Genome content reorganization in the non-model ciliate Chilodonella uncinata: insights into nuclear architecture, DNA content, and chromosome fragmentation during macronuclear development

Abstract

Ciliates are a model lineage for studies of genome architecture given their unusual genome structures. All ciliates have both somatic macronuclei (MAC) and germline micronuclei (MIC), both of which develop from a zygotic nucleus following sex (i.e., conjugation). Nuclear developmental stages are not well documented among non-model ciliates, including Chilodonella uncinata (class Phyllopharyngea), the focus of our work. Here, we characterize nuclear architecture and genome dynamics in C. uncinata by combining 4',6-diamidino-2-phenylindole (DAPI) staining and fluorescence in situ hybridization (FISH) techniques with confocal microscopy. We developed a telomere probe for staining, which alongside DAPI allows for the identification of fragmented somatic chromosomes among the total DNA in the nuclei. We quantify both total DNA and telomere-bound signals from more than 250 nuclei sampled from 116 individual cells, and analyze changes in DNA content and nuclear architecture across Chilodonella's nuclear life cycle. Specifically, we find that MAC developmental stages in the ciliate C. uncinata are different from those reported from other ciliate species. These data provide insights into nuclear dynamics during development and enrich our understanding of genome evolution in non-model ciliates.

Importance: Ciliates are a clade of diverse single-celled eukaryotic microorganisms that contain at least one somatic macronucleus (MAC) and germline micronucleus (MIC) within each cell/organism. Ciliates rely on complex genome rearrangements to generate somatic genomes from a zygotic nucleus. However, the development of somatic nuclei has only been documented for a few model ciliate genera, including Paramecium, Tetrahymena, and Oxytricha. Here, we study the MAC developmental process in the non-model ciliate, C. uncinata. We analyze both total DNA and the generation of gene-sized somatic chromosomes using a laser scanning confocal microscope to describe C. uncinata's nuclear life cycle. We show that DNA content changes dramatically during their life cycle and in a manner that differs from previous studies on model ciliates. Our study expands knowledge of genome dynamics in ciliates and among eukaryotes more broadly.

Keywords: DAPI; DNA content; FISH; chromosomes; ciliate; confocal imaging; genes; genome dynamics; macronuclei; nuclear cycle; single-celled microorganisms; telomere.

Fig 1

Representative images from each categorized developmental stage, DAPI-stained (blue) and Telomere-stained (green) nuclei, and overlay for each of C. uncinata cell. (I) Representative cells and nuclei from vegetative stage (a–f). (II) All the exceptional forms of nuclei that were captured during the study but not categorized. (III) Representative conjugating cells and their nuclei ( a–r). (IV) Representative developing cells and their nuclei; panels a–c, g–i, and m–o represent the early developing stage; panels d–f, j–l, and p–r represent the late developing stage. The rows (I) e and f, (II) i–l, (III) m–r, and (IV) m–r represent cells that contain both the nuclei within the same individual. We adjusted the brightness and contrast of these overlay images to enhance visualization of all overall cells. All raw images for this study are accessible as described in our Data Availability statement. Orange arrowheads indicate the MICs, and white arrowheads indicate the developing MACs. MICs, micronuclei; MACs, macronuclei. Scale bar = 5 µm.

Fig 2

Different stages and total intensity during the macronuclear development in C. uncinata of all categorized cells. The x-axis represents the different stages during the macronuclear development, and the y-axis (in log scale) represents the total intensity (i.e. DAPI or telomere probe). Cells are categorized by “Developing“ (including early and late developing stages), “Vegetative,” and “Conjugating“ stages from left to right on the x-axis. Solid blue circles are the newly developing MACs DNA data, and solid green circles are the newly developing MACs telomere data, respectively. Open blue circles are the old MACs DNA data, while open green circles are the old MACs telomere data, respectively, during early and late development. Solid orange circles indicate the MICs data on the plot. MICs, micronuclei; MACs, macronuclei.

Fig 3

Inferred life cycle of Chilodonella uncinata illustrated by cartoons based on images from Fig. 1 and Fig. S1 to S8. (a) MAC amitosis (asexual division). (b) Vegetative cells and nuclei. (c) Meiosis-I. (d–f) Conjugation, meiosis-II of MICs, and exchange of haploid MICs. (g and h) Zygotic nuclei and mitotic division of zygotic nuclei. (i and j) Early development of the new MAC occurs as the parental MAC shrinks (Fig. 1), yet DNA content remains relatively stable in the parental MAC at this stage (Fig. 2). (k–m) Late development of the new MACs occurs as the parental MAC disappears. MICs, germline micronuclei; MACs, somatic macronuclei.