Foraminifera as a model of eukaryotic genome dynamism

Abstract

In contrast to the canonical view that genomes cycle only between haploid and diploid states, many eukaryotes have dynamic genomes that change content throughout an individual's life cycle. However, the few detailed studies of microeukaryotic life cycles render our understanding of eukaryotic genome dynamism incomplete. Foraminifera (Rhizaria) are an ecologically important, yet understudied, clade of microbial eukaryotes with complex life cycles that include changes in ploidy and genome organization. Here, we apply fluorescence microscopy and image analysis techniques to over 2,800 nuclei in 110 cells to characterize the life cycle of Allogromia laticollaris strain Cold Spring Harbor (CSH), one of few cultivable foraminifera species. We show that haploidy and diploidy are brief moments in the A. laticollaris life cycle and that A. laticollaris nuclei endoreplicate up to 12,000 times the haploid genome size. We find that A. laticollaris reorganizes a highly endoreplicated nucleus into thousands of haploid genomes through a non-canonical mechanism called Zerfall, in which the nuclear envelope degrades and extrudes chromatin into the cytoplasm. Based on these findings, along with changes in nuclear architecture across the life cycle, we believe that A. laticollaris uses spatio-temporal mechanisms to delineate germline and somatic DNA within a single nucleus. The analyses here extend our understanding of the genome dynamics across the eukaryotic tree of life.IMPORTANCEIn traditional depictions of eukaryotes (i.e., cells with nuclei), life cycles alternate only between haploid and diploid phases, overlooking studies of diverse microeukaryotic lineages (e.g., amoebae, ciliates, and flagellates) that show dramatic variation in DNA content throughout their life cycles. Endoreplication of genomes enables cells to grow to large sizes and perhaps to also respond to changes in their environments. Few microeukaryotic life cycles have been studied in detail, which limits our understanding of how eukaryotes regulate and transmit their DNA across generations. Here, we use microscopy to study the life cycle of Allogromia laticollaris strain CSH, an early-diverging lineage within the Foraminifera (an ancient clade of predominantly marine amoebae). We show that DNA content changes significantly throughout their life cycle and further describe an unusual process called Zerfall, by which this species reorganizes a large nucleus with up to 12,000 genome copies into hundreds of small gametic nuclei, each with a single haploid genome. Our results are consistent with the idea that all eukaryotes demarcate germline DNA to pass on to offspring amidst more flexible somatic DNA and extend the known diversity of eukaryotic life cycles.

Keywords: amoebae; endoreplication; eukaryotic life cycles; genome evolution; microbial eukaryote; nuclear architecture; polyploidy; protists.

Fig 1

Stacked bar plot of life stage durations separated by the type of reproductive cell emerged from and life cycle characterization based on light microscopy. The life cycle of A. laticollaris CSH begins with juveniles that emerge from parental test and grow into adults, which are defined by their size, dark pigmentation, and complex pseudopodia. Adult cells transition into pre-reproductive cells and in the process retract their pseudopods. Pre-reproductive cells develop into either Type 1 or Type 2 reproductive cells that then give rise to juveniles; the outline of a next-generation juvenile is shown as a dashed circle. Each tile represents one cell on a given day after emergence, with tiles colored by life stage. Cells are ranked by their total life duration on the y-axis. (A) Life stage durations of Type 1 emergers (inferred to be uninucleate; see text). (C) Life stage durations of Type 2 emergers (inferred to be multinucleate; see text). (B) Example cells at each life cycle stage. Scale bars are 100 µm. Type 1 reproductive cells give rise to fewer, larger juveniles while Type 2 cells give rise to a greater number of smaller juveniles.

Fig 2

Proposed life cycle for Allogromia laticollaris CSH: the Allogromia life cycle includes brief haploid and diploid stages, with all other stages containing endoreplicated genomes. Cells are represented by black circles containing images of Hoechst-stained DNA captured by a confocal laser scanning microscope and depicted here in white (see File S4 for complete images). Blue colors are Type 2 cells where the euploidy is 2N (i.e., diploid), while warmer colors are Type 1 cells where the euploidy is N (i.e., haploid). Beginning with the haploid stage, amoeboid gametes fuse within the parent test (i.e., autogamy) to form diploid zygotic nuclei. These zygotic nuclei are compartmentalized to generate multinucleate juveniles, which, upon release from the parent, grow into adults with uniform V1 nuclei (see Table 1). Meiosis occurs within multinucleate adults, with centralized chromosomes forming meiotic bouquets (MB) that are surrounded by a chromatin ring. Meiosis appears asynchronous among nuclei within a cell, since cells in meiosis have heterogeneous nuclear architecture (see Fig. 3). Following meiosis, the resulting “haploid” nuclei (still endoreplicated to ~26N [Table 1]) are partitioned into uninucleate individuals that emerge from the parent test and grow into adults. Large uninucleate adults reset their genomes through Zerfall (see Fig. S5), a process that includes the elimination of the nuclear envelope to release chromatin into the cytoplasm and then the generation of threads that resolve into multiple haploid genome complements (Table 1). These haploid genomes are eventually surrounded with nuclear envelope and membranes to generate the amoeboid gametes that are typical of this species. Not shown here are life cycle stages in which haploid and diploid adults produce offspring of the same ploidy through a process called schizogony.

Fig 5

Details of the three types of Hoechst-positive structures observed in Zerfall cells. In order, panels A–C show three cells that represent our inferred progression of genome reorganization during Zerfall. Panels D–F show details of the three distinct Hoechst-positive structures within these three cells. DNA is shown in grayscale, and images have been enhanced for brightness and contrast (raw fluorescence measurements in File S3 and original images in File S4). Scale bars in panels A–C are 25 µm, and those in panels D–F are 5 µm. (A) Cell filled with globular chromatin structures. (B) Cell filled with threadlike DNA structures. (C) Cell filled with small spheres, inferred to be haploid genome complements. (D–F) Detailed regions of panels A–C, respectively. See Fig. S5 for additional images of Zerfall cells.

Fig 3

Inferred transitions in nuclear architecture during meiosis in A. laticollaris CSH. All nuclei derive from multinucleate adult cells with heterogeneous nuclear architecture. Nuclei in panels A and B derive from the same cell. All scale bars are 5 µm; see Fig. S4 for detailed breakdown of nuclear architecture in all multinucleate adult cells imaged in the study. (A) V2 nucleus with a condensed chromatin ring around a DNA-poor center with central chromosomes. We infer that V1 nuclei amitotically divide to form V2 nuclei. (B) MB nucleus with a condensed chromatin ring around a DNA-poor center with a meiotic chromosome bouquet. We infer that MB nuclei represent a meiotic prophase that begins in V2 nuclei. The V2 nucleus in panel A and the MB nucleus in panel B derive from the same cell (“adult with meiotic nuclei; Fig. 2). (C) H nucleus (top) with condensed chromatin and no DNA-poor region. We infer that H nuclei are the products of meiosis based on their genome content, which is reduced compared with that of other nuclear types in multinucleate adult cells and similar to that of developing uninucleate juveniles (Table 1). The bottom structure is an MB nucleus within the same cell.

Fig 4

Inverse relationship between concentrations of DNA and newly transcribed RNA in A. laticollaris CSH nuclei. A. laticollaris CSH cells were stained with Hoechst 33342 (blue) to visualize DNA and an azide molecule bound to an AlexaFluor 488 probe (green) which binds to modified uracils to visualize all newly transcribed RNA. Scale bars in panels A–C are 5 µm, and scale bars in panels D–F are 7.5 µm. Images are enhanced to increase brightness and contrast; original images are in File S4. (A) Hoechst-stained nucleus in a uninucleate cell, which shows greater intensity at the nuclear periphery. (B) AlexaFluor-stained nucleus in a uninucleate adult cell, which shows greater intensity at the nuclear center. (C) Overlay image. (D) Hoechst-stained nuclei in a multinucleate adult cell, which show greater intensity at the nuclear periphery. (E) AlexaFluor-stained nuclei in multinucleate cells, which show greater intensity at the nuclear center; the bright spot of extranuclear signal in the top center of the image likely comes from autofluorescence of partially digested algal food, whereas dim extranuclear signal likely represents cytoplasmic RNAs. (F) Overlay image.

Fig 6

Total fluorescence and estimated DNA contents of Allogromia laticollaris CSH nuclei across life cycle stages (colored) show endoreplication and can be compared with non-foraminifera standards (gray scale). Warm-toned colors denote haploid stages (Type 1 cells), and cool-toned colors denote diploid stages (Type 2 cells). The maroon-colored data refer to fluorescent spheres in Zerfall cells. (A and B) Scatterplots comparing the total fluorescence from Hoechst per nucleus to the total nuclear volume in A. laticollaris CSH cells (A) and standards (yeast, human, and onion) (B). Each point represents a nucleus, and points are colored by inferred life stage. Both axes are shown on a log scale. (C) Boxplots comparing estimated DNA content across life stages in A. laticollaris CSH cells and standards. Each boxplot contains data from all the nuclei measured during a particular life stage. The y-axis (log scale) represents DNA content relative to the median estimate of the A. laticollaris CSH haploid genome size, i.e., 1C is the haploid genome size. See File S3 for fluorescence measurements.