Department of Anatomy and Cell Biology, Columbia University, 630 West 168th St, New York, NY 10032, USA1
Author for correspondence: Richard H. Kessin. Tel: +1 212 305 5653. Fax: +1 212 305 3970. e-mail: rhk2{at}columbia.edu
Keywords: SCF complex, proteolysis, parasitism, slime mould, Botryllus
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Multi-cellularity by aggregation |
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Consider the life cycles of Dictyostelium discoideum and a developing prokaryote, Myxococcus xanthus, both of which live in a similar soil niche. Dictyostelium and related species of amoebae feed on many species of bacteria. Once the bacteria are consumed, the amoebae construct a chemotactic and motile machinery that brings them into aggregates of about 100000 cells. The chemotactic signal, cAMP in the best studied species, is relayed so that amoebae can collect from a fairly large area perhaps a cubic centimetre of soil. Within the D. discoideum aggregate, cell-type differentiation occurs (Fig. 1), giving rise to progenitors of stalk cells and spore cells, in a strict ratio of 1:4 (Raper, 1940
; Stenhouse & Williams, 1977
; Bonner & Slifkin, 1949
). The mound of cells forms a tip, which extends into a motile slug that orients along heat gradients and towards light. At a certain moment, the slug undergoes a process of culmination such that the spores are extended onto a long stalk. The cells that form the stalk die and it is this sacrifice that is important to the argument that follows.
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Myxobacteria are Gram-negative gliding bacteria and comprise a number of genera of the Proteobacteria (Dworkin, 1996 ). They are common in the soil, where they feed on other bacteria. Most of this feeding is done in packs and the rationale for this behaviour has been that the secretion of large quantities of digestive enzymes is most effectively done in groups. This is in contrast to Dictyostelium, which feeds by phagocytosis, a process that is not aided by sociality. The cooperativity of the myxobacteria extends to their development when their nutrients are consumed, they glide toward aggregation centres and produce a mound of cells (Fig. 2
), although the role of chemotaxis in this process is controversial (Kearns & Shimkets, 1998
). The complexity of the fruiting structures that result is highly variable, depending on which of the families of myxobacteria is being studied, but some, such as Chondromyces crocatus, have elaborate stalk structures, while the more thoroughly studied M. xanthus has a simpler fruiting body (Fig. 3
). As in the case of the dictyostelids, many cells, even a majority, die in the process of forming a fruiting body in M. xanthus (Dworkin, 1996
; Wireman & Dworkin, 1977
). Other species have not been as thoroughly examined. Whether the death of the majority of cells benefits those that form the myxospores is not as clearly established as is the case of stalk formation in Dictyostelium.
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The problem of parasitism |
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Dictyostelium and myxobacteria collect cells from a small area to form a collection that has a much larger mass than an individual cell. There are two major limitations to this means of creating multicellular structures. The first is size. The fruiting bodies of Dictyostelium and myxobacteria are limited by the number of cells in a particular volume of soil and the distances that the cells can migrate to enter an aggregate. Animals depend on a great evolutionary innovation they can retain their differentiated state while they feed, grow and develop, and do not suffer the size limitation of aggregative organisms. The second limitation of the collective life stems from the fact that increasing size by aggregation is a rather open procedure. Dictyostelium sacrifices 20% of its cells to form a dead supporting stalk and 90% of M. xanthus cells die during development of myxospores (Dworkin, 1996 ; Wireman & Dworkin, 1977
). Over evolutionary time spans, and given genetic diversity in the wild, we expect that strains would evolve that make no contribution to the stalk of Dictyostelium or the fruiting structures of the various myxobacteria. We call such strains cheaters because of their partial or complete evasion of the stalk-cell fate. The formation of chimeric colonies in which cheater mutants can function requires that isolated populations be brought into contact. We have known since Darwin studied earthworms that the soil is constantly churned and that therefore social amoebae with different genotypes would be brought into contact. Cheaters, we predict, would form only viable spores and upon co-aggregation with normal cells, might ignore signals to become supporting structures. If they were really good cheaters they might actively drive the wild-type to be stalk cells, perhaps by suborning a normal regulatory feature of development. Some evolutionary biologists muse that Dictyostelium should never have evolved because of its susceptibility to parasitism by non-cooperative mutants of its own species. Indeed, non-cooperating variants are a major difficulty when considering selection theories that would lead to cooperating structures like those of the dictyostelids or of the myxobacteria (Michod, 1999
; Zahavi & Ralt, 1984
).
There are several ways out of this dilemma. If new mutations leading to, or sustaining, cooperative behaviour appeared more frequently than non-cooperative cheater mutations, the sort of selection we see in the evolution of the dictyostelids could occur (Zahavi & Ralt, 1984 ). Since the elaboration of a Dictyostelium fruiting body requires hundreds of genes acting in concert and the construction of a non-cooperating strain requires only one mutation, this is not likely. Since Dictyostelium spores are often dispersed to give rise to clonal colonies (Kessin et al., 1996
), a non-cooperative mutant would have to be able to survive on its own and could not, in the process of becoming a parasite, sacrifice its own developmental capacity. The frequent imposition of clonal growth would select against a large class of potential parasites those that require a partner wild-type cell at least in the wild. The third way to avoid non-cooperating cells is to evolve incompatibility or policing mechanisms, or to change chemotactic molecules so that cheaters could not enter aggregates (Armstrong, 1984
; Buss, 1987
; Frank, 1995
). Finally, there is a species called Acytostelium leptomosum (Raper & Quinlan, 1958
) that produces a non-cellular stalk and therefore does not expend the normal 20% of cells in forming a stalk. Acytostelium, although perhaps still subject to cheating, indicates that there is more than one strategy for forming a stalked structure, as in the case of C. crocatus (Fig. 3
).
There have been two reports indicating that in freshly isolated uncloned populations of Dictyostelium amoebae, variants preferentially make spores in chimeras with normal fruiting cells. The first report was by Filosa (1962) , working with Dictyostelium mucoroides. Starting with an uncloned population, Filosa found individual colonies with variant morphologies. One, which formed slugs but no fruiting bodies, preferentially made spores when mixed with wild-type cells. The second observation of parasitism was by Buss (1982)
, also with D. mucoroides. Buss found a single very small soil sample which, when plated clonally, gave rise to normal colonies and colonies which only formed spores. The latter, in chimeras with the fruiting strain, could suppress the wild-type. It was Buss who first pointed out the potentially disruptive role of parasites on multi-cellularity by aggregation. One might imagine that some cells have mutated to secrete greater amounts of a factor that causes other cells to differentiate into stalk. A compound called differentiation-inducing factor (DIF), which has properties that could cause such an effect, has been described (Traynor & Kay, 1991
; Williams et al., 1989
). It is possible that DIF evolved not to mediate cell-type differentiation, but as an instrument of the competition among genetically diverse populations of D. discoideum. By this logic, combining wild-type isolates of Dictyostelium might reveal that one strain is overrepresented in the spore population. Such studies have been undertaken by Strassmann and colleagues, who made chimeras of genetically diverse amoebae that were isolated from the same locale. Microsatellite analysis revealed that these chimeric mixtures are often not fair and that often one clone contributes less than its proportional share to the stalk and more to the spores (J. Strassmann, personal communication). Whether the ability to achieve extra representation in the spore population is based on the actions of DIF is not known.
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Exploiting parasitism |
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In our laboratory we have isolated a cheater mutant of D. discoideum (denoted chtA) by a selection procedure that was based on the idea that a cheater mutant should increase its frequency in a chimeric population. A population of 14000 amoebae, each with a different insertional mutation, was allowed to form spores and then the spores were harvested and treated with detergent to kill any cell that was not a spore. The spores were then plated on lawns of bacteria, where they were allowed to feed by phagocytosis, aggregate and form spores again. The process was repeated a number of times and at the end, a single mutant made up an increasing portion of the population. This procedure only recovers mutants that have a distinct morphological phenotype. The recovered mutant was marked with a lacZ gene that is only expressed in cells destined to be spores. The marked strain was then mixed in a ratio of 1:1000 with unlabelled wild-type cells and the fruiting bodies were monitored for an increase in the number of mutant spores. This can be done by plating the spores clonally and observing the phenotypes of the colonies, or by staining the fruiting bodies for ß-galactosidase activity, as shown in Fig. 4.
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In slugs formed from chimeras, the mutant cells always populate the zones that are fated to give rise to spores. The wild-type cells always end up in the prestalk zone, even if they were originally in the rear of the slug a region normally fated to produce spores. The mutant causes wild-type cells that have begun to express spore-specific genes in the rear of the slug to migrate to the prestalk compartment of the developing organism. This conversion can be seen if the wild-type cells are marked with a prespore promoter expressing ß-galactosidase (Fig. 5). Trans-differentiation of the wild-type into a non-viable stalk-cell population allows the cheater cells to increase in the population. Under certain conditions of development, which are more controlled than those used in the original selection, they increase five- to sixfold in one cycle of development (Ennis et al., 2000
). An alternative explanation could be extra cell divisions of the chtA mutant cells. Our preliminary experiments have not detected a gross increase in chtA cell number (H. L. Ennis, unpublished). It should be remembered that these cells are starving, and this would mitigate against DNA synthesis and cell division.
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Recovering the chtA gene |
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Do equivalent phenomena occur in the myxobacteria? |
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Phase variation in M. xanthus could also lead to cheating. One classical example of phase variation is the alteration of flagellar antigens in Salmonella typhimurium. In the case of S. typhimurium, a site-specific recombination system causes the inversion of a genomic fragment, leading to expression of different flagellar structural proteins (Scott & Simon, 1982 ). The molecular basis of phase variation in M. xanthus is not known. M. xanthus has yellow and tan variants that appear by phase variation in all colonies, with the yellow form predominating and the tan making up 15% in the standard strains. Yellow colonies have a rough appearance and swarm, while tan colonies have a smooth, mucoid appearance. A freshly isolated tan colony has about 25% yellow variants, indicating that the variation occurs at high frequency and in both directions, and that all colonies are mixed. However, the frequency of the tan variants rises six- to sevenfold over one cycle of fruiting-body production and sporulation. Starting at 5% tan variants in the vegetative population, fruiting bodies end up with about 35% tan variants. There is speculation that the yellow cells lyse to provide essential inducers or nutrients for the encapsulation of the spores of the tan cells (Dworkin, 1996
; Wireman & Dworkin, 1977
).
Laue & Gill (1995) have asked what the developmental significance of these mixed colonies is by creating a variant that is locked almost completely in the tan phase. These cells form prespores, but do not make the final resistant spore cells unless some of the yellow variants are present. Recall that the prespores of the D. discoideum chtA mutant do not form encapsulated spores unless some of the wild-type are present. This is perhaps the best solution that the bacterial genome can provide to establish two essential cell types (Laue & Gill, 1995
). How is it that sporulation of one cell type (the tan or the cheater) is dependent on the presence of the second sacrificial cell type (the yellow or the stalk)? Given that mixed colonies are required for successful sporulation, high-frequency phase variation, whether genetic or epigenetic, assures that even after dispersal, a single yellow or tan cell would not suffer an inability to sporulate. A Dictyostelium cheater mutant would be counter-selected if dispersed into a single clonal colony, as would a phase-locked tan or yellow variant, such as that described by Laue & Gill (1995)
. There is one way in which phase variation could be exploited to achieve an advantage. If there are competing populations that form chimeric fruiting bodies, the cells that have a reduced frequency of variation in the direction tan to yellow would be selected. A phase-locked variant such as the one described by Laue & Gill would have an advantage as long as it sticks to wild-type myxospores. There could be other genes, having nothing to do with phase variation, which would give certain strains of myxobacteria an advantage in chimeric colonies. To our knowledge, the types of selection experiment performed in Dictyostelium have not yet been done with the myxobacteria.
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Does the cheating phenomenon extend to other multicellular organisms? |
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If we make the prediction that the germ line is important as an evolutionary innovation because it blocks the invasion of somatic-cell variants, there should be complex organisms that do not produce completely sequestered germ lines and suffer the consequences of competition. The phenomenon of cheating is seen in certain complex metazoans that do not set aside a germ line. These include the colonial protochordates, as most recently shown by Stoner et al. (1999) . This large class of organisms reproduce asexually, but with an occasional sexual generation. The zygotes of Botryllus schosseri mature into a tadpole larval stage with the body plan of a chordate. As Stoner and colleagues point out, metamorphosis results in the loss of the chordate body plan, including the notochord and the musculature, and results in a sessile oozooid with its own tissues and circulation. Individual oozooids fuse at their borders so that they have a common circulation. Eventually, ovaries and testes form and sexual reproduction takes place. The fusion of individuals is the interest here because sometimes there are colonies of different genotypes on the same substratum. Because these individuals have not yet constructed gonads, the primordial germ cells are not sequestered, but are free to move. When fusion occurs, an incompatibility mechanism is engaged and two genetically distinct individuals usually remain separate. The fusion and rejection mechanism is controlled by a single polymorphic locus. In certain cases, the incompatibility mechanism is not engaged and individuals with two different genotypes fuse. Using microsatellite markers, Stoner and colleagues showed that there is migration of cells with one genotype into somatic tissues with another to form a true chimera. Most important, the germ cells of one individual (there is a hierarchy) populate the gonads of a genetically distinct individual a situation similar to the cheater phenomenon described for Dictyostelium. For a further comparison, see the commentary by Buss (1999)
. Buss (1987)
and others cited by Stoner et al. (1999)
have postulated that the great variety of incompatibility mechanisms that we see in nature are an adaptation designed to limit cell-lineage competition.
Though Dictyostelium, Myxococcus and Botryllus have no direct relationship in phylogeny, they all confront a problem of parasitism by cells of their own species and this problem is probably at the root of the evolution of a variety of incompatibility mechanisms. In the case of the genetically tractable Dictyostelium, the tendency to parasitism can be exploited to investigate the normal regulation of development.
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ACKNOWLEDGEMENTS |
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