Regulation and function of developmentally controlled polyploidization

The development of a multicellular organism requires intricate regulation of cellular behaviour, especially on the cell division cycle. While we have a lot of knowledge on how cells make the decision to divide, insight on how cell cycle alterations are regulated in development and disease is lacking. Two main cell cycle variations, endoreplication and endomitosis, give rise to polyploid cells, containing more than two chromosome sets divided over one or two nuclei. Polyploidization is a process that occurs frequently in plants and animals, with endoreplication occurring often in plants and insects, while endomitosis is more prevalent in mammalian tissues. Although a general molecular mechanism that regulates endoreplication has been established, we do not yet understand what controls endomitosis. The scope of this thesis comprises the regulation and function of developmentally controlled polyploidization cycles in the C. elegans intestine. With this, we aim to broaden our understanding of the molecular mechanisms that control endomitosis and the functional significance of polyploidization in development, providing insights that aid the investigation of polyploidization in other model organisms and tissues as well. In chapter 2, we describe observed differences between canonical mitosis and endomitosis in the C. elegans intestine. We find that many cytokinesis and mitosis genes are downregulated on a transcriptional level during endomitosis. By inducing additional cell cycles in the intestinal lineage, we also establish that the switch from canonical cell cycle regulation to endomitosis is made during embryogenesis. Finally, we identify the transcriptional regulator SIN-3 as an important player in the regulation of endomitosis. In chapter 3, we investigate the transcriptional changes that occur during embryonic differentiation of the intestine. We use fluorescence activated cell sorting and single cell RNA sequencing to characterize expression profiles of intestinal cells throughout embryonic development and identify transcriptional regulators with temporal expression patterns as potential players in the regulation of endomitosis. In chapter 4, we describe our investigation of the functional difference between endomitosis and endoreplication. Using an auxin-inducible degradation system, we specifically block binucleation of the C. elegans intestine and find that this affects adult hermaphrodite tissue-specific gene expression needed to support progeny growth. Altogether, we show that binucleation of polyploid cells facilitates rapid upregulation of gene expression needed during development and upon heat stress induction. In chapter 5, we provide our perspective on the function of polyploid cells in development and disease. Based on the observation that polyploid cell types often function to support other, proliferating cells, we suggest that polyploidization is advantageous for this supporting role, possibly enhancing the capacity to provide nutrients and signals and form a barrier, thereby creating a (micro-)environment for dividing cells. Finally, the findings presented in this thesis are summarized and discussed in chapter 6, along with suggestions for possible future directions

Regulation and function of developmentally controlled polyploidization

The development of a multicellular organism requires intricate regulation of cellular behaviour, especially on the cell division cycle. While we have a lot of knowledge on how cells make the decision to divide, insight on how cell cycle alterations are regulated in development and disease is lacking. Two main cell cycle variations, endoreplication and endomitosis, give rise to polyploid cells, containing more than two chromosome sets divided over one or two nuclei. Polyploidization is a process that occurs frequently in plants and animals, with endoreplication occurring often in plants and insects, while endomitosis is more prevalent in mammalian tissues. Although a general molecular mechanism that regulates endoreplication has been established, we do not yet understand what controls endomitosis. The scope of this thesis comprises the regulation and function of developmentally controlled polyploidization cycles in the C. elegans intestine. With this, we aim to broaden our understanding of the molecular mechanisms that control endomitosis and the functional significance of polyploidization in development, providing insights that aid the investigation of polyploidization in other model organisms and tissues as well. In chapter 2, we describe observed differences between canonical mitosis and endomitosis in the C. elegans intestine. We find that many cytokinesis and mitosis genes are downregulated on a transcriptional level during endomitosis. By inducing additional cell cycles in the intestinal lineage, we also establish that the switch from canonical cell cycle regulation to endomitosis is made during embryogenesis. Finally, we identify the transcriptional regulator SIN-3 as an important player in the regulation of endomitosis. In chapter 3, we investigate the transcriptional changes that occur during embryonic differentiation of the intestine. We use fluorescence activated cell sorting and single cell RNA sequencing to characterize expression profiles of intestinal cells throughout embryonic development and identify transcriptional regulators with temporal expression patterns as potential players in the regulation of endomitosis. In chapter 4, we describe our investigation of the functional difference between endomitosis and endoreplication. Using an auxin-inducible degradation system, we specifically block binucleation of the C. elegans intestine and find that this affects adult hermaphrodite tissue-specific gene expression needed to support progeny growth. Altogether, we show that binucleation of polyploid cells facilitates rapid upregulation of gene expression needed during development and upon heat stress induction. In chapter 5, we provide our perspective on the function of polyploid cells in development and disease. Based on the observation that polyploid cell types often function to support other, proliferating cells, we suggest that polyploidization is advantageous for this supporting role, possibly enhancing the capacity to provide nutrients and signals and form a barrier, thereby creating a (micro-)environment for dividing cells. Finally, the findings presented in this thesis are summarized and discussed in chapter 6, along with suggestions for possible future directions

A dual transcriptional reporter and CDK-activity sensor marks cell cycle entry and progression in <i>C</i>. <i>elegans</i>

<div><p>Development, tissue homeostasis and tumor suppression depend critically on the correct regulation of cell division. Central in the cell division process is the decision whether to enter the next cell cycle and commit to going through the S and M phases, or to remain temporarily or permanently arrested. Cell cycle studies in genetic model systems could greatly benefit from visualizing cell cycle commitment in individual cells without the need of fixation. Here, we report the development and characterization of a reporter to monitor cell cycle entry in the nematode <i>C</i>. <i>elegans</i>. This reporter combines the <i>mcm-4</i> promoter, to reveal Rb/E2F-mediated transcriptional control, and a live-cell sensor for CDK-activity. The CDK sensor was recently developed for use in human cells and consists of a DNA Helicase fragment fused to eGFP. Upon phosphorylation by CDKs, this fusion protein changes in localization from the nucleus to the cytoplasm. The combined regulation of transcription and subcellular localization enabled us to visualize the moment of cell cycle entry in dividing seam cells during <i>C</i>. <i>elegans</i> larval development. This reporter is the first to reflect cell cycle commitment in <i>C</i>. <i>elegans</i> and will help further genetic studies of the mechanisms that underlie cell cycle entry and exit.</p></div

Sensor localization during S phase and CDK-1-dependent phosphorylation of the sensor.

<p>(A) Quantification of DNA content by propidium iodide staining in L1 larvae at 2, 3, 4 and 5 hours after hatching. Muscle cells were used as the reference for a 2N DNA content. Error bars indicate SEM. (B) Boxplots of ratios calculated from confocal fluorescence microscopy images of larvae staged at 2, 3 and 4 hours after hatching (n = 9 for single seam cells, n = 27 for average of cells). Avg. refers to the average ratio of V2, V3 and V4 together. The borders of the boxes are the 25^th and 75^th percentile, • indicates the mean, error bars correspond to 1.5× the interquartile range, outliers are shown. (C) Boxplots containing the quantification of cytoplasmic-to-nuclear fluorescence ratio in control and HU-treated larvae, 5 hours after hatching (n = 9). The borders of the boxes are the 25^th and 75^th percentile, the mean is indicated by <b>+</b>, error bars correlate to 1.5× the interquartile range. (D) Live-cell imaging of WT (top) and <i>cdk-1(he5)</i> mutant (bottom) larvae expressing the CDK sensor, at 300 minutes (5 hours) after hatching. (E) Spinning disk confocal fluorescence microscopy time-lapse movie analysis of sensor localization in control animals (n = 3 cells) and <i>cdk-1(he5)</i> mutants in L1 (n = 3). Control cells before anaphase in grey, control anterior cells in orange, control posterior cells in blue, <i>cdk-1(he5)</i> mutant cells in green. Average ratio is indicated by a bold line, individual cells are shown with thin lines. Dotted black lines indicate the time between anaphase and nuclear envelope reformation, where ratios could not be determined because of absence of the nucleus. (F) Comparison between the maximal calculated ratio’s in control (n = 10) and <i>cdk-1(he5)</i> (n = 14) animals during L1 development. The borders of the boxes are the 25^th and 75^th percentile, the mean is indicated by–, error bars correlate to 1.5× the interquartile range.</p

Dynamic localization of the CDK sensor in <i>C</i>. <i>elegans</i>.

<p>(A) The CDK-2 sensor consists of amino acids 994 till 1087 of Human DNA Helicase B with an NLS, NES, and four CDK target sites, and is fused to eGFP. Both the sensor and eGFP are codon optimized for use in <i>C</i>. <i>elegans</i>. (B) The phosphorylation status of the DHB fragment determines whether the sensor localizes to the nucleus or to the cytoplasm. As CDK-cyclin complexes are activated during cell cycle progression, the sensor becomes phosphorylated and accumulates in the cytoplasm. (C-H) Representative fluorescence microscopy images highlighting sensor expression and localization in a variety of tissues. (C) Expression of the sensor from the general <i>eft-3</i> promoter resulted in expression in all somatic cells of the animal. Arrow indicates seam cells in which the sensor is localized in the cytoplasm. (D-H) expression from the cell cycle regulated <i>mcm-4</i> promoter is specifically detected in cells that have the potential to divide, as shown for seam cells in an L3 larva (D). (E-H) Dynamic sensor localization was observed in seam cells (E), Q cells (F), vulval precursor cells (G), and to a lesser extent in intestinal cells (H).</p

Sensor dynamics in L3 asymmetric and L2 symmetric seam cell divisions.

<p>(A) Schematic overview of divisions in the Vn seam cell lineages (n = 1–4, 6). These seam cells undergo one asymmetric cell division during every larval stage (L1-L4). During these divisions the seam cells produce a posterior self-renewing daughter cell (Vn.p), and an anterior daughter cell that differentiates and fuses with the hyp7 syncytium (Vn.a; orange circles). In the second larval stage (L2), seam cells undergo one additional, symmetric cell division, producing two self-renewing seam daughter cells. (B) Still images and blow-ups from spinning disk confocal fluorescence microscopy time-lapse movies of larvae staged at 24 hours after hatching. From top to bottom, images show seam cells (V3.pap, anterior to the left) before anaphase, during nuclear envelope breakdown at prometaphase, and after anaphase (V3.papa and V3.papp). Note that in the two newly formed daughter cells, nuclear export of the sensor is observed in the anterior daughter cell before it is observed in its posterior sister cell. Scale bar indicates 20 μm. (C) Graph representing the cytoplasmic-to-nuclear ratio (cyt/nuc) of DHB-eGFP in seam cell L3 asymmetric division (n = 5). Anterior cells in orange, posterior cells in blue, average ratio is indicated by a black line. Asterisk indicates the moment where plotted ratios in the anterior cell start to deviate from the ratios in the posterior cell. The arrow indicates the moment where the difference in ratios is statistically significant (t = 44 min after anaphase). Cells are aligned at the moment of anaphase (t = 0). (D) Still images and blow-ups of spinning disk confocal microscopy movies of worms staged at 20 hours after hatching. From top to bottom, images show seam cells (highlighted: V2.p, anterior to the left) before anaphase, during nuclear envelope breakdown at prometaphase, and after division (V2.pa and V2.pp). Note that in the two newly formed daughter cells the rate of nuclear export of the sensor is similar. Anterior to the left, scale bar indicates 20 μm. (E) Graph representing the cyt/nuc ratio of DHB-eGFP in symmetric L2 seam division (n = 3). Anterior cells in orange, posterior cells in blue, average ratio is indicated by a black line. The cells are aligned at the moment of anaphase (t = 0).</p

Endomitosis controls tissue-specific gene expression during development

Polyploid cells contain more than 2 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 1 nucleus (mononucleation) or 2 or more nuclei (multinucleation). Despite the widespread occurrence of polyploidy, the functional significance of different types of polyploidy is largely unknown. Here, we assess the function of multinucleation in Caenorhabditis elegans intestinal cells through specific inhibition of binucleation without altering genome ploidy. Through single-worm RNA sequencing, we find that binucleation is important for tissue-specific gene expression, most prominently for genes that show a rapid up-regulation 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 up-regulation of intestine-specific gene expression during development, independently of genome ploidy, underscoring the importance of spatial genome organization for polyploid cell function

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage

Graphical Abstract Highlights d Individual cycling cancer cells display enhanced E2F target gene expression d E2F7/8 deletion or E2F3 overexpression overrides cell-cycle exit after DNA damage d Elevated levels of the E2F target Emi1 prevent DNA-damage-induced cell-cycle exit d The cell-cycle exit after DNA damage is transient and leads to endoreplicatio

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage

Graphical Abstract Highlights d Individual cycling cancer cells display enhanced E2F target gene expression d E2F7/8 deletion or E2F3 overexpression overrides cell-cycle exit after DNA damage d Elevated levels of the E2F target Emi1 prevent DNA-damage-induced cell-cycle exit d The cell-cycle exit after DNA damage is transient and leads to endoreplicatio