Functional significance and remodeling challenges of polyploidy across the lifetime of epithelial tissues

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Animal cells have characteristic structures that support their biological roles. For example, epithelial cells adhere tightly to their neighbors to form sheets of tissue, a structure that supports barrier functions such as with the skin as an outer protective layer, or the lining of the gut. One aspect of cell structure is the nucleus and its size. While most cells are diploid, with a copy of the genome from each parent, certain cells become polyploid, with many copies of the genome within a single nucleus. This leads to large nuclei and proportionately large cells. What are the benefits and costs of polyploidy?

Polyploid cells occur in the liver, muscles, and maternal and extra-embryonic tissues, such as those that support placental development at the mother-fetus interface. Benefits include barrier tissue maintenance and repair after wounding. Wound repair involves polyploid cells becoming even larger to help cover the wound site. Also, having many copies of the genome supports rapid, efficient production of needed proteins, such as from placental genes that promote maternal milk production. However, there is not always a clear correlation between DNA copy number and actual protein output, and under experimental conditions some tissues seem to also function without being polyploid. Thus, investigation of additional tissue types is needed to better understand the hypothesized benefits of polyploidy.

On the other hand, polyploidy also poses challenges to cells. In some cases, the entire cell's genome is not copied, but only DNA regions containing certain genes. This is an efficient way to support rapid production when only the specific proteins from those genes are needed. Yet, this partial increase in DNA copies is an unstable arrangement that usually triggers programmed cell death, known as apoptosis, as a form of biological quality control. Polyploid cells therefore require special genetic properties to suppress this. One key research system where this has been explored is the fruit fly Drosophila melanogaster, a powerful genetics model species. However, we discovered that critical genes for controlling programmed cell death, the so-called Grim Reaper genes, are a unique feature in flies. It remains unknown which genes serve this function in other animals. Also, one hallmark of cancer cells is that they have unstable DNA arrangements yet somehow evade programmed cell death. Research in new species on how polyploid cells handle the programmed cell death issue will identify critical genes and could help improve our understanding of genetics relevant to cancer treatment.

In this research project, we will test the hypothesized benefits (barrier tissue structure, rapid protein production) and examine the potential risks (unstable DNA arrangement, need to block cell death) of polyploidy in a novel biological system: the two extra-embryonic tissues of the flour beetle Tribolium castaneum. These tissues serve epithelial barrier functions by surrounding and protecting the embryo. The serosa surrounds the embryo and yolk while the amnion, like its namesake in mammals, forms a fluid-filled, inner cavity. The serosa also has a rapid production role to make a cuticle layer that reinforces the eggshell. Notably, these tissues are stable and polyploid for most of their lifetime, and then they actively uncover the embryo and precisely undergo cell death. We have developed methods to investigate polyploidy in the beetle, including genome sequencing and live imaging microscopy that reveals healthy and experimentally perturbed barrier tissue structure. The serosa and amnion offer an excellent comparative system. Our preliminary work suggests that the serosa is capable of increased polyploidy to compensate in genetic models of wound-like tissue impairment while the amnion is not. This paves the way to better understanding of tissue-specific features of polyploidy and to uncover new critical genes involved in polyploidy control.

Technical Summary

Polyploid cells have multiple copies of the genome, resulting in large cells with large nuclei, such as in tissues that support embryogenesis in mammals and insects. Polyploidy is hypothesized to aid epithelial barrier formation and its repair after wounding and to rapidly supply gene products by transcription from multiple gene copies. Yet polyploid genomic structure, with potentially incomplete replication, can pose challenges for cells' ability to undergo apoptosis. Investigating the costs and benefits of polyploidy is essential to understand tissue-specific development, homeostasis, and ageing.

An excellent model of polyploidy is the extraembryonic (EE) epithelia of insects. The serosa encloses the embryo and yolk and produces a protective cuticle. The amnion, analogous to its mammalian namesake, forms a fluid-filled inner cavity. Ultimately, both tissues reverse their barrier configuration and undergo apoptosis. In the beetle Tribolium castaneum, we have developed methods to assess nuclear size and tissue integrity by fluorescent live cell imaging, quantify gene expression throughout development (RT-qPCR, RNA-seq, FACS), and genetically challenge EE barrier organization and cell number (RNAi). Here, we will target our recently identified EE polyploidy factors and molecular readouts: fate map and tissue remodeling factors, polyploidy regulators, and cuticle markers. Importantly, our pilot work suggests that the EE tissues differ in their capacity for compensatory ploidy after genetic challenge.

This project will 1) characterize the developmental progression of polyploidy relative to barrier formation and rapid transcription, 2) test polyploidy function across the tissues' lifetimes, and 3) link tissue-specific genomic and transcriptomic features to apoptosis competence. Altogether, we will test long-standing hypotheses on polyploidy function and its end-stage implications in animal tissues.

Planned Impact

The non-academic beneficiaries of the proposed research are expected to include:

THE APPLIED BIOMEDICAL, QUANTITATIVE IMAGING, AND INVERTEBRATE RESEARCH COMMUNITIES
This project investigates genetic, cell, developmental, and lifecourse physiological aspects of tissue structure in relation to function in healthy and genetically challenged conditions. As noted in the Academic Beneficiaries section, this supports research spanning the development and ageing research communities, with the potential to generate new fundamental knowledge of relevance for cancer cell biology. In long-term application, genetic principles arising from our results could offer novel gene targets for personalized medicine approaches to maintaining genome stability and homeostasis in ageing cells and to targeting cancer cells that would otherwise evade apoptosis. We will also generate datasets and analysis computational pipelines for the integration of quantitative bioimaging data, which could ultimately provide big data training datasets for machine learning approaches to diagnosis of healthy and damaged epithelial cell parameters. Our sequencing and transgenic resources will provide tools and specific genetic targets for the insect and invertebrate research communities, including those dedicated to precision pest management strategies for disease vectors and agricultural pests.

FUNDAMENTAL CONTRIBUTION TOWARDS TISSUE ENGINEERING FOR REGENERATIVE MEDICINE
The proposed project has the potential to lay a foundation for improved design principles in the field of synthetic tissue engineering. Epithelial tissues form essential barriers, such as the skin or the lining of the gut, and the integrity of these barriers is required for healthy tissue homeostasis. Integrity can be lost due to wound damage or disease states that compromise the structure of individual cells or their ability to maintain cell-cell physical contact within the tissue sheet. Understanding the contribution of polyploidy to barrier tissue integrity will elucidate suites of linked cellular properties (relative and absolute cell size, rates of remodeling in dynamic or impaired/ repairing tissues). This quantitative understanding will contribute to the design of synthetic tissues with appropriate physical parameters for tissue engineering applications such as regenerative medicine. For example, tissues with the capacity for self-repair, modeled on compensatory growth properties of polyploid epithelial cells, could offer new therapeutic approaches to limit re-grafting and scarring after tissue damage such as from burns or abrasions.

THE PUBLIC, PARTICIPATING RESEARCH STAFF, AND FUTURE SCIENTISTS
This project addresses a critical, accessible issue: which physical features (polyploidy, large nuclear size) support the function of protective tissue layers? Communicating the results of this project will engage the public in understanding a key biomedical issue on how healthy tissue function relates to tissue structure, and how impaired tissue morphogenesis can result in birth defects. Our live imaging movie data offers a visually compelling entry point to promote education and discussion on these topics. Furthermore, the project integrates a diverse range of experimental and computational research approaches. This will provide a wide array of training with advanced research techniques for bioimaging and next-generation sequence analysis to the PDRA and SRT, with international research travel integrated into the training of the PDRA. Opportunities for student research in our lab under these auspices will also extend communication about the research topic and its approaches in hands-on, practical fashion.