Broken chromosomes heading into mitosis: More than one way to patch a flat tire.

A cell dealing with a broken chromosome in mitosis is like a driver dealing with a flat tire on the highway: damage repair must occur under non-ideal circumstances. Mitotic chromosome breaks encounter problems related to structures called micronuclei. These aberrant nuclei are linked to cell death, mutagenesis, and cancer. In the last few years, a flurry of studies illuminated two mechanisms that prevent mitotic problems related to micronuclei. One mechanism prevents micronuclei from forming during mitosis and involves DNA Polymerase Theta, a DNA repair regulator that patches up broken mitotic chromosomes. A second mechanism is activated after micronuclei form and then rupture, and involves CIP2A and TOPBP1 proteins, which patch micronuclear fragments to promote their subsequent mitotic segregation. Here, we review recent progress in this field of mitotic DNA damage and discuss why multiple mechanisms exist. Future studies in this exciting area will reveal new DNA break responses and inform therapeutic strategies.

Conserved chamber-specific polyploidy maintains heart function in Drosophila.

Developmentally programmed polyploidy (whole-genome duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. Here, in both Drosophila larvae and human organ donors, we reveal distinct polyploidy levels in cardiac organ chambers. In Drosophila, differential growth and cell cycle signal sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume and cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic human cardiomyopathies. Our results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest that precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery.

Conserved Chamber-Specific Polyploidy Maintains Heart Function in Drosophila.

Developmentally programmed polyploidy (whole-genome-duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. Here, we reveal roles for precise polyploidy levels in cardiac tissue. We highlight a conserved asymmetry in polyploidy level between cardiac chambers in Drosophila larvae and humans. In Drosophila , differential Insulin Receptor (InR) sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume, cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic systemic human heart failure. Using human donor hearts, we reveal asymmetry in nuclear volume (ploidy) and insulin signaling between the left ventricle and atrium. Our results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery.

Measuring Cellular Ploidy In Situ by Light Microscopy.

Determining cellular DNA content is valuable in the study of numerous biological processes, including organ development and injury repair. While FACS analysis of dissociated cells is a widely used method for assaying ploidy in a tissue cell population, for many tissue samples, it is possible and convenient to measure ploidy in situ using light microscopy. Here, we present two protocols for measuring cellular ploidy in tissues. These protocols are based on our studies in Drosophila melanogaster, but these are applicable to other settings as well. We present example results from Drosophila hindgut, midgut, and wing imaginal disc as examples. The first protocol focuses on measuring DNA content from decondensed interphase nuclei, while the second protocol details the visualization of condensed chromosomes for ploidy determination, either from mitotic cells or from interphase cells with drug-induced chromosome condensation. These techniques can be completed in 1 day and require standard lab supplies as well as a fluorescence light microscope.

Looking on the horizon; potential and unique approaches to developing radiation countermeasures for deep space travel.

Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

DNA Damage Responses during the Cell Cycle: Insights from Model Organisms and Beyond.

Genome damage is a threat to all organisms. To respond to such damage, DNA damage responses (DDRs) lead to cell cycle arrest, DNA repair, and cell death. Many DDR components are highly conserved, whereas others have adapted to specific organismal needs. Immense progress in this field has been driven by model genetic organism research. This review has two main purposes. First, we provide a survey of model organism-based efforts to study DDRs. Second, we highlight how model organism study has contributed to understanding how specific DDRs are influenced by cell cycle stage. We also look forward, with a discussion of how future study can be expanded beyond typical model genetic organisms to further illuminate how the genome is protected.

Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation.

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

Communal living: the role of polyploidy and syncytia in tissue biology.

Multicellular organisms are composed of tissues with diverse cell sizes. Whether a tissue primarily consists of numerous, small cells as opposed to fewer, large cells can impact tissue development and function. The addition of nuclear genome copies within a common cytoplasm is a recurring strategy to manipulate cellular size within a tissue. Cells with more than two genomes can exist transiently, such as in developing germlines or embryos, or can be part of mature somatic tissues. Such nuclear collectives span multiple levels of organization, from mononuclear or binuclear polyploid cells to highly multinucleate structures known as syncytia. Here, we review the diversity of polyploid and syncytial tissues found throughout nature. We summarize current literature concerning tissue construction through syncytia and/or polyploidy and speculate why one or both strategies are advantageous.

Polyploidy: A Biological Force From Cells to Ecosystems.

Polyploidy, resulting from the duplication of the entire genome of an organism or cell, greatly affects genes and genomes, cells and tissues, organisms, and even entire ecosystems. Despite the wide-reaching importance of polyploidy, communication across disciplinary boundaries to identify common themes at different scales has been almost nonexistent. However, a critical need remains to understand commonalities that derive from shared polyploid cellular processes across organismal diversity, levels of biological organization, and fields of inquiry - from biodiversity and biocomplexity to medicine and agriculture. Here, we review the current understanding of polyploidy at the organismal and suborganismal levels, identify shared research themes and elements, and propose new directions to integrate research on polyploidy toward confronting interdisciplinary grand challenges of the 21st century.

Physiology, Development, and Disease Modeling in the Drosophila Excretory System.

The insect excretory system contains two organ systems acting in concert: the Malpighian tubules and the hindgut perform essential roles in excretion and ionic and osmotic homeostasis. For over 350 years, these two organs have fascinated biologists as a model of organ structure and function. As part of a recent surge in interest, research on the Malpighian tubules and hindgut of Drosophila have uncovered important paradigms of organ physiology and development. Further, many human disease processes can be modeled in these organs. Here, focusing on discoveries in the past 10 years, we provide an overview of the anatomy and physiology of the Drosophila excretory system. We describe the major developmental events that build these organs during embryogenesis, remodel them during metamorphosis, and repair them following injury. Finally, we highlight the use of the Malpighian tubules and hindgut as accessible models of human disease biology. The Malpighian tubule is a particularly excellent model to study rapid fluid transport, neuroendocrine control of renal function, and modeling of numerous human renal conditions such as kidney stones, while the hindgut provides an outstanding model for processes such as the role of cell chirality in development, nonstem cell-based injury repair, cancer-promoting processes, and communication between the intestine and nervous system.

Polyploidy and Mitotic Cell Death Are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells.

Prior studies have found that HIV, through the Vpr protein, promotes genome reduplication (polyploidy) in infection-surviving epithelial cells within renal tissue. However, the temporal progression and molecular regulation through which Vpr promotes polyploidy have remained unclear. Here we define a sequential progression to Vpr-mediated polyploidy in human renal tubule epithelial cells (RTECs). We found that as in many cell types, Vpr first initiates G2 cell cycle arrest in RTECs. We then identified a previously unreported cascade of Vpr-dependent events that lead to renal cell survival and polyploidy. Specifically, we found that a fraction of G2-arrested RTECs reenter the cell cycle. Following this cell cycle reentry, two distinct outcomes occur. Cells that enter complete mitosis undergo mitotic cell death due to extra centrosomes and aberrant division. Conversely, cells that abort mitosis undergo endoreplication to become polyploid. We further show that multiple small-molecule inhibitors of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including those that target ATR, ATM, and mTOR, indirectly prevent Vpr-mediated polyploidy by preventing G2 arrest. In contrast, an inhibitor that targets DNA-dependent protein kinase (DNA-PK) specifically blocks the Vpr-mediated transition from G2 arrest to polyploidy. These findings outline a temporal, molecularly regulated path to polyploidy in HIV-positive renal cells.IMPORTANCE Current cure-focused efforts in HIV research aim to elucidate the mechanisms of long-term persistence of HIV in compartments. The kidney is recognized as one such compartment, since viral DNA and mRNA persist in the renal tissues of HIV-positive patients. Further, renal disease is a long-term comorbidity in the setting of HIV. Thus, understanding the regulation and impact of HIV infection on renal cell biology will provide important insights into this unique HIV compartment. Our work identifies mechanisms that distinguish between HIV-positive cell survival and death in a known HIV compartment, as well as pharmacological agents that alter these outcomes.

Interorgan regulation of Drosophila intestinal stem cell proliferation by a hybrid organ boundary zone.

The molecular identities and regulation of cells at interorgan boundaries are often unclear, despite the increasingly appreciated role of organ boundaries in disease. Using Drosophila as a model, we here show that a specific population of adult midgut organ-boundary intestinal stem cells (OB-ISCs) is regulated by the neighboring hindgut, a developmentally distinct organ. This distinct OB-ISC control occurs through proximity to a specialized transition zone between the endodermal midgut and ectodermal hindgut that shares molecular signatures of both organs, which we term the hybrid zone (HZ). During homeostasis, proximity to the HZ restrains OB-ISC proliferation. However, injury to the adult HZ/hindgut drives upregulation of unpaired-3 cytokine, which signals through a Signal transducer and activator of transcription (STAT) protein to promote cell division only in OB-ISCs. If HZ disruption is severe, hyperplastic OB-ISCs expand across the interorgan boundary. Our data suggest that interorgan signaling plays an important role in controlling OB-ISCs in homeostasis and injury repair, which is likely to be crucial in prevention of disease.

Polyteny: still a giant player in chromosome research.

In this era of high-resolution mapping of chromosome territories, topological interactions, and chromatin states, it is increasingly appreciated that the positioning of chromosomes and their interactions within the nucleus is critical for cellular function. Due to their large size and distinctive structure, polytene chromosomes have contributed a wealth of knowledge regarding chromosome regulation. In this review, we discuss the diversity of polytene chromosomes in nature and in disease, examine the recurring structural features of polytene chromosomes in terms of what they reveal about chromosome biology, and discuss recent advances regarding how polytene chromosomes are assembled and disassembled. After over 130 years of study, these giant chromosomes are still powerful tools to understand chromosome biology.

Proliferation of Double-Strand Break-Resistant Polyploid Cells Requires Drosophila FANCD2.

Conserved DNA-damage responses (DDRs) sense genome damage and prevent mitosis of broken chromosomes. How cells lacking DDRs cope with broken chromosomes during mitosis is poorly understood. DDRs are frequently inactivated in cells with extra genomes (polyploidy), suggesting that study of polyploidy can reveal how cells with impaired DDRs/genome damage continue dividing. Here, we show that continued division and normal organ development occurs in polyploid, DDR-impaired Drosophila papillar cells. As papillar cells become polyploid, they naturally accumulate broken acentric chromosomes but do not apoptose/arrest the cell cycle. To survive mitosis with acentric chromosomes, papillar cells require Fanconi anemia proteins FANCD2 and FANCI, as well as Blm helicase, but not canonical DDR signaling. FANCD2 acts independently of previous S phases to promote alignment and segregation of acentric DNA produced by double-strand breaks, thus avoiding micronuclei and organ malformation. Because polyploidy and impaired DDRs can promote cancer, our findings provide insight into disease-relevant DNA-damage tolerance mechanisms.

Distinct responses to reduplicated chromosomes require distinct Mad2 responses.

Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without separation frequently produces polytene chromosomes, a barrier to accurate mitosis. Chromosome reduplication occurs in many contexts, including: polytene tissue development, polytene tumors, and following treatment with mitosis-blocking chemotherapeutics. However, mechanisms responding to or resolving polyteny during mitosis are poorly understood. Here, using Drosophila, we uncover two distinct reduplicated chromosome responses. First, when reduplicated polytene chromosomes persist into metaphase, an anaphase delay prevents tissue malformation and apoptosis. Second, reduplicated polytene chromosomes can also separate prior to metaphase through a spindle-independent mechanism termed Separation-Into-Recent-Sisters (SIRS). Both reduplication responses require the spindle assembly checkpoint protein Mad2. While Mad2 delays anaphase separation of metaphase polytene chromosomes, Mad2's control of overall mitotic timing ensures efficient SIRS. Our results pinpoint mechanisms enabling continued proliferation after genome reduplication, a finding with implications for cancer progression and prevention.

The expanding implications of polyploidy.

Polyploid cells, which contain more than two genome copies, occur throughout nature. Beyond well-established roles in increasing cell size/metabolic output, polyploidy can also promote nonuniform genome, transcriptome, and metabolome alterations. Polyploidy also frequently confers resistance to environmental stresses not tolerated by diploid cells. Recent progress has begun to unravel how this fascinating phenomenon contributes to normal physiology and disease.

Indispensable pre-mitotic endocycles promote aneuploidy in the Drosophila rectum.

The endocycle is a modified cell cycle that lacks M phase. Endocycles are well known for enabling continued growth of post-mitotic tissues. By contrast, we discovered pre-mitotic endocycles in precursors of Drosophila rectal papillae (papillar cells). Unlike all known proliferative Drosophila adult precursors, papillar cells endocycle before dividing. Furthermore, unlike diploid mitotic divisions, these polyploid papillar divisions are frequently error prone, suggesting papillar structures may accumulate long-term aneuploidy. Here, we demonstrate an indispensable requirement for pre-mitotic endocycles during papillar development and also demonstrate that such cycles seed papillar aneuploidy. We find blocking pre-mitotic endocycles disrupts papillar morphogenesis and causes organismal lethality under high-salt dietary stress. We further show that pre-mitotic endocycles differ from post-mitotic endocycles, as we find only the M-phase-capable polyploid cells of the papillae and female germline can retain centrioles. In papillae, this centriole retention contributes to aneuploidy, as centrioles amplify during papillar endocycles, causing multipolar anaphase. Such aneuploidy is well tolerated in papillae, as it does not significantly impair cell viability, organ formation or organ function. Together, our results demonstrate that pre-mitotic endocycles can enable specific organ construction and are a mechanism that promotes highly tolerated aneuploidy.

Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium.

BACKGROUND: Reestablishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how postmitotic diploid cells contribute to repair. RESULTS: Puncture wounds to the adult Drosophila epidermis close initially by forming a melanized scab. We found that epithelial cells near the wound site fuse to form a giant syncytium, which sends lamellae under the scab to re-epithelialize the damaged site. Other large cells arise more peripherally by initiating endocycles and becoming polyploid, or by cell fusion. Rac GTPase activity is needed for syncytium formation, while the Hippo signaling effector Yorkie modulates both polyploidization and cell fusion. Large cell formation is functionally important because when both polyploidization and fusion are blocked, wounds do not re-epithelialize. CONCLUSIONS: Our observations indicate that cell mass lost upon wounding can be replaced by polyploidization instead of mitotic proliferation. We propose that large cells generated by polyploidization or cell fusion are essential because they are better able than diploid cells to mechanically stabilize wounds, especially those containing permanent acellular structures, such as scar tissue.

Endoreplication and polyploidy: insights into development and disease.

Polyploid cells have genomes that contain multiples of the typical diploid chromosome number and are found in many different organisms. Studies in a variety of animal and plant developmental systems have revealed evolutionarily conserved mechanisms that control the generation of polyploidy and have recently begun to provide clues to its physiological function. These studies demonstrate that cellular polyploidy plays important roles during normal development and also contributes to human disease, particularly cancer.

Error-prone polyploid mitosis during normal Drosophila development.

Endopolyploidy arises during normal development in many species when cells undergo endocycles-variant cell cycles in which DNA replicates but daughter cells do not form. Normally, polyploid cells do not divide mitotically after initiating endocycles; hence, little is known about their mitotic competence. However, polyploid cells are found in many tumors, and the enhanced chromosomal instability of polyploid cells in culture suggests that such cells contribute to tumor aneuploidy. Here, we describe a novel polyploid Drosophila cell type that undergoes normal mitotic cycles as part of a remodeling process that forms the adult rectal papillae. Similar polyploid mitotic divisions, but not depolyploidizing divisions, were observed during adult ileum development in the mosquito Culex pipiens. Extended anaphases, chromosome bridges, and lagging chromosomes were frequent during these polyploid divisions, despite normal expression of cell cycle regulators. Our results show that the switch to endocycles during development is not irreversible, but argue that the polyploid mitotic cycle is inherently error-prone, and that polyploid mitoses may help destabilize the cancer genome.