Local assembly of long reads enables phylogenomics of transposable elements in a polyploid cell line

Animal cell lines cultured for extended periods often undergo extreme genome restructuring events, including polyploidy and segmental aneuploidy that can impede de novo whole-genome assembly (WGA). In Drosophila, many established cell lines also exhibit massive proliferation of transposable elements (TEs) relative to wild-type flies. To better understand the role of transposition during long-term animal somatic cell culture, we sequenced the genome of the tetraploid Drosophila S2R+ cell line using long-read and linked-read technologies. Relative to comparable data from inbred whole flies, WGAs for S2R+ were highly fragmented and generated variable estimates of TE content across sequencing and assembly technologies. We therefore developed a novel WGA-independent bioinformatics method called "TELR" that identifies, locally assembles, and estimates allele frequency of TEs from long-read sequence data (https://github.com/bergmanlab/telr). Application of TELR to a ~130x PacBio dataset for S2R+ revealed many haplotype-specific TE insertions that arose by somatic transposition in cell culture after initial cell line establishment and subsequent tetraploidization. Local assemblies from TELR also allowed phylogenetic analysis of paralogous TE copies within the S2R+ genome, which revealed that proliferation of different TE families during cell line evolution in vitro can be driven by single or multiple source lineages. Our work provides a model for the analysis of TEs in complex heterozygous or polyploid genomes that are not amenable to WGA and yields new insights into the mechanisms of genome evolution in animal cell culture.

Blocking cell fusion inhibits age-induced polyploidy and maintains epithelial organization in Drosophila

A characteristic of normal aging and age-related diseases is the remodeling of a tissue's cellular organization through polyploid cell growth. Polyploidy arises from an increase in nuclear ploidy or the number of nuclei per cell. However, it is not known whether age-induced polyploidy is an adaption to stressors or a precursor to degeneration. Here, we find that the adult fruit fly's abdominal epithelium becomes polyploid with age through generation of large multinucleated cells that make up more than 40% of the tissue area. The syncytia arise by cell fusion, not endomitosis. Epithelial multinucleation is also a characteristic of macular degeneration, including Ctnna1tvrm5, a mouse model for pattern dystrophy. Similarly, we find that the knockdown of alpha-catenin enhances multinucleation in the fly epithelium. We further show that age-induced polyploidy can be suppressed by inhibiting cell fusion revealing a means to maintain tissue organization in older animals.

Endomitosis controls tissue-specific gene expression during development

SummaryPolyploid cells contain more than two copies of the genome and are found in many plant and animal tissues. Different types of polyploidy exist, in which the genome is confined to either one nucleus (mononucleation) or two or more nuclei (multinucleation). Despite the widespread occurrence of polyploidy, the functional significance of different types of polyploidy are largely unknown. Here, we assess the function of multinucleation in C. elegans intestinal cells through specific inhibition of binucleation without altering genome ploidy. Through single worm RNA sequencing, we find that binucleation is important for tissuespecific gene expression, most prominently for genes that show a rapid upregulation at the transition from larval development to adulthood. Regulated genes include vitellogenins, which encode yolk proteins that facilitate nutrient transport to the germline. We find that reduced expression of vitellogenins in mononucleated intestinal cells leads to progeny with developmental delays and reduced fitness. Together, our results show that binucleation facilitates rapid upregulation of intestine-specific gene expression during development, independently of genome ploidy, underscoring the importance of spatial genome organization for polyploid cell function.

Wound-induced polyploidization is dependent on integrin-yki signaling

A key step in tissue repair is to replace lost or damaged cells. This occurs via two strategies: restoring cell number through proliferation or increasing cell size through polyploidization. Studies in Drosophila and vertebrates have demonstrated that polyploid cells arise in adult tissues, at least in part, to promote tissue repair and restore tissue mass. However, the signals that cause polyploid cells to form in response to injury remain poorly understood. In the adult Drosophila epithelium, wound-induced polyploid cells are generated by both cell fusion and endoreplication, resulting in a giant polyploid syncytium. Here, we identify the integrin focal adhesion complex as an activator of wound-induced polyploidization. Both integrin and focal adhesion kinase are upregulated in the wound-induced polyploid cells and are required for Yorkie induced endoreplication and cell fusion. As a result, wound healing is perturbed when focal adhesion genes are knocked down. These findings show that conserved focal adhesion signaling is required to initiate wound-induced polyploid cell growth.

Single nuclei RNA-seq of mouse placental labyrinth development

The placenta is the interface between mother and fetus in all eutherian species. However, our understanding of this essential organ remains incomplete. A substantial challenge has been the syncytial cells of the placenta, which have made dissociation and independent evaluation of the different cell types of this organ difficult. Here, we address questions concerning the ontogeny, specification, and function of the cell types of a representative hemochorial placenta by performing single nuclei RNA sequencing (snRNA-seq) at multiple stages of mouse embryonic development focusing on the exchange interface, the labyrinth. Timepoints extended from progenitor-driven expansion through terminal differentiation. Analysis by snRNA-seq identified transcript profiles and inferred functions, cell trajectories, signaling interactions, and transcriptional drivers of all but the most highly polyploid cell types of the placenta. These data profile placental development at an unprecedented resolution, provide insights into differentiation and function across time, and provide a resource for future study.

Features of regenerative processes in case of extreme factors influencing the body

Changes in the specific liver mass, number of single and binucleate cells of different ploidy, mitotic index and alteration index, DNA content and inclusion of 3H-thymidine in DNA of Wistar rats and CBA mice in the following conditions: at 0°C for 3, 7, 14 days for 23 hours per day, and in the highlands conditions (3, 200 m above sea level) for 3 and 30 days. Activation of regenerative processes has been established — proliferation of hepatocytes (mainly diploid) in combination with polyploid cell share reduction, as well as cellular hypertrophy development. Cell dynamics shifts persisted throughout the observation duration and were opposite in direction when growing up and aging.

Wound-induced polyploidization is driven by Myc and supports tissue repair in the presence of DNA damage

Tissue repair requires either polyploid cell growth or cell division, but the molecular mechanism promoting polyploidy and limiting proliferation remains poorly understood. Here we find that injury to the adult Drosophila epithelium causes cells to enter the endocycle through the activation of Yorkie dependent genes (myc, e2f1, or cycE). Myc is even sufficient to induce the endocycle in the post-mitotic epithelium. As result, epithelial cells enter S phase but mitosis is blocked by inhibition of mitotic gene expression. The mitotic cell cycle program can be activated by simultaneously expressing the mitotic activator, Stg, while genetically depleting fzr. However, forcing cells to undergo mitosis is detrimental to wound repair as the adult fly epithelium accumulates DNA damage and mitotic errors ensue when cells are forced to proliferate. In conclusion, we find that wound-induced polyploidization enables tissue repair when cell division is not a viable option.

Polyploid monolayer Ishikawa endometrial cells form unicellular hollow spheroids capable of migration

The results in this paper demonstrate that Ishikawa endometrial monolayer cells become multinucleated by a process of nuclear “donation” from neighboring cells. As the resulting polyploid cell detaches from the colony in which it was formed, it is possible to detect mitonucleon(s) in the center of the cell. The mitonucleon is a transient mitochondrial superstructure surrounding aggregated chromatin (Fleming et al. 1998) with characteristics of the family of mitochondrial superstructures that are sometimes called spheroids or cup-shaped mitochondria (Fleming, 2016a). As was recently demonstrated gas vacuoles form within mitonucleons (Fleming, 2018). In the free-floating single cell, the retained gas creates a central vacuole, and the cell becomes a spheroid that floats above the monolayer. It resembles a “signet ring cell” in being characterized by a central vacuole and chromatin compressed against the vacuole membrane. The resulting structure is a spheroids that is hollow and unicellular, albeit polyploid. But whereas signet ring cells are assumed to be undergoing apoptosis, that is not the case for unicellular spheroids. Complete spheres with chromatin and cytosolic cell contents compressed against the cell membrane can be found floating independently above Ishikawa monolayers. When an isolated sphere settles back onto the surface of the petri dish, it is possible to observe dissipating gas bubbles within the now flattened sphere for a short period of time. When the gas is discharged the resulting cell looks like a typical giant polyploid cell.

Polyploid monolayer Ishikawa endometrial cells form unicellular hollow spheroids capable of migration

The results in this paper demonstrate that Ishikawa endometrial monolayer cells become multinucleated by a process of nuclear “donation” from neighboring cells. As the resulting polyploid cell detaches from the colony in which it was formed, it is possible to detect mitonucleon(s) in the center of the cell. The mitonucleon is a transient mitochondrial superstructure surrounding aggregated chromatin (Fleming et al. 1998) with characteristics of the family of mitochondrial superstructures that are sometimes called spheroids or cup-shaped mitochondria (Fleming, 2016a). As was recently demonstrated gas vacuoles form within mitonucleons (Fleming, 2018). In the free-floating single cell, the retained gas creates a central vacuole, and the cell becomes a spheroid that floats above the monolayer. It resembles a “signet ring cell” in being characterized by a central vacuole and chromatin compressed against the vacuole membrane. The resulting structure is a spheroids that is hollow and unicellular, albeit polyploid. But whereas signet ring cells are assumed to be undergoing apoptosis, that is not the case for unicellular spheroids. Complete spheres with chromatin and cytosolic cell contents compressed against the cell membrane can be found floating independently above Ishikawa monolayers. When an isolated sphere settles back onto the surface of the petri dish, it is possible to observe dissipating gas bubbles within the now flattened sphere for a short period of time. When the gas is discharged the resulting cell looks like a typical giant polyploid cell.