Using Mosaic Cell Labeling to Visualize Polyploid Cells in the Drosophila Brain
Abstract
Traditional methods used to study endoreplication have limitations when used to identify rare events of polyploidization in complex, densely-packed tissues. Here, we describe a method to identify and visualize polyploid cells in situ using an existing mosaic, multicolor labeling technique named "CoinFLP" (Bosch et al., Development 142(3):597-606, 2015). CoinFLP allows easy visualization of polyploid cells in situ and can be combined with other techniques such as immunofluorescence for cell-type-specific labeling and flow cytometry to perform quantifications and can also be used for genetic manipulations. Further, by modifying the time of labeling, this technique can also be used to distinguish events of cell fusion from endocycle (Nandakumar et al., eLife 25:9, 2020)-allowing one to infer the method of polyploidization.
... were observed via confocal microscopy (Nandakumar & Buttitta, 2023). ...
The Drosophila melanogaster male accessory gland is a functional analog of the mammalian prostate and seminal vesicles containing two secretory epithelial cell types, termed main and secondary cells. This tissue is responsible for making and secreting seminal fluid proteins and other molecules that contribute to successful reproduction. The cells of this tissue are bi-nucleate and polyploid, due to variant cell cycles that include endomitosis and endocycling during metamorphosis. Here we provide evidence of additional cell cycle variants in this tissue. We show that main cells of the gland are connected by ring canals that form after the penultimate mitosis and we describe an additional post-eclosion endocycle required for gland maturation that is dependent on juvenile hormone signaling. We present evidence that the main cells of the Drosophila melanogaster accessory gland undergo a unique cell cycle reprogramming throughout organ development that results in step-wise cell cycle truncations culminating in cells containing two octoploid nuclei with under-replicated heterochromatin in the mature gland. We propose this tissue as a model to study developmental and hormonal temporal control of cell cycle variants in terminally differentiating tissues.
Various insect species serve as valuable model systems for investigating the cellular and molecular mechanisms by which a brain controls sophisticated behaviors. In particular, the nervous system of Drosophila melanogaster has been extensively studied, yet experiments aimed at determining the number of neurons in the Drosophila brain are surprisingly lacking. Using isotropic fractionator coupled with immunohistochemistry, we counted the total number of neuronal and non-neuronal cells in the whole brain, central brain, and optic lobe of Drosophila melanogaster . For comparison, we also counted neuronal populations in three divergent mosquito species: Aedes aegypti , Anopheles coluzzii and Culex quinquefasciatus . The average number of neurons in a whole adult brain was determined to be 199,380 ±3,400 cells in D . melanogaster , 217,910 ±6,180 cells in Ae . aegypti , 223,020 ± 4,650 cells in An . coluzzii and 225,911±7,220 cells in C . quinquefasciatus . The mean neuronal cell count in the central brain vs. optic lobes for D . melanogaster (101,140 ±3,650 vs. 107,270 ± 2,720), Ae . aegypti (109,140 ± 3,550 vs. 112,000 ± 4,280), An . coluzzii (105,130 ± 3,670 vs. 107,140 ± 3,090), and C . quinquefasciatus (108,530 ±7,990 vs. 110,670 ± 3,950) was also estimated. Each insect brain was comprised of 89% ± 2% neurons out of its total cell population. Isotropic fractionation analyses did not identify obvious sexual dimorphism in the neuronal and non-neuronal cell population of these insects. Our study provides experimental evidence for the total number of neurons in Drosophila and mosquito brains.
Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins, and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.
Multiple nuclei sharing a common cytoplasm are found in diverse tissues, organisms, and diseases. Yet, multinucleation remains a poorly understood biological property. Cytoplasm sharing invariably involves plasma membrane breaches. In contrast, we discovered cytoplasm sharing without membrane breaching in highly resorptive Drosophila rectal papillae. During a six-hour developmental window, 100 individual papillar cells assemble a multinucleate cytoplasm, allowing passage of proteins of at least 62kDa throughout papillar tissue. Papillar cytoplasm sharing does not employ canonical mechanisms such as incomplete cytokinesis or muscle fusion pore regulators. Instead, sharing requires gap junction proteins (normally associated with transport of molecules 1kDa), which are positioned by membrane remodeling GTPases. Our work reveals a new role for apical membrane remodeling in converting a multicellular epithelium into a giant multinucleate cytoplasm.
Long-lived cells such as terminally differentiated postmitotic neurons and glia must cope with the accumulation of damage over the course of an animal’s lifespan. How long-lived cells deal with ageing-related damage is poorly understood. Here we show that polyploid cells accumulate in the adult fly brain and that polyploidy protects against DNA damage-induced cell death. Multiple types of neurons and glia that are diploid at eclosion, become polyploid in the adult Drosophila brain. The optic lobes exhibit the highest levels of polyploidy, associated with an elevated DNA damage response in this brain region. Inducing oxidative stress or exogenous DNA damage leads to an earlier onset of polyploidy, and polyploid cells in the adult brain are more resistant to DNA damage-induced cell death than diploid cells. Our results suggest polyploidy may serve a protective role for neurons and glia in adult Drosophila melanogaster brains.
Screens in mosaic Drosophila tissues that use chemical mutagenesis have identified many regulators of growth and patterning. Many of the mutant phenotypes observed were contingent upon the presence of both wild-type and mutant cells in the same tissue. More recently, large collections of RNAi lines or cDNAs expressed under Gal4/UAS control have been used to alter gene expression uniformly in specific tissues. However, these newer approaches are not easily combined with the efficient generation of genetic mosaics. The CoinFLP system described here enables mosaic screens in the context of gene knockdown or overexpression by automatically generating a reliable ratio of mutant to wild-type tissue in a developmentally controlled manner. CoinFLP-Gal4 generates mosaic tissues composed of clones of which only a subset expresses Gal4. CoinFLP-LexGAD/Gal4 generates tissues composed of clones that express either Gal4 or LexGAD, thus allowing the study of interactions between different types of genetically manipulated cells. By combining CoinFLP-LexGAD/Gal4 with the split-GFP system GRASP, boundaries between genetically distinct cell populations can be visualized at high resolution.
© 2015. Published by The Company of Biologists Ltd.
5-Bromo-2'-deoxyuridin (BrdU) is frequently used in anaylsis of neural stem cell biology, in particular to label and to fate-map dividing cells. However, up to now, only a few studies have addressed the question as to whether BrdU labeling per se affects the cells to be investigated. Here, we focused on the potential impact of BrdU on neurosphere cultures derived from the adult rat brain and on proliferation of progenitors in vivo. In vitro, neurospheres were pulsed for 48 h with BrdU, and cell proliferation, cell cycle, differentiation, survival and adhesion properties were subsequently analyzed. BrdU inhibited the expansion of neural progenitors as assessed by MTS assay and increased the fraction of cells in the G0/G1-phase of the cell cycle. Moreover, BrdU increased cell death and dose-dependently induced adherence of NPCs. Cell adherence was accompanied by a reduced amount of active matrix-metalloproteinase-2 (MMP-2). Furthermore, BrdU repressed neuronal and oligodendroglial differentiation, whereas astroglial fate was not affected. In contrast to the in vitro situation, BrdU apparently did not influence endogenous proliferation of NPCs or neurogenesis in concentrations that are typically used for labeling of neural progenitors in vivo. Our results reveal so far uncharacterized effects of BrdU on adult NPCs. We conclude that, because of its ubiquitous use in stem cell biology, any potential effect of BrdU of NPCs has to be scrutinized prior to interpretation of data.
Thymidine analogs (TAs) are synthetic nucleosides that incorporate into newly synthesized DNA. Halogenated pyrimidines (HPs), such as bromodeoxyuridine (BrdU), are a class of TAs that can be detected with antibodies and are commonly used for birthdating individual cells and for assessing the proliferative index of cell populations. It is well established that HPs can act as radiosensitizers when incorporated into DNA chains, but they are generally believed not to impair normal cell function in the absence of secondary stressors. However, we and others have shown that HP incorporation leads to a sustained suppression of cell cycle progression in mammalian cells, resulting in cellular senescence in somatic cells. In addition, we have shown that HP incorporation results in delayed tumor progression in a syngeneic rat model of glioma. Here we examine ethynyldeoxyuridine (EdU), a newly developed and alkylated TA, for its anti-cancer activity, both in vitro and in vivo. We show that EdU, like HPs, leads to a severe reduction in the proliferation rate of normal and transformed cells in vitro. Unlike HPs, however, EdU incorporation also causes DNA damage resulting in the death of a substantial subset of treated cells. When administered over an extended time as a monotherapy to mice bearing subcutaneous xenografts of human glioblastoma multiforme tumors, EdU significantly reduces tumor volume and increases survival without apparent significant toxicity. These results, combined with the fact that EdU readily crosses the blood-brain barrier, support the continued investigation of EdU as a potential therapy for malignant brain tumors.
The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to
reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase
as well as an equilibrative nucleoside transporter. By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal
kinetics at low thymidine concentrations. Using this novel approach, we show that depletion of a single deoxyribonucleoside
causes reversible arrest of cells in S phase with concomitant phosphorylation and activation of the S phase checkpoint kinase,
Rad53. We show that this strain also efficiently incorporates the thymidine analogue, BrdU, into DNA and can be used for pulse–chase
labelling.
Neural stem cells (NSCs) are found in a tailored, intricate cellular microenvironment, the niche, which supports and regulates their activity. Whilst niche architecture is indissociable from its function, the morphogenetic aspects of niche development have been poorly explored. Here, we use the formation of the cortex glia (CG) network in Drosophila as a paradigm of acquisition of architectural complexity of a NSC niche. CG are essential for normal neurogenesis and build a reticular network spanning the entire central nervous system while encasing each NSC linage. We first show that individual CG cells grow tremendously to enwrap several NSC linages, ultimately covering and tiling the entire tissue. Several proliferative mechanisms, including endoreplication and mitosis, in part acytokinetic, support such growth and result in the formation of multinucleated, syncytial CG cells, that we call units. We then reveal that CG units are able to fuse to each other, resulting in the exchange of several subcellular compartments, such as membrane, cytoplasm and organelles. This process relies on well-known molecular players of cell fusion, involving cell surface communication molecules and actin regulators, while being atypical by its extent, dynamics and partial nature. Ultimately, the coordination in time and space of growth, proliferation and fusion mechanisms is required for the remarkable, multi-level architecture of the Drosophila NSC niche.
The identity of cellular populations that drive liver regeneration after injury is the subject of intense study, and the contributions of polyploid hepatocytes to organ regeneration and homeostasis have not been systematically assessed. Here, we developed a multicolor reporter allele system to genetically label and trace polyploid cells in situ. Multicolored polyploid hepatocytes undergo ploidy reduction and subsequent re-polyploidization after transplantation, providing direct evidence of the hepatocyte ploidy conveyor model. Marker segregation revealed that ploidy reduction rarely involves chromosome missegregation in vivo. We also traced polyploid hepatocytes in several different liver injury models and found robust proliferation in all settings. Importantly, ploidy reduction was seen in all injury models studied. We therefore conclude that polyploid hepatocytes have extensive regenerative capacity in situ and routinely undergo reductive mitoses during regenerative responses.