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Historical Perspectives and Tetrahymena Epigenetics
A century ago biological scientists began assembling of the Chromosome Theory of Inheritance by juxtaposing the mendelian rules of gene transmission on the cytological observations made possible by improved microscopic techniques. That synthesis, utilizing a highly successful reductionistic approach, bypassed temporarily the incipient union of understandings about the phenomena of heredity and development and opened a breach between genetics and developmental biology that is still apparent. Genetics, stripped of its unresolved problems in physiology and development, soon found accommodation with the stream of darwinian thought. The Modern Evolutionary Synthesis combined bean bag biology, chromosome behavior and natural selection, and was accounted a great success. But many biologists, particularly developmental biologists, were dissatisfied because of the unfilled gap between genotype and phenotype. Sometimes a simple relationship was found between genes and phenes, a one to one relationship between a chromosomal element and an organismic trait, and these examples collected in text books and classrooms.
The most important intermediate connection between genes and phenes was the ?One gene - one enzyme hypothesis?, which was later corrected to the ?one gene - one polypeptide hypothesis?. Even this amended doctrine was recognized as inadequate to account for the activity of genes. All genes are not involved in protein synthesis, or in the synthesis of only one polypeptide, for example. Importantly, developmental biologists continued to ask about the patterns of gene expression in different parts of organisms. Because formal genetics provided no convincing answer to this question, the adequacy of the chromosome theory was doubted. An important manifestation of this dissatisfaction was the emergence of a contending theory of ?cytoplasmic Inheritance.?
Cytoplasmic inheritance was never considered as an alternative to the chromosome theory, but as an addendum, an explanation for phenomena not accounted for in the classical chromosome theory. Tracy Sonneborn, the leading American proponent of cytoplasmic inheritance, often referred to partners of the gene or of mechanisms beyond the gene (See Sapp, 1980). It is noteworthy that, in addition to many embryologists, many of the sympathizers with the doctrine of cytoplasmic inheritance were workers with microorganisms. Sonneborn was drawn to cytoplasmic explanations because available genetic theory provided no explanations for his observations on mating types, serotypes and killer traits in paramecium. Boris Ephrussi, the leading European theorist supporting cytoplasmic inheritance came from studies in Drosophila genetics to explore respiratory mutants in yeasts, and also found inexplicable phenomena.
The resolution to this discord in theoretical biology came, predominantly through the rapidly expanding studies on microorganisms. The filamentous fungus Neurospora was the work-horse for the gene-enzyme analysis. Lederberg and Tatum, and Luria and Delbruck, in the 1940s established the methods for bacterial and virus genetics. When investigators began to explore the conditional expression of genetic capabilities, particularly in the phenomena of induced enzyme synthesis, a previously unrecognized cybernetic system responsible for the integration of environmental signals and genetic responses began to emerge (Nanney, 1957; Ephrussi, 1958). The lac operon, in the hands of Monod and Jacob (1961;Jacob and Monod,1961) became the first thoroughly explored and widely accepted demonstration of the genetic regulatory system, a system accessible through the mutational and biochemical dissection of a microbial model. It was assumed to apply in principle to eukaryotes and to explain the phenomena of developmental differentiation.
Saccaromyces cerevisiae, a cellular fungus, has become the main eukaryotic racehorse for exploring mechanisms of gene expression. It has all the basic eukaryotic machinery, it is easily grown in the lab , and it has acquired a large and aggressive following. Coming up on the outside, however, is another eukaryotic protist that may have some significant contributions to make in genetic regulation, particularly in morphogenesis and in evolutionary responses.
The nearly uniform morphology of laboratory tetrahymenas easily misleads those who work with them to misunderstand their diverse molecular composition and their distinctive roles in nature. They are misled partly because of a gene-phene doctrine, derived primarily from studies on multicellular plants and animals, that is inappropriate in eukaryotic protists. We need to survey briefly the evolutionary and epigenetic diversity that is imposed on a rigidly preserved morphotypology.
First, consider the standard morphology of the vegetative laboratory tetrahymena (Fig.2). The cortex is the definitive feature of a ciliates anatomy. Tetrahymenas cortex is composed of kineties, long rows of cortical units bearing the epinominal feature of its phylum. The complex and still unexplained details of the cortical unit revealed by silver staining and ultrastructural analysis are beyond our consideration here, except to note their apparent invariance. Variance begins to appear in the arrangement of the cortical units. The number of longitudinal rows may vary, even within a clonal culture, but tends to be preserved over large numbers of divisions. A strain is not characterized by a specific number of rows, but rather by a range and a pattern of transition probabilities. Ciliary units are added to ciliary rows as the animal grows, developing just anterior to preexisting ciliary units. Oddly enough, the number of ciliary units in the animal as a whole is more constant than the number of ciliary rows. As the number of ciliary rows increases within a strain, the number of ciliary units per row declines, so as to maintain a constant number of ciliary units at a particular stage in the division cycle.
The cortex incorporates three essential organelles in a complex asymmetric geometry. The oral apparatus is composed of four ciliated membranelles and lies near the anterior end of the animal, terminating the ciliary rows that abut upon it. The interrupted ciliary row to the animals left is referred to as the stomatogenic row, and is the site of formation of a new oral apparatus as the organism prepares to divide. This row, near the posterior end of the animal, provides the site of the cytoproct, where waste materials are egested. The third kind of organelle located on the cortex is the contractile vacuole pore. CVPs are usually paired organelles located on adjacent ciliary rows about 25% of the organisms diameter to the right of the stomatogenic kinety. The number of CVPs ranges from 1-3 in a single culture, but their mean location tends to be geometrically constant, at about 25% of the circumference (90o from the stomatogenic meridian), even though the number of rows varies. In one strain studied the CVPs were located further around the animals' circumference. This observation led to the characterization of its species as T. hyperangularis, even though not enough strains were studied to establish this variant geometry as a species, as opposed to a strain difference.
The most dramatic morphological variant in tetrahymena cultures is the doublet, the side by side union of twinned animals, with separate organelles and independent cytogeometric domains. The growth of homopolar doublets is coordinated so that the twinned state may be maintained for hundreds of cell divisions. Ciliate doublets are one of the best studied of a class of epigenetic homeostatic phenomena. Beisson and Sonneborn (1965) provided the most critical analysis of these phenomena in paramecium. Joe Frankels (1990) account is comprehensive and masterful. No evidence has been found to indicate that any kind of genetic change is involved in the origin or maintenance of the morphological variants.
The doublet condition is an extreme example of the epigenetic plasticity that tetrahymena manifests usually in more subtle ways. Under some circumstances tetrahymenas may undergo a transformation from their usual global feeding condition to an elongated streamlined form and a more rapid rate of movement ( Williams 1960 ; Buhse 1966 a, b). A long caudal cilium develops that may aid in locomotion. The streaker morphotype, is rationalized as a tactic which aids escape from unsatisfactory living conditions. Occasionally caudal cilia have been observed in cultures of various origin, and have sometimes been considered distinctive enough to characterize species.
Another morphological change has been observed under more controlled conditions. When mature T. thermophila cells in proper physiological condition are mixed with cells of a complementary mating type, a distinctive morphological transformation occurs. The anterior end is transformed into a rostrum which seems to faciliate cell union and conjugation ( Wolfe and Grimes, 1979).
A morphological transformation that has been studied more systematically is that from the small-mouthed bactivorous microstome to the macrostome form capable of engulfing larger eukaryotic prey, including decrepit tetrahymenas. This epigenetic morphological transition is viewed as an adaptation to carnivory or cannibalism. It appears to be triggered by a pherome produced by prey (Buhse 1966a, b). Williams 1984) was unable to demonstrate any changes in the molecules composing the oral apparatus. All tetrahymenas are not capable of macrostome formation, and the ability was considered as an important phyletic character. The evolutionary tree, however, provides no support for this notion. Macrostome formers are distributed widely among the evolutionary clusters (See particularly, Struder-Kypke et al., 2002). This epigenetic capability seems to be easy-come easy-go on an evolutionary time scale.
Most strains of tetrahymena in exhausted laboratory cultures undergo a gradual diminution in size. The cell volume is reduced, the number of cortical units declines. Eventually some cells shrink to a small fraction of their normal size, and show only scattered arrays of disordered cilia. Other strains, however, form spores when their food supply is exhausted or the cultures dry out. This differential epigenetic modification is probably an adaptation to life in temporary streams or ponds. The ability to sporulate seems to be characteristic of particular species, but like macrostome formation, it appears in evolutionarily scattered species. The genetic capability for the epigenetic transition comes and goes as part of partitioning of the ecosystem, providing an adaptive edge for new species exploiting a particular habitat.
The facultative morphological transformations that tetrahymenas sometimes manifest are only a small fraction of a very wide epigenetic repertoire. One of the most studied systems of epigenetic variations in ciliates is that controlling the mating types. The systems are responsible for the most basic distinction among sibling species - their genetic incompatibility. Beyond the exclusion of strangers from their unique gene pools, the mating systems also influence the degree of relationships of conjugants within a species, the circumstances appropriate for mating, and the time between mating events (Nyberg and Dini).
T. thermophila may serve as an example of one, not necessarily typical mating system. The species contains seven mating types that will mate in any combination under appropriate conditions. Every conjugating pair produces a pair of identical zygotes capable of manifesting a range of mating types. The restriction of that range to the eventual manifestation of a single mating type is accomplished through a still incompletely resolved epigenetic redaction (Orias). The epigenetic choices made at the time of macronuclear differentiation are not expressed until the clonal calendar signals maturity, and environmental clues - including a suitable mate - allow conjugation to proceed.
Most tetrahymenas brought into the laboratory are not known to mate. Many of them have no micronuclei, though they may have had germinal nuclei and genetic capability before they came into the lab. Some manifest uncontrolled mating (selfing) within a culture, though such matings are often lethal. Extrapolating from observations made under laboratory conditions to the natural world is hazardous. Those species that are compatible with laboratory conditions manifest considerable diversity in the ways the regulate their mating. T. thermophila and T. malaccensis, for example, show caryonidal inheritance of their multiple mating specificities, but they are the only species of tetrahymena known to behave in this way. The other species that have been studied exhibit peck-order dominance of multiple mating alleles and lack epigenetic control of mating type choice.
Mating phenomena are not the only traits under epigenetic control. The best studied of these are transformations of surface antigens, which permit them to survive under environmental challenges. T. thermophila, like most ciliates (and many other eukaryotic protists) manifests different surface antigens when grown in specific antiserum, at different temperatures, or in media with different salt concentrations (Grass, Doerder, ). In fact many proteomic transitions occur when culture media and growth conditions are changed, but most of these have never been reported in detail. Their epigenetic controls permit a considerable physiological plasticity, and probably permit individual ciliates to explore diverse niches (Nyberg, 1974).
Some tetrahymenas have apparently abandoned the ability to cope with diversified habitats, and have become restricted to special niches. Such an evolutionary pathway may have led to species such as Ichthyopteris multifiliis - the aquarium denizen affectionately referred to as ick. The dispersal forms of Ick are indistinguishable from those of other tetrahymenids ( ), whose basic form and way of life was established long before fish appeared on the scene. Ribosomal sequences agree with vegetative morphology in placing Ick fairly close to a particular evolutionary cluster. Placing ick in a separate genus no longer seems like a good idea. A similar case is provided by Lambornella clarkii, a tetrahymena specialized to parasitize anopholine mosquitoes that live in tree stumps in California (Edgeter, et al, 1986). Some other tetrahymenids were similarly named because of the habitats in which they were first collected. T. chironomus was so-named because Corliss found them commonly in the larvae of chironomid flies. Their vegetative forms and their ribosomal signatures place all these distinctive tetrahymenids well within the Tetrahymena clade, but clearly they are genetically isolated and exploit distinctive niches.
More impressive, perhaps, than the facultative morphogenetic changes possible within a ciliate life cycle, are the physiological and synthetic changes that may occur under a variety of circumstances throughout the life cycle and/or in response to particular environmental cues (Doerder, et al. 1995). The mating type distributions within a clone can be explained as a device for governing the breeding economy. The transition from immaturity to maturity is regulated apparently to assure the mating of ciliates of appropriate relatedness and is governed by both hereditary and environmental factors. The most fully studied phenotypes generated in response to environmental stimuli (Preer) are the antigenic types that may be initiated by temperature or feeding cues, or the presence of dilute homologous antiserum. The chromosomal changes effected by the stimuli are quasi-hereditary within the vegetative life cycle, and have long been recognized as epigenetic mechanisms (1957), at least analogous to those employed in developmental programs in higher organisms. Jenuwein (2002) refers to the system of regulation as the epigenome and cites particularly the studies on tetrahymena carried out in the laboratories of Martin Gorovsky and David Allis (Moon et al., 2002; Taverna et al. 2002).
These observations deny the equivalence of tetrahymena species. They represent an ancient lineage that has become providentially fragmented physically and genetically through the agencies of time and space. The genus Tetrahymena is an enormously plastic, evolutionarily active clade of ciliates, that is frozen, curiously enough, only in the elaborately decorated epitopes of the cortical architecture of its dispersive morphotype.