Cell differentiation in a temperature-sensitive stalkless mutant of Dictyostelium discoideum

Transcription

1 /. Embryol. exp. Morph. 74, (1983) 235 Printed in Great Britain The Company of Biologists Limited 1983 Cell differentiation in a temperature-sensitive stalkless mutant of Dictyostelium discoideum By AIKO AMAGAI 1, SHUJI ISHIDA AND IKUO TAKEUCHI From the Department of Botany, Faculty of Science, Kyoto University, Japan SUMMARY A temperature-sensitive aggregateless and stalkless mutant was isolated from Dictyostelium discoideum NC-4. The mutant cells cannot aggregate at 27 C, but aggregate and form normal fruiting bodies at 21 C. When the temperature was shifted to 27 C after aggregation at 21 C, almost all of the cells in the aggregate differentiated into spores. Neither stalk cells nor stalk tubes formed at 27 C. Inhibition of stalk formation was not lifted by addition of cyclic AMP. Nevertheless, the proportion of prespore to total cells within the mutant slugs was normal, at both 21 C and 27 C. At 27 C, a slug was transformed into a spherical cell mass at the end of migration, within which pre-existing prespore cells differentiated into spores. The remaining prestalk cells were then converted to prespore cells which later became spores. As the celltype conversion continued, formation of a spore mass resulted. The development of the mutant is thus consistent with the idea that the presumptive cell differentiation is directly related to the terminal cell differentiation. During migration at 27 C, the number of prestalk cells decreased in the anterior part of the slug but instead increased at the foot or the rear part, whereas the prestalk-prespore pattern remained normal at 21 C. The fact that a normal proportion of prespore cells was maintained in spite of their deranged distribution at 27 C indicates that the regulation of proportion is independent of the formation of pattern. INTRODUCTION After the vegetative stage, the cells of the cellular slime mould, Dictyostelium discoideum aggregate and form a cell mass which assumes the shape of a slug. After a series of morphogenetic movements, each slug eventually forms a fruiting body consisting of stalk cells and spores. During this process, the anterior part of the slug becomes stalk cells, while the posterior part becomes spores (Raper, 1940). Prespore cells in the slug are distinguished from prestalk cells by holding prespore-specific vacuoles (Hohl & Hamamoto, 1969; Maeda & Takeuchi, 1969), which contain prespore antigen stainable with fluoresceinconjugated antispore serum (Takeuchi, 1963). The prespore-prestalk pattern observed in the slug is generally regarded as representing an initial step of differentiation toward the final spore-stalk pattern in the fruiting body (for review, see Mac Williams & Bonner, 1979). Recently, however, the idea was 1 Author's address: Department of Botany, Faculty of Science, Kyoto University, Kyoto 606, Japan.

2 236 A. AMAGAI, S. ISHIDA AND I. TAKEUCHI questioned by Morrissey, Farnsworth & Loomis (1981), on the grounds that stalky and stalkless mutants which form only stalks and spores respectively in the fruiting bodies are nevertheless normal in the prespore-prestalk pattern at the slug stage. In our attempt to isolate temperature-sensitive aggregateless mutants for the studies of aggregation, we have found one which cannot aggregate at 27 C but aggregate and form fruiting bodies at 21 C. By shifting the cell masses to 27 C after aggregation at 21 C, differentiation of the mutant could occur but only in an abnormal fashion, i.e. stalk differentiation did not occur and almost all of the cells in the aggregate differentiated into a mass of spores. By closely examining differentiation pattern of prespore cells in this mutant, we found that the spore mass was derived from prespore cells which not only pre-existed in the slug but also were converted from the remaining prestalk cells through proportion regulation. MATERIALS AND METHODS 1. Cultures Dictyostelium discoideum NC-4 and a mutant KYH-13 isolated from it were used. Wild-type cells were cultured with Escherichia coli on nutrient agar (Bonner, 1947), at 21 C. For the cultures of the mutant, mutant spores and E. coli were put together in a 300 ml Erlenmyer flask which contained 100 ml of a lactose-peptone medium (5g lactose, 5g peptone, 1000ml distilled water). The flask was shaken, at 21 C, on a rotary shaker (120r.p.m./min). Growthphase cells were collected and washed by centrifugation, twice with Bonner's salt solution (Bonner, 1947) and once with 20mM-phosphate buffer (ph6-l). Washed cells were resuspended in the phosphate buffer and spread on nonnutrient or salt (lower pad solution (LPS): Ellingson, Telser & Sussman, 1971) agar (2 %) at a density of 5 x 10 5 cells/cm 2. After the cells were settled, excess water was removed. The plates were incubated at 21 C or 27 C in the dark. 2. Immunocytochemical and vital staining Prespore cells were identified by staining with fluorescein-isothiocyanate (FITC)-conjugated serum globulin produced against spores of Dictyostelium mucoroides. The preparation of the serum and the staining with it were conducted according to Takeuchi (1963). The proportions of prespore to total cells within cell masses were determined by the method of Hayashi & Takeuchi (1976). To know the distribution of prespore cells, slugs were fixed in cold methanol, embedded in paraffin and sectioned. Sections and cells stained with the conjugated globulin were observed under a Nikon fluorescence microscope (OPTIPHOT). Vital staining of cells with neutral red was conducted by the method of Yamamoto & Takeuchi (1983).

3 A temperature-sensitive stalkless mutant ofd. discoideum 237 RESULTS 1. Isolation of the mutant The isolation of mutants was conducted by the method of Fukui & Takeuchi (1971) using nitrosoguanidine as a mutagen. Temperature-sensitive mutants which could not aggregate at 27 C, but aggregate at 21 C were selected. Among them, one was found to undergo abnormal differentiation at 27 C, after it aggregated at 21 C. This mutant, KYH-13, was used for the work described in this paper. 2. Development at 21 C When plated on non-nutrient agar, KYH-13 aggregated and usually formed normal fruiting bodies, at 21 C. Occasionally, during slug migration, some cells were left in the trail and became stalk cells. The remaining cells differentiated into a mass of spores. When the mutant cells were allowed to develop on high salt (LPS) agar, they always formed normal fruiting bodies without a migration phase. 3. Development at 27 C The mutant did not aggregate at 27 C, but when the temperature was shifted to 27 C after aggregation at 21 C, the cells formed slugs. After some hours of migration, a slug stopped and formed a spherical cell mass with a tip-like protrusion on the original anterior side. Most cells within the cell mass differentiated into spores (Fig. 1). The cells which remained undifferentiated at this stage later became spores as well, thus completing formation of a spore mass (Fig. 2). In some cases, however, the tip portion remained undifferentiated, or was separated from the spore mass and undifferentiated. In any case, the mutant formed neither stalk cells nor stalk tubes at 27 C, indicating that it is temperature sensitive for stalk formation as well as aggregation. The mutant took much longer to form the spore mass than the fruiting body. During migration, some slugs divided longitudinally or transversely and each portion produced a spore mass. Dividing also occurred at 21 C, but more frequently at 27 C. Occasionally, during migration, many cells were left behind inside the slime trail. Although these cells became stalk cells at 21 C, they remained undifferentiated at 27 C. The effects of the timing of the temperature shift on the development were examined. After starvation, cells were allowed to develop at 21 C and shifted to 27 C every 2h. Normally, cells kept at 21 C became aggregation competent (judged by their elongate form) after 8 h and began to aggregate after 10 h. Cells shifted to 27 C before 8h did not aggregate, while those shifted after 8h aggregated and produced spore masses. The results suggest that acquisition of aggregation competence is temperature sensitive in this mutant. EMB74

4 238 A. AMAGAI, S. ISHIDA AND I. TAKEUCHI Fig. 1. Culmination of a KYH-13 slug at 27 C. Cells stained with neutral red were allowed to aggregate at 21 C and then shifted to 27 C. A phase-contrast photomicrograph of a squashed spherical cell mass formed at the end of migration, within which spores formed. The tip and the opposite end contained amoeboid cells. v No stalk formation was observed. x590. Fig. 2. A phase-contrast photomicrograph of a squashed spore mass formed at 27 C. It comprised only spores. Neither stalk cells nor stalk tubes formed. X The effects of cyclic AMP Since cyclic AMP is known to induce stalk-cell differentiation (Bonner, 1970; Town, Gross & Kay, 1976), the effects of cyclic AMP on the development of the mutant was examined at both 21 C and 27 C. At 21 C, cyclic AMP had no effect when cells were incubated after starvation on agar containing 1 mm-cyclic AMP and LPS. Hence, cells were first incubated, at 21 C, on a thin layer of nonnutrient agar until cell aggregates produced tips (this was the most effective stage to cyclic AMP), and then transferred onto cyclic AMP agar and incubated at either 21 C or 27 C. When incubated at 21 C, (Fig. 3), a majority of cells in the aggregate became stalk cells and the rest were undifferentiated. At 27 C, however, the cells in the aggregate remained undifferentiated and only a few spore masses formed. Thus, cyclic AMP induced the mutant to form stalk cells at 21 C, but not at 27 C. At either temperature, wild-type cells formed globular cell masses, within which the majority of the cells became stalk cells and the rest were either undifferentiated or became spores. 5. The proportion of prespore cells We examined the proportion of prespore to total cells within the mutant slugs, since they eventually produced spore masses. Cells disaggregated from slugs were stained with fluorescent antispore serum globulin and the number of stained and

5 A temperature-sensitive stalkless mutant ofd. discoideum 239 Fig. 3. A KYH-13 aggregate on cyclic AMP agar. An aggregate formed at 21 C was transferred to cyclic AMP (1 mm) agar and incubated at 21 C. The majority of cells in the aggregate were stalk cells. x620. Table 1. Changes in proportion of prespore {spore) to total cells during the development of KYH-13 Stages standing slugs 3 h migrating slugs 6h migrating slugs spherical cell masses spherical cell masses after spore formation Incubation temperature 21 C 27 C 80-0 ± ± ± ± ± ± ±1-2* /prespores 10-8 ± 4-5\ Vspores 81-3 ±4-6/ Cells were allowed to develop at 21 C until the standing slug stage, when half of the plates were shifted up to 27 C. At the stages indicated, cells disaggregated from cell masses were stained with FITC-conjugated antispore serum globulin and stained and unstained cells were counted. The values indicate the percentages of prespore (stained) cells with standard deviations, except for the asterisked value which shows the sum of prespores and spores. Over 20 cell masses were used for one determination. The values represent the average of six determinations. unstained cells were scored. Table 1 showed that prespore proportion within the standing slugs was approximately 80 %, a value equivalent to that of wild-type slugs (Hayashi & Takeuchi, 1976). The proportion did not change during migration for at least 6h. Almost the

6 240 A. AMAGAI, S. ISHIDA AND I. TAKEUCHI same proportion was also observed in the spherical cell masses formed at 27 C at the end of migration (Table 1). Proportions of spores and prespore cells were determined with spherical cell masses after spores formed. In these cell masses, many additional prespore cells were found besides mature spores (whose ratio was equivalent to that of prespore cells in the slugs), resulting in a considerable increase in the ratio of stained cells (spores plus prespores) (Table 1). This suggests that after spores formed from pre-existing prespore cells, the remaining prestalk cells redifferentiated into prespore cells. 6. The distribution of prespore cells Changes in the distribution of prespore cells during the development of the mutant were examined, both at 21 and 27 C. A standing slug of the mutant formed at 21 C showed the same staining pattern as that of wild type: the anterior part of the slug was unstained, whereas the posterior part stained except for the rear-most part. This staining pattern did not change during migration at 21 C(Fig.4). On the other hand, slugs which had been shifted up to 27 C at the standing slug stage showed different staining pattern. During 3 to 6 h of migration, the area of the anterior prestalk region of these slugs decreased greatly, while prestalk cells accumulated at the foot (Fig. 5) or the rear (Fig. 6) of the slugs. The mutant slugs stained with neutral red showed a staining pattern complementary to that of immunocytochemical staining (Fig. 7). At the end of migration, a slug was transformed into a spherical cell mass, in which prespore cells differentiated into spores. The cell mass at this stage contained groups of cells which were unstained by fluorescent antispore serum globulin but stained by neutral red, i.e., prestalk cells (Bonner, 1952). However, sections of cell masses at a later stage revealed the appearance of prespore cells with prespore antigen among the prestalk cells (Fig. 8). During the process such prestalk cells were never observed dying on and being eliminated from the cell mass. It was concluded from this and the above sections' results that after the Fig. 4. A section of a KYH-13 slug migrating at 21 C. The section was stained with FITC-conjugated antispore serum globulin. The prestalk (unstained)-prespore (stained) pattern was the same as in wild type. X270. Figs 5, 6. Sections of KYH-13 slugs migrating at 27 C, immunocytochemically stained as in Fig. 4. Prestalk cells (unstained) decreased in the anterior part (toward the left) of the slug, but accumulated at the foot (Fig. 5) or the rear (Fig. 6). x300 (Fig. 5), X270 (Fig. 6). Fig. 7. A squashed KYH-13 slug migrating at 27 C, vitally stained by neutral red. Prestalk cells in the anterior (toward the left) and rear parts of the slug were stained. X270. Fig. 8. A section of a spherical cell mass containing groups of prestalk cells which remained amoeboid after the majority of cells had become spores. The section was immunocytochemically stained, as in Fig. 4. Spores and prespore cells were stained. The latter were observed among unstained prestalk cells. x520.

7 A temperature-sensitive stalkless mutant ofd. discoideum 241

8 242 A. AMAGAI, S. ISHIDA AND I. TAKEUCHI prespore cells pre-existing in the slug had differentiated into spores, the prestalk cells regulated to become prespore cells. These prespore cells later became mature spores. DISCUSSION A temperature-sensitive aggregateless and stalkless mutant, KYH-13 could aggregate and form fruiting bodies at 21 C, but not at 27 C. When temperature was shifted to 27 C after aggregation at 21 C, the mutant formed masses of spores. Formation of neither stalk cells nor stalk tubes was observed. In this respect, the mutant differs from a stalkless mutant, KY19 (Ashworth & Sussman, 1967), which forms small stalk tubes containing unvacuolized amoebae (Morrissey & Loomis, 1981). The inhibition of stalk cell differentiation in KYH- 13 at 27 C was not lifted by cyclic AMP, although it induced stalk cell formation at 21 C. Since vital staining of the mutant slugs indicated differentiation of prestalk cells at 27 C, the blockage must occur in the maturation of stalk cells. Although KYH-13 formed only spores at the terminal developmental stage, the proportion of prespore cells within the slug was the same as in wild-type. Similar observations have been made not only with KY19 but also with several stalky mutants which form only stalks (Morrissey, Farnsworth & Loomis, 1981). From these findings, they questioned the idea that the pattern of differentiation at the slug stage is related to the pattern of terminal differentiation. In the case of KYH-13, however, the present work revealed that after the prespore cells pre-existing in the slug differentiated into spores, the remaining prestalk cells were converted to prespore cells which then became spores. This result indicates that even in this mutant, prestalk-prespore differentiation precedes and corresponds to terminal stalk-spore differentiation and hence is consistent with the aforementioned idea. The conversion probably occurred through the mechanism of proportion regulation analogous to that working in a prestalk isolate of a slug. Since the mutant cells are unable to become stalk cells but only form spores at 27 C, the cell-type conversion could continue until almost all the cells differentiate into spores. This explains the fact that the mutant took much longer to form spore masses than fruiting bodies. It is possible that a similar type of continual regulation occurs during fruiting body formation of stalky or stalkless mutants examined by Morrissey et al. (1981) as well, especially since those mutants take far longer to culminate than the wild type. The present work showed that during 3 to 6 h of migration at 27 C, the distribution pattern of prespore and prestalk cells in the mutant slug was considerably deranged. Because the proportion of prespore cells did not change during this period, the pattern changes presumably resulted from redistribution of prespore and prestalk cells. This suggests some alterations at 27 C of cell properties involved in cell sorting, such as adhesiveness or motive force of cells.

9 A temperature-sensitive stalkless mutant o/d. discoideum 243 The fact that the proportion of prespore cells remained normal in spite of their deranged distribution indicates that the regulation of proportion is independent of the formation of pattern. This was originally suggested by Forman & Garrod (1977) and is consistent with the recent finding of Oyama, Okamoto & Takeuchi (1983) that dissociated aggregative and slug cells shaken in a medium containing glucose, albumin and cyclic AMP form small cell clumps, in which prespore proportion can be regulated in the complete absence of the normal prestalkprespore pattern as observed in slugs. We are grateful to Dr. M. Filosa of University of Toronto for critically reading the manuscript. This work was supported in part by a grant ( ) from the Ministry of Education of Japan. REFERENCES ASHWORTH, J. M. & SUSSMAN, M. (1967). The appearance and disappearance of uridine diphosphate glucose pyrophosphorylase activity during differentiation of the cellular slime mold Dictyostelium discoideum. J. biol. Chem. 242, BONNER, J. T. (1947). 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. (1952). The pattern of differentiation in amoeboid slime molds. Amer. Nat. 86, BONNER, J. T. (1970). Induction of stalk cell differentiation by cyclic AMP in the cellular slime mold Dictyostelium discoideum. Proc. natn. Acad. Sci., U.S.A. 65, ELLINGSON, J. S., TELSER, A. & SUSSMAN, M. (1971). Regulation of functionally related enzymes during alternative developmental programs. Biochim. Biophys. Ada TAA, FORMAN, D. & GARROD, D. R. (1977). Pattern formation in Dictyostelium discoideum: II. Differentiation and pattern formation in non-polar aggregates./. Embryol. exp. Morph. 40, FUKUI, Y. & TAKEUCHI, I. (1971). Drug resistant mutants and appearance of heterozygotes in the cellular slime mould Dictyostelium discoideum. J. gen. Microbiol. 67, HAYASHI, M. & TAKEUCHI, I. (1976). Quantitative studies on cell differentiation during morphogenesis of the cellular slime mold Dictyostelium discoideum. Devi Biol. 50, HOHL, H. R. & HAMAMOTO, S. T. (1969). Ultrastructure of spore differentiation in Dictyostelium: the prespore vacuole. J. Ultrastruct. Res. 26, MACWILLIAMS, H. K. & BONNER, J. T. (1979). The prestalk-prespore pattern in cellular slime molds. Differentiation 14, MAEDA, Y. & TAKEUCHI, I. (1969). Cell differentiation and fine structures in the development of the cellular slime molds. Devi. Growth & Differ. 11, MORRISSEY, J. H., FARNSWORTH, P. A. & LOOMIS, W. F. (1981). Pattern formation in Dictyostelium discoideum: An analysis of mutants altered in cell proportioning. Devi Biol. 83,1-8. MORRISEY, J. H. & LOOMIS, W. F. (1981). Parasexual genetic analysis of cell proportioning mutants of Dictyostelium discoideum. Genetics 99, OYAMA, M., OKAMOTO, K. & TAKEUCHI, I. (1983). Proportion regulation without pattern formation in Dictyostelium discoideum. J. Embryol. exp. Morph. (in press). RAPER, K. B. (1940). Pseudoplasmodium formation and organization in Dictyostelium discoideum. J. Elisha Mitchell Sci. Soc. 56, TAKEUCHI, I. (1963). Immunochemical and immunohistochemical studies on the development of the cellular slime mold Dictyostelium mucoroides. Devi Biol. 8, TOWN, C. D., GROSS, J. D. & KAY, R. R. (1976). Cell differentiation without morphogenesis in Dictyostelium discoideum. Nature 262, YAMAMOTO, A. & TAKEUCHI, I. (1983). Vital staining of autophagic vacuoles in differentiating cells of Dictyostelium discoideum. Differentiation (in press). (Accepted 18 November 1982)

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