Tetrahymena as a Unicellular Model Eukaryote: Genetic and Genomic Tools

Abstract

Tetrahymena thermophila is a ciliate model organism whose study has led to important discoveries and insights into both conserved and divergent biological processes. In this review, we describe the tools for the use of Tetrahymena as a model eukaryote, including an overview of its life cycle, orientation to its evolutionary roots, and methodological approaches to forward and reverse genetics. Recent genomic tools have expanded Tetrahymena's utility as a genetic model system. With the unique advantages that Tetrahymena provide, we argue that it will continue to be a model organism of choice.

Keywords: Tetrahymena thermophila; amitosis; ciliates; genetics; model organism; somatic polyploidy.

Figure 1

Tetrahymena cell image, illustrating its crystal-like organization of ciliary units. A single Tetrahymena cell labeled for basal bodies (α-centrin, red) (Stemm-Wolf et al. 2005), kinetodesmal fibers (5D8, green) (Jerka-Dziadosz et al. 1995), and DNA (Hoerscht-33342, blue). Note that basal bodies are packed together in the four ciliary “membranelles” of the oral apparatus, for which the genus is named. Bar, 10 μm.

Figure 2

Tetrahymena areas of research. Fields of study that Tetrahymena has impacted.

Figure 3

Tetrahymena conjugation. The life cycle of Tetrahymena includes a sexual phase called conjugation. Cells are represented as ovals with a micronucleus (small circle) and a macronucleus (large circle) whose DNA content (ploidy) is indicated. Images of cells at corresponding steps are shown on the periphery of the illustration and are lettered accordingly. To begin the sexual phase of the life cycle, two cells of different mating types (A), homozygous for black and white alleles, respectively, undergo pairing (B). Completion of two rounds of meiosis (C) leads to the production of four haploid products (half circles, indicated as 1n). One of these meiotic products is positioned at the anterior cytoplasm of each conjugant, while the remaining three are targeted for elimination (red outline) at the conjugant’s posterior end. Subsequently, mitosis of the surviving meiotic product generates two gamete pronuclei (D). Each migratory pronucleus is transferred to the opposite conjugant in a process called pronuclear exchange (E). The incoming migratory pronuclei fuse with the stationary pronuclei (pronuclear fusion), restoring the diploid character of the MIC and thus generating the fertilization (or zygote) nucleus (F). Thus, each fertilization nucleus gets one haploid genome from each parent (black and white semicircles). The fertilization nucleus undergoes two postzygotic mitoses (G), leading to the production of four genetically identical diploid nuclei in each conjugant. (H) The two anterior nuclei (MAC anlagen) develop into new MACs, while the two posterior nuclei become the new MICs. New MAC maturation involves ploidy increase and programmed DNA rearrangement (see text). The parental MACs are eliminated apoptotically (red outline) and contribute no DNA to the progeny. (I) The exconjugant cells separate and one of the two MICs is eliminated. When the exconjugants divide, one of the two fully developed MACs and a mitotic copy of the surviving MIC in each exconjugant are segregated to their daughter cells, called karyonides because each one gets an independently developed MAC (J). Having completed the conjugation process, these cells must sexually mature through vegetative cell division before they can conjugate.

Figure 4

Evolutionary relationship of Tetrahymena to other eukaryotes. (Top) Eukaryotic supergroups and their subgroups. Adapted from Lynch et al. (2014). (Bottom) Expanded tree of the Alveolates showing the lineage leading to Tetrahymena (red lines) (Cavalier-Smith 1993). Line lengths do not represent evolutionary distances.

Figure 5

Amitotic macronuclear division and phenotypic assortment. (A) Phenotypic assortment: During vegetative growth, the MIC undergoes mitotic division, whereas the MAC divides amitotically, meaning that chromosome copies are randomly distributed to the daughter macronuclei. When the MAC is heterozygous, this leads to unequal segregation of allelic genetic information, a phenomenon called phenotypic assortment, illustrated by the all-white or all-black MACs, the two alternative endpoints of the assortment process. The mitotically dividing MIC remains heterozygous. (B) Amitosis leads to phenotypic assortment: A vegetatively growing cell whose MAC is represented in (a) one of the chromosome copies has a mutant allele, represented by a star. (For simplicity of illustration, only one MAC chromosome is shown, and the G1 ploidy is 4 instead of 45 copies.) One possible sequence of events is illustrated. (b) The number of copies has doubled during S phase. The two mutant copies can be distributed in one of two equally probable ways: one mutant copy goes to each daughter MAC (no change, not shown), or both mutant copies go to the same daughter (c), so that the other daughter becomes fixed for the wild-type allele. Additional cell cycles (d and e) generate MACs fixed for the mutant allele (f). Once a cell with a pure MAC is generated, every MAC in the resulting subclone remains fixed. Thus, the fraction of cells with pure MACs continuously increases in the clone. At steady state, the probability that a cell with a mixed MAC will generate a daughter with a pure MAC is 1 / (2n − 1), where n is the ploidy at G1 stage, or 0.011/fission for n = 45. The initial allele ratio in the progenitor cell’s MAC (a) is called the input ratio. The output ratio is the ultimate ratio of cells whose MACs have become pure for either allele after many fissions. In the absence of selection, the output ratio should be identical to the input ratio. Heterozygous MACs produced by crossing, as in Figure 3, will generally have an ∼1:1 input ratio. In contrast, when MAC transformation is carried out, the initial transformant is likely to have an input ratio highly biased against the transforming allele, which is compensated for by introducing selection for the transformed allele.

Figure 6

Useful heterokaryons. (A) Drug resistance heterokaryon: often used to eliminate unmated parental cells (drug sensitive) and recover only true progeny (drug resistant) in a mass cross. (B) Heterokaryon for a lethal mutation: used to propagate a homozygous lethal mutation in the MIC, while “covered” by a wild-type MAC. The phenotype is expressed when two such cell lines are crossed to one another. (C) Nullisomic heterokaryons: the lethal MIC genotype is the absence of both copies of one MIC chromosome or chromosome arm (arrow); such heterokaryons are useful for genetically mapping mutations and DNA polymorphisms to chromosome locations. (D) Gene knockout: the lethal mutation is a deleterious or lethal gene KO, in which the coding sequence of the KO’s gene has been replaced by a cassette expressing drug resistance (to paromomycin, related to neomycin). The KO comes to expression in the progeny when such heterokaryons are crossed while the neo cassette allows elimination of wild-type parental and untransformed progeny cells.

Figure 7

Genetic approaches to generate whole-genome homozygotes from a heterozygote. Symbols are as in Figure 3. Asterisk denotes a mutation or allele of interest, such as a drug resistance gene. (A) Genomic exclusion. A cell containing a heterozygous MIC (represented by black and white) for a mutation in the MIC (asterisk) is crossed to a cell line with a defective MIC—called a “star” strain—which is incapable of contributing germline DNA to sexual progeny (round I). At meiosis, the remnant of its MIC disintegrates without cytological trace (stage b). The MIC of the nonstar mate undergoes normal meiosis, gametogenic mitosis, and gamete pronucleus transfer (stages c and d). The gamete pronuclei diploidize and the exconjugants separate without undergoing postzygotic nuclear differentiation (stage e). The two exconjugants are heterokaryons: their MICs are both whole-genome homozygotes derived from the single, diploidized meiotic product, and their MACs are those of the two parental cells that remain unchanged. The two types of whole-genome homozygotes are obtained in 1:1 ratio (only the white, mutated is shown). If two exconjugants from the same round 1 pair are crossed to one another (round II; stage f), whole-genome homozygous homokaryon progeny are obtained (stage g). (B) UPC. In this method, a cell heterozygous for a mutation is crossed to a star cell (stages a and b are identical to stages a and b in genomic exclusion), but gamete pronucleus transfer is blocked by a suitably timed hyperosmotic shock (between stages c and d). As a consequence, the two gamete pronuclei fuse to one another, generating a diploid, whole-genome homozygous zygote nucleus (stage d). Unlike round I of the genomic exclusion cross, postzygotic nuclear differentiation and old MAC destruction occur normally. The star mate dies for lack of a MIC and MAC (stage e). The resulting cell line has MICs and MACs that are whole-genome homozygous for the haploid genome of the surviving meiotic product at stage b (homokaryons). UPC is the method of choice to isolate mutants homozygous for recessive mutations after mutagenesis.