Dependence of post-aggregative cell fate on early phenotypicdifferences with respect to cellular calcium and cell cycle phase in Dictyostelium discoideum

Abstract

The social amoeba Dictyostelium discoideum is an excellent model system to study cell differentiation and embryonic development. Its life cycle consists of a unicellular phase involving feeding, growth, and multiplication, followed by a multicellular phase whose onset is triggered by starvation. Multicellular behavior begins with aggregation via chemotaxis in response to the chemoattractant cyclic AMP. It continues through a facultative slug-shaped embryonic stage and culminates in a fruiting body consisting of spores held aloft on a stalk of dead cells. It is possible to draw a 'fate map' of the slug: prestalk (presumptive stalk) cells constitute approximately the anterior 20%, and prespore (presumptive spore) cells occupy the posterior 80%.

Early cellular correlates of cell cycle phase and nutritional status have been shown to influence post-aggregative cell fate. Using fluorescence-activated cell sorting (FACS), we have found (not part of this thesis) that there are naturally occurring variations of sequestered calcium between pre-aggregation amoebae which anticipate future prestalk-prespore differences (Saran et al., 1994a). However, the relevant intracellular pool of Ca² that functions as a "second messenger" and a mediator of most downstream effects of Ca² is the free, or cytoplasmic, pool. The present thesis deals with the role of early phenotypic differences with respect to Ca² (unless stated otherwise "Ca²" will mean cytoplasmic Ca²) and cell cycle phase in determining the initial cell-type choice and its subsequent maintenance during post-aggregative development. The following questions are investigated:

(a) Do vegetative amoebae of D. discoideum differ in a functional sense depending on their levels of Ca² (b) Do early phenotypic differences with respect to cellular Ca² persist during development, and do they manifest in the slug as an in vivo prestalk-prespore difference of Ca² (c) Is there any correlation between early heterogeneity with respect to Ca² and cell cycle phase, and do they act conjointly or independently in predicting the post-aggregative cell fate (d) How do Ca² and the cell cycle interact to influence cell fate

The work carried out is summarized as follows: When freshly starved amoebae of D. discoideum are loaded with the Ca²-specific dye indo-1/AM and analyzed using FACS, they exhibit a quasi-bimodal distribution of fluorescence. Cell sorting, secondary labeling, and mixing experiments show that amoebae with high (/low) cellular Ca² display a prestalk (/prespore) tendency. Simultaneous monitoring of Ca²-indo-1 and Ca²-chlortetracycline fluorescence shows that, by and large, the same cells tend to have high (/low) levels of both cytoplasmic and sequestered Ca². Polysphondylium violaceum, a cellular slime mold that does not possess prestalk and prespore cells, also does not display a Ca²-dependent heterogeneity at the vegetative stage or in slugs.

Independent experiments with Differentiation-Inducing Factor (DIF), an inducer of prestalk genes, show that DIF application causes a rapid increase (approximately twofold) in the relative fraction of amoebae falling in the high cellular Ca² class. A major part of the increase is caused by Ca² influx from the extracellular medium. Also, in parallel with the cellular heterogeneity with respect to Ca² content, there is a heterogeneity in the response to DIF, which appears to be restricted to cells of the low Ca² class. On the basis of observations made using the calcium-sensitive fluorescent dyes fura-2 and chlortetracycline, it is shown that genetically defined anterior prestalk cells (pstA, pstAB) contain significantly higher levels of calcium than the posterior prespore cells. These findings are further substantiated in vitro in a fine glass capillary using the technique of Bonner et al. (1995).

Ca² and cell cycle phase are both shown to have a determinative influence on initial cell-type choice in D. discoideum. Simultaneous monitoring of cell cycle phase and relative Ca² content by FACS reveals that freshly starved amoebae in S and early G2 (/mid to late G2) phases also have relatively high (/low) cellular Ca² and display a prestalk (/prespore) tendency after starvation. The use of cell cycle inhibitors (hydroxyurea and nocodazole) influences the proportions of amoebae containing high or low Ca² as expected on the basis of this correlation. In the mutant rtoA, which upon differentiation generates an abnormally high proportion of stalk cells, the initial cell-type choice is independent of cell cycle position at starvation (Wood et al., 1996). Correspondingly, in rtoA, a disproportionately large fraction of amoebae display high cellular Ca².

Thus, under normal conditions, the initial cell-type choice is determined by a cell cycle-dependent mechanism with the phase of the cell cycle influencing the level of cellular Ca², and also by a cell cycle-independent and Ca²-dependent pathway that can bias cell fate. Studies involving the effect of inhibitors hydroxyurea and nocodazole on fruiting body morphology and prestalk-prespore proportioning (as judged by neutral red and -galactosidase staining pattern in ecmA-lacZ transformants) confirm that Ca² is a downstream element to the cell cycle and show that it acts as a mediator of the cell cycle-dependent cell fate mechanism by regulating cell-type-specific gene expression. At the molecular level, artificially increasing the level of cellular Ca² with the help of the calcium ionophore A23187 significantly decreases the cyclin B (clb1) mRNA level. The cdc2 mRNA level shows a marginal decrease under similar conditions. This suggests that a primary effect of Ca² on cell fate might be exerted at the level of transcription. I infer from these results that a combination of early heterogeneities of cellular Ca² and cell cycle phase is an early indicator of post-aggregative cell fate.