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Endomitosis controls tissue-specific gene expression during development

['Lotte M. Van Rijnberk', 'Hubrecht Institute', 'Royal Netherlands Academy Of Arts', 'Sciences', 'University Medical Center Utrecht', 'Utrecht', 'The Netherlands', 'Ramon Barrull-Mascaró', 'Reinier L. Van Der Palen', 'Erik S. Schild']

Date: 2022-06 

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.

(A) Three types of cell cycles that take place during intestinal development in C. elegans. (B) Overview of intestinal cell cycles in wild-type/control animals and upon intestinal depletion of CDK-1 or KNL-1. In wild-type C. elegans, 20 intestinal cells are formed during embryogenesis by canonical cell cycles. During the first larval stage (L1) intestinal cells undergo one round of endomitosis creating binucleated cells. Thereafter, intestinal cells undergo one round of endoreplication at the end of each larval stage, leading to adult intestinal cells with two 32C nuclei (and thus a cellular ploidy of 64C DNA content). Upon inhibition of either CDK-1 or KNL-1 during endomitosis using the auxin-inducible degradation system, endomitotic binucleation is blocked, and a mononucleated polyploid cell is generated. Importantly, cellular ploidy remains the same as wild-type conditions (64C). (C) Schematic overview of experimental procedure. Mixed stage embryos are isolated from adult hermaphrodites containing intestinally expressed TIR1 (Pges-1::TIR1) and either AID::cdk-1 or AID::knl-1 and starved overnight to yield a synchronized population of arrested L1 animals. The population of starved L1 animals is split into 2 conditions: an auxin (+) condition and a control (ct) condition. For the auxin condition, animals are grown on plates containing auxin for the first 24 hours of postembryonic development, when endomitosis normally occurs, and transferred to plates without auxin after this period. Animals in control conditions are grown on plates without auxin for the first 24 hours of development and transferred to plates containing auxin for 24 to 48 hours of postembryonic development, when intestinal endomitosis has already occurred and neither KNL-1 or CDK-1 are required in the intestine. (D) DAPI stainings of adult hermaphrodites containing Pges-1::TIR1; AID::knl-1 and grown under auxin or auxin-control conditions. Intestinal nuclei are outlined in magenta. Scale bar is 50 μm. (E) Number of intestinal nuclei in adult hermaphrodites containing Pges-1::TIR1; AID::knl-1 and grown under auxin (+) or auxin-control (ct) conditions. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. (F) Fluorescent images of intestinal H2B-mCherry (DNA) and GFP-PH (membrane) in intestinal ring 3 of Pges-1::TIR1; AID::knl-1 animals grown on auxin or auxin-control conditions. Dashed line indicates cell outline. Scale bar is 20 μm. (G) Number of binucleated intestinal cells in adult Pges-1::TIR1; AID::knl-1 hermaphrodites grown on auxin (+) or auxin-control (ct) conditions. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. (H) Quantification of intestinal nuclear ploidy in binucleated control (ct, n = 49 for Pges-1::TIR1; AID::cdk-1 strain and n = 28 for Pges-1::TIR1; AID::knl-1 strain) or mononucleated intestinal cells (+ auxin, n = 29 for Pges-1::TIR1; AID::cdk-1 strain and n = 26 cells for Pges-1::TIR1; AID::knl-1 strain). Ploidy is measured by total fluorescence intensity of propidium iodide (PI) DNA staining in intestinal ring 3 nuclei (Int3D and Int3V) and normalized to proximal 2C nuclei. Each dot represents the average ploidy of individual Int3D/V nuclei in one animal. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. Underlying data can be found in S1 Data . AID, auxin-inducible degron.

The Caenorhabditis elegans intestine provides an ideal model system to study distinct types of polyploidy, as intestinal cells undergo both endomitosis and endoreplication cycles in a highly tractable manner at defined moments during larval development ( Fig 1A and 1B ) [ 19 ]. Such consecutive cycles of endomitosis and endoreplication give rise to large, binucleated polyploid cells that make up the adult intestine. Here, we develop a method to inhibit intestinal endomitosis using auxin-inducible degradation of key mitotic regulators, allowing us to study the function of binucleation at both the cellular and tissue level. We find that animals with mononucleated instead of binucleated intestinal cells have decreased fitness due to defects in the expression of a group of tissue-specific genes, including the vitellogenin genes. Vitellogenin genes are normally rapidly expressed during the maturation of intestinal cells at the end of larval development, indicating that rapid up-regulation of tissue-specific genes requires binucleation of polyploid cells. Importantly, this rapid up-regulation of intestine-specific genes is important to support progeny development. Together, our results show that binucleation is important for correct functioning of a polyploid tissue and that partitioning of genomes into multiple nuclei allows efficient and rapid up-regulation of gene expression during development.

Somatic polyploidy can arise either by cell fusion or by noncanonical cell cycles in which cells replicate their DNA but do not divide. Two types of noncanonical cell cycles that result in polyploidy have been described: endoreplication and endomitosis [ 17 ]. In endoreplication, M phase is skipped, resulting in cycles of DNA replication (S phase) and gap (G) phases without intervening mitosis and cytokinesis. Endoreplicative cell cycles result in large, mononucleated cells. In endomitosis, cells do enter mitosis, but do not undergo cell division, resulting in polyploid cells with either a single nucleus or 2 nuclei, depending on whether M phase is aborted before or after initiation of sister chromosome segregation (which normally occurs during anaphase) [ 7 , 11 , 18 ]. The existence of multiple types of polyploid cells (e.g., mononucleated or binucleated) suggests that there may be a functional difference between these different types of polyploidy. However, testing the functional significance of multinucleation has been challenging due to the complexity of many polyploid tissues and a lack of tools to specifically alter noncanonical cells cycles without affecting any other cells or tissues.

Polyploidization occurs in many plant and animal cells as part of a developmental program, where it is crucial to increase cell size and metabolic output, as well as to maintain the barrier function of certain tissues. For example, polyploidization of the giant neurons of Limax slugs allows them to reach the enormous size they need to transmit signals over large distances, and polyploidization of the Drosophila subperineurial glial cells ensures the integrity of the blood–brain barrier during organ growth [ 1 , 2 ]. In vertebrates, polyploid cells are present in many organs, such as the liver, blood, skin, pancreas, placenta, and mammary glands, where they play important functions in tissue homeostasis, regeneration, and in response to damage [ 3 – 13 ]. Interestingly, polyploid cells can either be mononucleated, such as megakaryocytes and trophoblast giant cells [ 14 , 15 ], or they can be multinucleated, such as cardiomyocytes and mammary epithelial cells [ 8 , 16 ], but how genome partitioning in either 1 or multiple nuclei affects polyploid cell function is yet unknown.

Results

Auxin-inducible degradation of mitotic regulators prevents binucleation To investigate the function of binucleation, we developed a system in which we can specifically perturb intestinal endomitosis, without affecting other cell cycles within developing C. elegans larvae. We employed 2 mechanistically distinct approaches to block binucleation of the intestine; we either depleted CDK-1, which is essential for mitotic entry, or KNL-1, a conserved kinetochore protein that is required for chromosome segregation during M phase. CDK-1 inhibition is known to be a key mechanism to initiate endoreplication, and degrading CDK-1 essentially converts the endomitosis cycle to an endoreplication cycle, preventing mitotic entry and subsequent binucleation [14,17,20,21]. In contrast, KNL-1 inhibition does not affect mitotic entry but prevents chromosome segregation and partitioning of sister chromatids into 2 nuclei [22,23]. To deplete CDK-1 and KNL-1 specifically in intestinal cells and only during the time when endomitosis occurs, we made use of the auxin-inducible degradation system. In this system, proteins tagged with an auxin-inducible degron (AID) are degraded only in the presence of auxin and the F Box protein TIR1 [24–26]. By using a strain that expresses TIR1 under control of the intestine-specific Pges-1 promoter and exposing animals to auxin only during the time that endomitosis occurs, we can specifically inhibit intestinal binucleation without affecting canonical cell cycles of intestinal or other cells. We generated AID knock-ins on the cdk-1 and knl-1 genes using CRISPR-mediated gene targeting and tested whether auxin induced depletion of CDK-1 or KNL-1 during endomitosis was able to block binucleation. In wild-type animals, endomitosis takes place at the end of the first larval stage in 12 to 14 of the 20 intestinal cells, resulting in 20 intestinal cells with 32 to 34 nuclei [19]. To control for nonspecific effects of auxin on development, we included an auxin control in each experiment, in which animals were treated with auxin for the same duration, but during a time in development in which CDK-1 or KNL-1 are not required in the intestine (Fig 1B and 1C). To assess the effect of auxin treatment on intestinal binucleation, we analyzed animals grown with auxin during endomitosis (hereafter referred to as auxin-treated animals or “+”) or in the auxin-control condition (hereafter referred to as auxin-control animals or “ct”) and found that auxin-treated animals contain 20 to 22 intestinal nuclei, in contrast to the 32 to 34 nuclei that are present in auxin-control animals (Fig 1D and 1E). Of the 12 to 14 intestinal cells that are known to undergo endomitosis and become binucleated in wild-type intestines, we found on average 13 cells to be binucleated in auxin-control animals compared to 0 binucleated cells in auxin-treated animals (Fig 1F and 1G). We next performed a quantitative DNA staining using PI and found that auxin-treated AID::cdk-1 and AID::knl-1 animals have a 64C DNA content, which is exactly double the nuclear ploidy of controls (Fig 1H). Thus, by inhibiting CDK-1 and KNL-1 during endomitosis, we can block binucleation and generate animals with mononucleated intestinal cells with the same cellular ploidy as wild-type binucleated intestinal cells. Depletion of KNL-1 is known to prevent chromosome segregation in anaphase by blocking formation of kinetochore microtubule attachments [22]. To exclude that KNL-1 knockdown results in formation of micronuclei caused by chromosome missegregations, which can lead to DNA damage, cellular stress, and potentially arrest cells in the following cell cycle [27–30], we performed live-cell imaging of fluorescently labeled chromosomes in KNL-1 depleted cells. In all cells, KNL-1 depletion prevented chromosome segregation and resulted in mononucleation, but did not extend the duration of mitosis or result in the formation of micronuclei (S1A and S1B Fig, S1 and S2 Movies). After endomitosis, cells go through multiple rounds of endoreplication in which newly formed replicated chromosomes remain clustered together. Wild-type intestinal cells that have undergone endomitosis contain 2 nuclei, each containing 2 sets of chromosome clusters, which can be visualized by chromosomal LacO/LacI tagging (S1C Fig). As expected, we observed on average 4 chromosome clusters in nuclei of mononucleated KNL-1 depleted cells (S1D Fig), indicating that all copies of the labeled chromosome were present in a single nucleus. Finally, using a fluorescent cell cycle marker (Pges-1:: CYB-1DB::mCherry), we found that preventing binucleation did not affect the timing of the subsequent endoreplicative S phase (S1E Fig). Taken together, our results indicate that KNL-1 depletion prevents binucleation without inducing detectable chromosomal or cell cycle aberrancies. Thus, this system provides a unique opportunity to study the function of binucleation, without altering the ploidy or number of cells in the tissue of interest.

Intestinal mononucleation decreases the nuclear surface-to-volume ratio, but does not affect cell size or morphology To investigate whether mononucleation influences intestinal cell size or morphology, we used fluorescent markers in the AID::knl-1 strain to visualize the cell membranes and intestinal lumen. We found no effect of intestinal mononucleation on animal size, cell size, or lumen morphology (Fig 2A–2H). Because polyploidization has also been shown to influence nuclear morphology [31], we investigated the effect of binucleation on nuclear geometry using a fluorescently labeled nuclear pore protein (NPP-9::mCherry) as a marker for the nuclear membrane (Fig 2I and 2J). We measured nuclear size and found that the nuclear volume was increased with a factor of approximately 2.5 in mononucleated cells, indicating that per cell, the total nuclear volume has more than doubled (Fig 2K). The nuclear surface area was also increased in mononucleated cells, but to a lesser extent, with a factor of 2 (Fig 2L). Consequently, the surface-to-volume ratio decreased, indicating that less surface area is available per volume in mononucleated cells (Fig 2M). To test whether this effect is compensated to any extent by shape alterations or membrane invaginations that enlarge nuclear surface area, such has been observed in other polyploid cells [31], we also measured the ratio between the circumference and area of nuclear sections, but observed a similar effect (S2A–S2D Fig), indicating that there are no large invaginations that compensate for the amount of available nuclear surface area per volume. Thus, although mononucleation does not affect intestinal cell size or morphology, it produces nuclei that are more than twice as big and have an altered surface-to-volume ratio, which could potentially influence nuclear functions. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Intestinal cell mononucleation does not influence worm size, cell size, or intestinal morphology, but generates nuclei that are more than twice as big. (A, B) Total body length (A) and width (B) of Pges-1::TIR1; AID::knl-1 young adults grown under control conditions (ct, n = 30) or in the presence of auxin (+, n = 32). Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. NS, not significant (P > 0.05, Student t test). (C) Representative Z-stack projections of intestinal H2B-mCherry (DNA) and GFP-PH (membrane) in intestinal ring 3 of Pges-1::TIR1; AID::knl-1 animals grown on auxin or control conditions, used for quantifications of cell length and width. Scale bar is 20 μm. (D, E) Cellular length (D) and width (E) of Int3D/V cells of Pges-1::TIR1; AID::knl-1 young adults grown under control conditions (ct, n = 58) or in the presence of auxin (+, n = 24). Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. NS, not significant (P > 0.05, Student t test). (F) Representative Z-stack projections of intestinal GFP-PH (membrane) in the apical membrane of intestinal ring 3 of Pges-1::TIR1; AID::knl-1 animals that were grown under control conditions or in the presence of auxin, used for quantifications of brush border and luminal width. Scale bar is 10 μm. (G, H) Intestinal brush border (G) and lumen (H) width in intestinal ring 3 of young adult worms grown under control (ct, n = 40) or auxin (+, n = 34) conditions. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. NS, not significant (P > 0.05, Mann–Whitney test). (I) Fluorescent images of intestinal NPP-9::mCherry localization in the nuclear membranes of Int3 cells of Pges-1::TIR1; AID::knl-1 adults grown under control or auxin conditions, corresponding to a 32C and 64C DNA content, respectively. Scale bar represents 10 μm. (J) Schematic depicting an ellipsoid shape and radii a, b, and c used to calculate nuclear volume and surface area. (K–M) Boxplots depicting the average nuclear volume (K), surface area (L) and surface-area-to-volume ratio (M) in binucleated (ct, n = 60 cells, 18 animals) or mononucleated (+, n = 56 cells, 30 animals) cells of Pges-1::TIR1; AID::knl-1 animals. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. P values were calculated by Mann–Whitney (K, L) or unpaired Student t test (M). Underlying data can be found in S1 Data. AID, auxin-inducible degron. https://doi.org/10.1371/journal.pbio.3001597.g002

Binucleation is important for adult intestinal transcription To investigate possible transcriptional differences between young adults with mononucleated or binucleated intestines, we performed single-worm RNA sequencing of AID::knl-1 and AID::cdk-1 auxin-treated and auxin-control animals. Since the intestine is one of the most transcriptionally active and largest tissues in the worm, making up roughly one-third of the animals volume [32], we anticipated that changes in transcription in the intestine would be detectable in whole worm sequencing. We performed differential gene expression analysis for 68 (AID::knl-1) and 75 (AID::cdk-1) young adult (72 hours) auxin-treated or auxin-control animals (see Methods for details). For the AID::knl-1 animals alone, 16 percent of genes included in the analysis showed significant and substantial differential expression between worms with a mononucleated or binucleated intestine (420/2,638 genes, Fig 3A, S3A Fig). Sequencing of AID::cdk-1 animals revealed fewer differentially expressed genes between animals with mononucleated or binucleated intestines, which is likely due to a lower complexity of these samples (S3B–S3E Fig). Although it is unclear why the AID::cdk-1 samples had lower complexities, we focused on the overlap in differential gene expression between the AID::knl-1 and AID::cdk-1 strains to identify genes that are differentially expressed in animals with mononucleated intestines (S3F Fig). We found a strong enrichment (P = 2e-7) in the overlap between genes significantly and substantially down-regulated in both strains, consisting of 15 genes (Fig 3B and 3C). One of the genes that stood out was vit-2, 1 of the 6 C. elegans vitellogenin genes whose levels have been shown to correlate strongly with the growth and fitness traits of C. elegans progeny [33]. Vitellogenins are highly expressed in adult intestines and are essential to mobilize lipids in the intestine for transport to the developing oocytes in the germline, where they contribute to progeny development [34–37]. Because vitellogenins have previously been shown to function redundantly, and down-regulation of individual vit genes often leads to up-regulation of others [34], we analyzed the expression of all 6 vit genes in our dataset and found that all of them were down-regulated in worms with a mononucleated intestine (S4A–S4F Fig). Moreover, the sum of all vit gene expression values showed a stronger decrease than any gene alone (S4G Fig), suggesting that in mononucleated animals, the down-regulation of vit-2 is not compensated by the transcriptional up-regulation of other vitellogenins. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Blocking binucleation causes transcriptional down-regulation of intestinally expressed genes in young adult C. elegans. (A) Volcano plot of RNA sequencing data depicting the transcriptional gene up- and down-regulation in worms with a mononucleated intestine, compared to worms with a binucleated (wild type) intestine in Pges-1::TIR1; AID::knl-1 animals (n = 68). After filtering for coverage, batch consistency, and intestinal expression, 2,638 genes were analyzed for differential gene expression using DEseq. Red dots represent genes significantly differentially expressed (adjusted P value < 0.05, top 25% absolute log2(foldchange)). Genes significantly differentially expressed in both Pges-1::TIR1; AID::knl-1 and Pges-1::TIR1; AID::cdk-1 comparisons were individually annotated with their gene name (excluding genes without a gene name). (B) Venn diagram of the overlap of significantly down-regulated genes in Pges-1::TIR1; AID::knl-1 and Pges-1::TIR1; AID::cdk-1 animals. (C) Overview of genes that are found to be significantly down-regulated in worms in which intestinal binucleation is blocked, both by degradation of CDK-1 and degradation of KNL-1, including a short description and previously established associated phenotypes as described on WormBase [38]. Underlying data are available at the Gene Expression Omnibus, identifier GSE169330, and in S1 Data. AID, auxin-inducible degron. https://doi.org/10.1371/journal.pbio.3001597.g003

Binucleation of the intestine promotes vitellogenin expression and lipid loading into oocytes To confirm that vit-2 expression is down-regulated in animals with mononucleated intestinal cells, we generated a Pvit-2::NLSGFP transcriptional reporter to measure vit-2 promoter activity at different moments of development. Since vitellogenin expression is absent during larval development and drastically up-regulated at the L4-to-adult transition [34], expression levels are still relatively low at 48 hours of development, around the end of the L4 stage (Fig 4A). Upon adulthood, vit-2 expression levels increase considerably in both auxin-treated and auxin-control animals. However, vit-2 promoter activity shows a significant reduction in auxin-treated animals at 72 hours of development. This difference in vit-2 expression levels is no longer present in older adults, when vit-2 expression is peaking and becomes similar between animals with mononucleated and binucleated intestinal cells (Fig 4A, 96 and 120 hour time points). To further investigate whether mononucleated intestinal cells have reduced ability to express vit-2 during early stages of adulthood, we performed single molecule fluorescence in situ hybridization (smFISH) at 54 hours of development, when vit-2 starts becoming expressed in intestinal cells and it is possible to count single vit-2 mRNA molecules in intestinal cells. Our analyses revealed that animals with mononucleated intestinal cells had on average fewer vit-2 mRNAs per cell and reduced nascent transcription in their nuclei (Fig 4B–4D). Consistent with a transcriptional down-regulation of vit-2 in animals with mononucleated intestinal cells, we also found lower levels of VIT-2 protein in embryos derived from mothers with mononucleated intestines (Fig 4E). Again, VIT-2 levels were significantly lower in embryos derived from auxin-treated 72-hour old adults and, similar to the vit-2 promoter activity, the difference between auxin-treated and control animals was no longer present on subsequent days of adult development. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Perturbation of binucleation decreases yolk protein expression and lipid transport to the germline. (A) Fluorescence images and normalized total fluorescence intensities of a Pvit-2::NLSGFP transcriptional reporter at different moments during adult development for Pges-1::TIR1; AID::knl-1 animals grown under control (ct, n = 53 to 111) or auxin (+, n = 71 to 111) conditions, in 5 replicate experiments. Each data point represents the normalized fluorescence intensity of one worm. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. P values were calculated by Student t test. NS, not significant (P > 0.05). (B–D) smFISH analyses of vit-2 mRNAs in Pges-1::TIR1; AID::knl-1 animals at 54 hours of development, grown under control (ct) or auxin (+) conditions. (B) Z-stack projections of vit-2 smFISH (magenta) in Int3D/V cells. Animals contain an intestinal membrane marker (Pges-1::GFP-PH) that was used to determine cell boundaries (dashed line). Scale bar represents 10 μm. (C) Quantification of number of cellular vit-2 mRNAs in Int3D/V cells of animals grown under control (ct, n = 62) or auxin (+, n = 88) conditions in 2 replicate experiments. Scatter plot with median (middle line) and 95% confidence interval (error bars). Each dot represents the number of vit-2 mRNAs in a single cell. P value was calculated by Mann–Whitney test. (D) Quantification of number of nascent vit-2 transcripts per cell in Pges-1::TIR1; AID::knl-1 animals grown under control (ct) or auxin (+) conditions. Scatter plot with median (middle line) and 95% confidence interval (error bars). Each dot represents the number of nascent vit-2 mRNAs in a single cell. P value was calculated by Mann–Whitney test. (E) Fluorescence images and boxplots showing total endogenous VIT-2::GFP fluorescence intensity in early embryos (1-cell stage to 4-cell stage) derived from control (ct, n = 38 to 71) or auxin (+, n = 36 to 71) Pges-1::TIR1; AID::knl-1 animals, in 2 replicate experiments. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. P values were calculated by Mann–Whitney test. NS, not significant (P > 0.05). (F) Z-stack projection of fluorescence images and boxplots of normalized total fluorescence intensities of BODIPY lipid staining of early embryos isolated from young adult (72 hours) Pges-1::TIR1; AID::knl-1 animals grown under control (ct, n = 56) or auxin (+, n = 77) conditions in 3 replicate experiments. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. P values were calculated by Mann–Whitney test. (G) Z-stack projection images and boxplots of normalized fluorescence intensities of BODIPY lipid staining of Pges-1::TIR1; AID::knl-1 young adults (72 hours) that were grown under control (ct, n = 62) or auxin (+, n = 63) conditions in 2 replicate experiments. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. P values were calculated by Mann–Whitney test. Underlying data can be found in S1 Data. AID, auxin-inducible degron; smFISH, single molecule fluorescence in situ hybridisation. https://doi.org/10.1371/journal.pbio.3001597.g004 Because vitellogenins are required to transport lipids from the intestine to the germline, we investigated whether decreased vitellogenin levels resulted in a reduction of lipid loading into oocytes. For this, we used BODIPY staining to quantify lipid levels in embryos and young adult worms. We observed lower lipid levels in embryos from auxin-treated worms, consistent with reduced lipid loading into oocytes of animals with mononucleated intestines (Fig 4F). Moreover, we found higher amounts of lipids in the intestines of adults with mononucleated intestines (Fig 4G), indicating that specifically the transport of lipids is impaired, rather than lipid production or uptake. The effect of mononucleation on lipid levels was more striking than the decrease that we observed in Pvit-2::NLSGFP expression, which is consistent with the notion that vitellogenins function in 2 distinct yolk complexes, and that multiple vitellogenins are down-regulated in mononucleated animals. Together, our findings indicate that expression of vitellogenins is hampered by blocking binucleation in the intestine, resulting in reduced lipid loading into developing oocytes.

Intestinal binucleation enhances C. elegans fitness Abundant expression of vitellogenins in the adult maternal C. elegans intestine is important to support postembryonic development and fertility of the offspring [33,36]. To assess whether intestinal binucleation is functionally important to generate sufficient vitellogenins to promote progeny fitness, we performed a competitive fitness assay with animals that have either mononucleated or binucleated intestinal cells. To this end, we generated strains with either a Pmyo-2::mCherry or a Pmyo-2::GFP pharyngeal marker in addition to the AID::knl-1 or AID::cdk-1 alleles. This allowed us to follow the progeny of animals with mononucleated or binucleated cells over several generations. By mixing equal amounts of animals with mononucleated (auxin-treated, +) or binucleated intestines (control condition, ct) on plates and counting the proportion of GFP and mCherry positive progeny after several generations, we could determine how binucleation influences reproductive fitness (Fig 5A). It is important to note that auxin was only added during development of the parental worms and that progeny were not treated with auxin and their intestinal cells were thus binucleated. To control for fitness differences due to the Pmyo-2::mCherry or Pmyo-2::GFP markers, we included experiments with reversed fluorescent markers and used measurements of the fitness difference between Pmyo-2::mCherry and Pmyo-2::GFP control worms to normalize our data (see Methods for details). Using the AID::knl-1 strain, we found that after one generation, animals with a wild-type intestine had given rise to an average of 57.0% (±14.4%) of the population, while animals with a mononucleated intestine only gave rise to 43.0% (±14.4%) of the population (Fig 5B). Similar numbers were obtained with the AID::cdk-1 strain (Fig 5C). These results demonstrate that animals with binucleated intestines have a significant fitness advantage over animals with mononucleated intestines. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Inhibition of intestinal binucleation reduces relative reproductive fitness. (A) Overview of the relative fitness assay. L4 animals grown under auxin (+) or control (ct) conditions carrying a pharyngeal marker in either red (Pmyo-2::mCherry) or green (Pmyo-2::GFP) are transferred in equal amounts to a single plate. Worms are grown until the plate is full or starved, after which a random subset of worms are transferred to a new plate, effectively diluting the population, after which the proportion of mCherry+ and GFP+ progeny is counted. In replicate experiments, pharyngeal markers were swapped for the auxin and control conditions. Reproductive fitness measurements shown in panels B and C were made after one plate dilution, whereas reproductive fitness measured over consecutive plate dilutions is shown in panel G. (B, C) Boxplots showing the normalized proportion of progeny originating from worms with a wild-type binucleated (ct, n = 40 plates/280 worms and 34 plates/238 worms) or mononucleated (+, n = 40 plates/280 worms and 34 plates/238 worms) intestine in relative fitness assays using either the AID::cdk-1 (B) or AID::knl-1 alleles (C) to block binucleation, in 2 replicate experiments. Measurements were made after one plate dilution. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. (D) Boxplot showing total brood size of Pges-1::TIR1; AID::knl-1 animals grown under control conditions (ct, n = 43 plates) or in the presence of auxin (+, n = 43 plates), in 3 replicate experiments. Amounts of progeny were counted for 3 days of egg laying. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. (E) Violin boxplots depicting progeny growth rates from worms with increasing maternal age, grown under control conditions (ct, n = 9 plates) or in the presence of auxin (+, n = 9 plates), in 3 replicate experiments. For each condition, the timing of the L3 molt was quantified (see Methods for details), and the average timing in the control condition for each maternal age was used to calculate the developmental delay. Horizontal lines indicate the median and 25th to 75th percentile, violin plots extend to min and max values and individual values are shown as dots. (F) Scatter plots of brood size in the second generation of Pges-1::TIR1; AID::knl-1 animals (F2) that originated from animals grown under control (ct, n = 70 plates) or auxin (+, n = 71 plates) conditions. Amounts of animals were counted for 3 days of egg laying. Line and error bars represent mean and SEM. (G) Exponential growth model assuming a intergenerational (green) or transgenerational (orange) effect of blocking binucleation on reproductive fitness. Measurements of the mean reproductive fitness that were obtained over consecutive plate dilutions are shown in black, and error bars depict SEM. P values were calculated by Mann–Whitney test. NS, not significant (P > 0.05). Underlying data can be found in S1 Data. AID, auxin-inducible degron. https://doi.org/10.1371/journal.pbio.3001597.g005 To understand how perturbation of binucleation affects fitness, we investigated several aspects of worm growth and reproduction upon inhibition of binucleation. When analyzing AID::knl-1 and AID::cdk-1 animals, we noticed that the AID::cdk-1 strain had smaller brood sizes and increased embryonic lethality compared to wild-type animals, both in the presence or absence of auxin, suggesting that the AID tag compromises CDK-1 function resulting in a weak hypomorph (S5A–S5C Fig). Nonetheless, when comparing animals with mononucleated or binucleated intestines in either the AID::cdk-1 or AID::knl-1 background, we found no reduction in brood sizes in animals with mononucleated intestines (Fig 5D, S5C Fig). However, we did find a significant delay in progeny growth when examining eggs that were laid between 72 and 96 hours of development, corresponding to the first and second day of adulthood (Fig 5E). This delay was not due to growing animals on auxin or the presence of TIR1, as worms lacking the AID::knl-1 or AID::cdk-1 alleles did not show developmental delays or reproductive defects (S5D and S5E Fig). Moreover, animals derived from mothers with mononucleated intestines showed a mild decrease in brood sizes compared to controls (Fig 5F). To test whether these developmental effects could account for the observed decreases in fitness, we used an exponential growth model to predict the relative fitness of worms with a mononucleated intestine based on our growth and reproduction measurements. In this model, we differentiated the possibilities of an intergenerational effect of blocking binucleation in the intestine, where only the first generation of progeny is affected, and a transgenerational effect that lasts for several generations. Comparing this model with our experimental data revealed 2 things. First, the plateau in the proportion of progeny coming from mothers with a mononucleated intestine indicates that there is an intergenerational rather than a transgenerational effect on progeny fitness (Fig 5G). Second, the fitness decrease that we measured closely matches the model, suggesting that decreases in progeny growth and reproduction fully explain the difference in relative fitness. Taken together, our data show that intestinal binucleation is important for reproductive fitness and that blocking binucleation decreases progeny growth and reproduction.

Binucleation of the C. elegans intestine is important for the rapid up-regulation of gene expression To understand why mononucleated cells have decreased levels of vit-2 gene expression compared to binucleated cells, we performed in-depth analysis of our single-worm RNA sequencing data to identify similarities between genes that are affected by binucleation. First, we found no correlation between expression levels and differential gene expression in animals with a mononucleated intestine, indicating that binucleation is not solely important for the expression of highly or lowly expressed genes (S3A and S3C Fig). Next, we used sequencing data from wild-type worms at different stages of development, available from ModEncode [38], and analyzed the developmental expression profiles of the genes that we identified in our sequencing of animals with mononucleated intestinal nuclei. Overall, genes that are down-regulated in worms with a mononucleated intestine show an increase in gene expression throughout wild-type development, with a strong increase in expression from the L4 to adult stage (Fig 6A). In contrast, genes either unaffected by perturbation of binucleation or genes that are intestinally expressed showed a more constant expression profile during wild-type development. In addition, we found a substantial enrichment (P = 6.4e-8) of down-regulated genes located on the X chromosome (S6A Fig). The X chromosome–enriched down-regulation is not restricted to vitellogenin genes, of which 5 out of 6 are located on the X chromosome, but rather a global repression of all X chromosomal genes (S6B Fig). Possibly, an altered X chromosome organization within the nucleus contributes to the decreased expression in mononucleated cells. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Binucleation allows rapid up-regulation of transcription. (A) Mean expression for down-regulated (n = 96 genes), unaffected (n = 113 genes), or intestinally expressed (n = 80 genes) subsets of genes in different developmental stages from ModEncode FPKM gene expression data. Area around curve shows confidence interval. (B) Schematic overview of heat shock–inducible NLS-GFP transgene. smFISH probes were designed against the sfGFP sequence. (C) Schematic overview of smFISH and imaging experiments following heat shock induction in Pges-1::TIR1; AID::knl-1 animals. Young L4 stage animals grown under control or auxin conditions were heat shocked in a water bath at 33°C for 30 minutes, followed by 5 minutes in a 37°C air incubator. Animals were left to recover at 25°C. Samples were taken after 15 minutes for smFISH analyses or every hour, for the analysis of nuclear GFP accumulation. (D) Total nuclear fluorescence intensities of intestinal nuclei at different time points after heat shock for animals grown under control (ct, n = 13 to 44) or auxin (+, n = 18 to 35) conditions, in 3 replicate experiments. Boxplots indicate the median and 25th to 75th percentile, error bars indicate min to max values, and individual values are shown as dots. P values were calculated by Student t test. (E) Representative smFISH images and quantifications of cellular GFP mRNA concentration in animals grown under auxin (+, n = 113) or control (ct, n = 112) conditions 15 minutes after heat shock, in 2 replicate experiments. Scatter plot with median (middle line) and 95% confidence interval (error bars). Each dot represents mRNA concentration in a single cell. P value was calculated by Mann–Whitney test. Scale bar represents 5 μm. Underlying data can be found in S1 Data. AID, auxin-inducible degron; FPKM, fragments per kilobase of exon per million mapped fragments; smFISH, single molecule fluorescence in situ hybridisation. https://doi.org/10.1371/journal.pbio.3001597.g006 To test whether binucleation is important for the rapid up-regulation of gene expression in general, or specifically for expression of genes that are up-regulated upon adulthood, we measured the up-regulation of a heat shock–inducible GFP-NLS reporter in animals with mononucleated or binucleated intestines. In these experiments, L4 stage animals were heat shocked, and nuclear GFP intensities were measured at multiple time points after heat shock (Fig 6B and 6C). Interestingly, mononucleated intestinal cells showed a delay in the up-regulation of nuclear GFP after heat shock compared to binucleated controls, whereas the accumulation of GFP signal in body wall muscle nuclei was similar between the 2 conditions (Fig 6D, S7 Fig). To confirm that this delay in up-regulation of heat shock–inducible expression arises at the transcriptional level, we quantified the cellular GFP mRNA density 15 minutes after heat shock induction using smFISH. Consistent with a delay in GFP up-regulation, we observed a significant decrease in GFP mRNA levels in mononucleated compared to binucleated intestinal cells (Fig 6E). Together, these results show that rapid up-regulation of transcription is impaired in cells with one rather than two nuclei.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001597

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