G. MNBY, Morphology and fine structure of the slime mold Dictyostelium discoideum. Exp. Cell Res., 12, RATANO, S., T. TOTSUKA, and F. OOSAWA, Polymerization of plasmodium actin. Biochem. Biophys. Acta, 140, HOHL, H. R., S. T. HAMAMOTO, and D. E. HEMMES, Ultrastructual aspects of cell elongation, cellulose synthesis, and spore differentiation in Acytostelium leptoso mum, a cellular slime mold. Amer. J. Bot., 55, HOHL, H. R. and S. T. HAMAMOTO, Ultrastructure of spore differentiation in Dictyostelium : the prespore vacuole. J. Ultrastruct. Res., 26, KOMNICK, H. and K. E. WOHLFARTH-BOTTERMANN, Das Grundplasma und die Plasmafilamente der Amiibe, Chaos chaos nach enzymatisher Behandlung der ZelImembran. Z. Zellforsch., 66, MAEDA, Y., Electronmicroscopic studies on the morphogenesis of the cellular slime molds. M. S. thesis, Osaka Univ. MAEDA, Y. and I. TAKEUCHI, Electronmicroscopic studies on the development of the cellular slime molds. Jap. J. Exp. Morph., 21, 509. MERCER, E. H. and B. M. SHAFFER, Electron microscopy of solitary and aggrs gated slime mould cells. J. Biophys. Biochem. Cytol., 7, MUHLETHALER, K., Electron microscopic study of the slime mold Dictyostelium discoideum. Amer. J. Bot., 43, NAGAI, R. and L. 1. REBHUN, Cytoplasmic microfilaments in streaming Nitella cells. J. Ultrastruct. Res., 14, TAKEUCHI, I., The correlation of cellular changes with succinic dehydrogenase and cytochrome oxidase activities in the development of the cellular slime molds. Develop. Biol., 2, TAKEUCHI, I., Immunochemical and immunohistochemical studies on the develop ment of the cellular slime mold Dictyostelium mucoroides. Develop. Biol., 8, (Manuscript received: Oct. 23, 1969)