Decision letter: Visualizing the metazoan proliferation-quiescence decision in vivo (2020)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cell proliferation and quiescence are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these key events of the cell cycle in Caenorhabditis elegans and zebrafish through live-cell imaging. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from quiescent cells in G0, revealing a possible commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. Finally, we derive a predictive model of future proliferation behavior in C. elegans based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell-cycle control in a wide-range of developmental contexts. eLife digest All living things are made up of cells that form the different tissues, organs and structures of an organism. The human body, for example, is thought to consist of some 37 trillion cells and harbor over 200 cell types. To maintain a working organism, cells divide to create new cells and replace the ones that have died. Cell division is a tightly controlled process consisting of several steps, and cells continuously face a Shakespearean dilemma of deciding whether to continue dividing (also known as cell proliferation) or to halt the process (known as quiescence). This difficult balancing act is critical during all stages of life, from embryonic development to tissue growth in an adult. Problems in the underlying pathways can result in diseases such as cancer. Cell division is driven by proteins called CDKs, which help cells to complete their cell cycle in the correct sequence. To gain more insight into this complex process, scientists have developed tools for monitoring CDKs. One such tool is a fluorescent biosensor, a molecule that can be inserted into cells that glows and moves in response to CDK activity. The biosensor can be studied and measured in each cell using a microscope. Adikes, Kohrman, Martinez et al. adapted and optimized an existing CDK biosensor to help study cell division and the switch between proliferation and quiescence in two common research organisms, the nematode Caenorhabditis elegans and the zebrafish. Analysis of this biosensor showed that CDK activity at the end of cell division is higher if the cells will divide again but is low if the cells are going to become quiescent. This could suggest that the decision of a cell between proliferation and quiescence may happen earlier than expected. The optimized biosensor is sensitive enough to detect these differences and can even measure variations that influence proliferation in a region on C. elegans that was once thought to be unchanging. The development of this biosensor provides a useful research tool that could be used in other living organisms. Many research questions relate to cell division and so the applications of this tool are wide ranging. Introduction Organismal development requires a delicate balance between cell proliferation and cell cycle exit. In early embryos, the emphasis is placed on rapid cell proliferation, which is achieved by omitting gap phases (G1 and G2) and establishing a biphasic cell cycle that rapidly alternates between DNA synthesis (S phase) and mitosis (M phase) (Edgar and O'Farrell, 1989; Newport and Kirschner, 1982). After several rounds of embryonic cell division, the gap phases are introduced, coincident in many organisms with cell fate decisions and the execution of morphogenetic cell behaviors (Foe, 1989; Grosshans and Wieschaus, 2000). These gap phases are believed to function as commitment points for cell-cycle progression decisions. The earliest point of commitment occurs during G1, which is the focus of this study. Cells either engage in cell-cycle progression and enter S phase, or they exit the cell cycle altogether and enter a cell-cycle phase referred to as G0 and undergo quiescence or terminal differentiation (Sun and Buttitta, 2017). Although the location of the G1 commitment point in yeast (Start) and cultured mammalian cells (Restriction Point) has in large part been spatiotemporally mapped and molecularly characterized (Hartwell et al., 1974; Pardee, 1974; Spencer et al., 2013), when cells make this decision in living organisms while integrating intrinsic and the extrinsic cues of their local microenvironment during development remains poorly understood. A cell-cycle sensor that is amenable to such in vivo studies can shed new light on this four-decade-old biological phenomenon. In 2008, Sakaue-Sawano and colleagues engineered a multicolor fluorescent ubiquitination-based cell-cycle indicator (FUCCI) for mammalian cell culture (Sakaue-Sawano et al., 2008). FUCCI has since been adapted for many research organisms (Özpolat et al., 2017; Zielke and Edgar, 2015). However, FUCCI on its own cannot distinguish between a cell residing in G1 that will cycle again upon completing mitosis and a cell that is poised to enter G0 (Oki et al., 2015). Separating G1 from G0 is an essential first step to understanding mechanisms controlling cell cycle exit during quiescence or terminal differentiation. To distinguish G1 from G0 in mammalian cell culture, Hanh, Spencer and colleagues developed and implemented a single-color ratiometric sensor of cell-cycle state composed of a fragment of human DNA helicase B (DHB) fused to a fluorescent protein that is phosphorylated by CDKs (Hahn et al., 2009; Schwarz et al., 2018; Spencer et al., 2013). Notably, through quantitative measurements of CDK activity, this sensor provided new insights into the proliferation-quiescence decision in cultured mammalian cells by identifying cycling cells that exit mitosis in a CDK-increasing (CDKinc) state and quiescent cells that exit mitosis in a CDK-low (CDKlow) state (Spencer et al., 2013). Nonetheless, a DHB-based CDK sensor has not been utilized to evaluate the proliferation-quiescence decision in vivo. In this study, we investigate the proliferation-quiescence decision in Caenorhabditis elegans and zebrafish, two powerful in vivo systems with radically different modes of development. We generate transgenic CDK sensor lines in each organism to examine this decision live at mitotic exit. By quantifying CDK activity, or DHB ratios, at mitotic exit, we are able to predict future cell behavior across several embryonic and post-embryonic lineages. Despite cells generally exiting mitosis with decreased CDK-activity levels, we reliably distinguish cycling cells that exit mitosis into G1, in a CDKinc state, from quiescent cells that exit mitosis into G0, in a CDKlow state. To gain insights into cell-cycle progression commitment, we examine the activity of C. elegans cki-1, a cyclin-dependent kinase inhibitor (CKI) of the Cip/Kip family, demonstrating that endogenous CKI-1 levels are anti-correlated with CDK activity during the proliferation-quiescence decision. We propose that integration of CKI-1 levels in the mother cell and high CKI-1, low CDK activity at mitotic exit mediate this decision. By utilizing the CDK sensor to predict future cell behavior, we uncover a cryptic stochasticity that occurs in a temperature-dependent fashion in the C. elegans vulva, an otherwise invariant and well-characterized lineage. Finally, we reveal cell-cycle dynamics in zebrafish, an organism that lacks a defined cell lineage, demonstrating that quiescent embryonic tissues display DHB ratios that correlate with those observed in G0 cells in C. elegans. Together, we present a tool for visualizing G1/G0 dynamics in vivo during metazoan development that can be used to study the interplay between cell proliferation and quiescence. Results Design and characterization of a live C. elegans CDK sensor to define interphase states We synthesized a C. elegans codon-optimized fragment of human DHB composed of amino acids 994–1087 (Hahn et al., 2009; Spencer et al., 2013). The fragment contains four serine residues that are consensus CDK phosphorylation sites (Moser et al., 2018; Spencer et al., 2013). These serines flank a nuclear localization signal (NLS) that is adjacent to a nuclear export signal (NES) (Figure 1A). When CDK activity is low, the NLS is strong, the NES is weak and DHB localizes to the nucleus. However, when CDK activity increases during cell-cycle entry, the NLS is masked and DHB re-localizes to the cytoplasm (Figure 1B). Using this DHB fragment, we generated two CDK sensors by fusing green fluorescent protein (GFP) or two copies of a red fluorescent protein, mKate2 (2xmKate2), to the DHB C-terminus (Figure 1A). To visualize the nucleus, we co-expressed his-58/histone H2B fused to 2xmKate2 or GFP, respectively, which is separated from DHB by a P2A self-cleaving viral peptide (Ahier and Jarriault, 2014). We drove the expression of each CDK sensor via a ubiquitous rps-27 promoter (Ruijtenberg and van den Heuvel, 2015). Figure 1 with 3 supplements see all Download asset Open asset Design and characterization of a live C. elegans CDK sensor to define interphase states. (A) Schematic of the CDK sensor fused to GFP (top) or 2xmKate2 (bottom) and a nuclear mask (H2B::FP) separated by a self-cleaving peptide (P2A). Inset: nuclear localization signal (NLS), nuclear export signal (NES), and consensus CDK phosphorylation sites on serine (S) residues. (B) Schematic of CDK sensor translocation during the cell cycle. (C) Representative fluorescence overlay (bottom), H2B (top), and DHB::2xmKate2 (middle) time series images during embryo cell divisions (see Figure 1—video 1). Orange arrowheads follow the division of a single blastomere. (D) Confocal micrograph montage of CDK sensor expression in a C. elegans L3 stage larva. (D’) Three somatic tissues are highlighted (inset, dashed orange box) shown at higher magnification with pseudo-colored nuclei (magenta) depicting cells of interest. (E) Schematic of quantification and equation used to obtain the cytoplasmic:nuclear ratio of DHB. (F) Representative images of sensor expression in vulval precursor cells (VPCs) at peak G2 and 20 min after anaphase during G1 in DHB::GFP (gray) and DHB::2xmKate2 (magenta). Orange arrowheads indicate the VPCs. (G) Dot plot depicting G2 and G1 DHB ratios of the two CDK sensor variants in the VPCs (n ≥ 15 cells per phase). (H) Plot of DHB ratios in VPCs during one round of cell division, measured every 5 min (n ≥ 11 mother cells per strain). Dotted line indicates time of anaphase. Error bars and shaded error bands depict mean ± SD. (I) Representative images of sensor and PCNA expression in VPCs during G1 and S phase. Orange arrowheads indicate the VPCs. Blue arrowheads indicate S phase PCNA puncta. (J) Dot plot depicting G1 and S phase DHB::2xmKate2 ratios based on absence or presence of PCNA puncta (n ≥ 10 cells per phase). **p≤0.01, ****p≤0.0001. Significance determined by statistical simulation; p-values in Supplementary file 1. Scale bar = 10 µm (except in D: 20 µm and F, I: 5 µm). Figure 1—source data 1 Source data for Figure 1. Raw data of DHB ratios used to generate Figure 1G, H and J in Microsoft Excel format. https://cdn.elifesciences.org/articles/63265/elife-63265-fig1-data1-v2.xlsx Download elife-63265-fig1-data1-v2.xlsx Figure 1—source data 2 Source data for Figure 1—figure supplement 1. Raw data of DHB ratios used to generate Figure 1—figure supplement 1G, H and J in Microsoft Excel format. https://cdn.elifesciences.org/articles/63265/elife-63265-fig1-data2-v2.xlsx Download elife-63265-fig1-data2-v2.xlsx To test both the GFP (Figure 1—figure supplement 1A and B) and 2xmKate2 (Figure 1C, Figure 1—figure supplement 1B and C) versions of our CDK sensor, we began by examining cell divisions in the C. elegans embryo and germline (Figure 1—video 1). First, we visualized cells in the embryonic intestine, which is clonally derived from the E blastomere, as these are the first cells in the embryo to have gap phases (Edgar and McGhee, 1988). The E blastomere goes through four rounds of divisions to give rise to 16 descendants (E16 cells) about 4 hr after first cleavage. While 12 of the E16 cells have completed their embryonic divisions at this stage (Leung et al., 1999), four cells called E16* star cells divide once more to generate the 20-celled intestine (E20) (Rasmussen et al., 2013; Yang and Feldman, 2015). Thus, we wondered whether our CDK sensor could be used to distinguish between cycling E16* star cells and quiescent E16 cells. To accomplish this, we tracked E16* star cell division from the E16–E20 stage and observed that DHB::GFP localizes in a cell-cycle-dependent fashion during these divisions, with DHB::GFP translocating from the nucleus to the cytoplasm and then re-locating to the nucleus at the completion of E16* star cell division (Figure 1—figure supplement 1A). Consistent with our observations using the GFP version of our CDK sensor in mid-embryogenesis, DHB::2xmKate2 also dynamically translocates from the nucleus to the cytoplasm during cell divisions in the early embryo (Figure 1C). Second, we examined the localization of DHB::GFP and DHB::2xmKate2 in the adult C. elegans germline (Figure 1—figure supplement 1B and C). Here we detected a gradient of live CDK activity, from high in the distal mitotic progenitor zone to low in the proximal meiotic regions, as described with EdU incorporation and phospho-histone H3 staining (Kocsisova et al., 2018). Together, these results demonstrate that our CDK sensor is dynamic during cell-cycle progression in the C. elegans embryo and germline. The ability to distinguish cycling cells from quiescent cells in the embryo made us wonder whether we could also distinguish these cellular states post-embryogenesis. Therefore, we examined our CDK sensor in several post-embryonic somatic lineages that undergo proliferation followed by cell cycle exit (Sulston and Horvitz, 1977). Specifically, we selected the sex myoblasts (SM), the somatic sheath (SS) and ventral uterine (VU) cells of the somatic gonad, and the vulval precursor cells (VPCs) (Figure 1D and D’). To define each phase of the cell cycle while these lineages are proliferating, we combined static and time-lapse imaging approaches to measure cytoplasmic:nuclear DHB ratios for G1, S, and G2 (Figure 1E–J, Figure 1—figure supplement 1D–M). First, we quantified DHB ratios following time-lapse of VPC (Figure 1F–H), SM (Figure 1—figure supplement 1D, F and G) and uterine (Figure 1—figure supplement 1E, F and H) divisions to determine peak values of G2 CDK activity. All lineages exhibited the same CDK sensor localization pattern during peak G2 (i.e. maximal nuclear exclusion). We then RNAi depleted the sole C. elegans CDK1 homolog, cdk-1, to induce a penetrant G2 phase arrest in the SM cells to corroborate these results. Quantification of DHB ratios following cdk-1 RNAi treatment showed a mean ratio of 1.00 ± 0.28 and 2.36 ± 0.70 in the GFP and 2xmKate2 versions of our CDK sensor, respectively (Figure 1—figure supplement 1I and J). Next, for each lineage (Figure 1F, Figure 1—figure supplement 1D and E), we quantified DHB ratios 25 min after anaphase from our time-lapses to determine a threshold for G1 phase CDK activity. In G1, DHB::GFP and DHB::2xmKate2 were nuclear localized after mitotic exit with mean ratios of 0.35 ± 0.14 and 0.58 ± 0.32 in VPCs, 0.59 ± 0.11 and 0.97 ± 0.20 in SMs, and 0.67 ± 0.10 and 1.13 ± 0.17 in uterine cells (Figure 1G and H, Figure 1—figure supplement 1F–H). Finally, we paired DHB::2xmKate2 with a reporter for S phase, fusing GFP to the sole C. elegans proliferating cell nuclear antigen (PCNA) homolog, pcn-1, expressed under its own endogenous promoter at single copy. Although nuclear localized throughout the cell cycle, PCNA forms sub-nuclear puncta only in S phase (Brauchle et al., 2003; Dwivedi et al., 2019; Strzyz et al., 2015; Zerjatke et al., 2017). Analysis of time-lapse data found that punctate expression of PCN-1::GFP correlated with mean DHB::2xmKate2 ratios of 1.02 ± 0.22 in VPC (Figure 1I and J), 0.89 ± 0.16 in SM (Figure 1—figure supplement 1K and M; Figure 1—video 2), and 1.00 ± 0.10 in uterine (Figure 1—figure supplement 1L and M) lineages. Despite individual lineages showing differences in CDK activity (Figure 1—figure supplement 1F and M–O), primarily in G1, we can establish DHB ratios for each interphase state (G1/S/G2) across several post-embryonic somatic lineages using our CDK sensor paired with a PCNA reporter. We next wondered if we could distinguish G1 from G0 as these somatic lineages exit their final cell division; therefore, allowing us to visibly and quantitatively detect cellular quiescence in vivo. We mainly chose the DHB::GFP version of our CDK sensor to conduct the following experiments as it was more photostable. CDKlow activity after mitotic exit is predictive of cell cycle exit In asynchronously dividing MCF10A epithelial cell lines, cells that exited mitosis into a CDK2low state had a high probability of staying in G0 compared to cells that exited at a CDK2inc state (Spencer et al., 2013). We therefore wanted to determine whether the cytoplasmic:nuclear ratio of DHB::GFP following an in vivo cell division could be used to predict if a cell will enter G1 and divide again or enter G0 and undergo quiescence. Taking advantage of the predictable cell lineage pattern of C. elegans, we quantitatively correlated DHB::GFP ratios with the decision to proliferate or exit the cell cycle. We first quantified DHB::GFP ratios from time-lapse acquisitions of SM cell divisions. The SM cells undergo three rounds of cell division during the L3 and L4 larval stages before exiting the cell cycle and differentiating into uterine muscle (um) and vulval muscle (vm) (Figure 2A; and Horvitz, 1977). Quantification of DHB::GFP in this lineage that after the first and divisions, CDK activity increases after mitotic exit from an which we as a CDKinc state (Figure and Figure CDK activity following the and terminal division remains low, which we as a CDKlow state. a in DHB::GFP ratios between (CDKinc) and terminal divisions but not divisions (Figure supplement We then quantified DHB::GFP ratios during the division of two somatic the and cells. and cells undergo several rounds of division during the L3 larval stage and exit the cell cycle in the early L4 stage (Figure and Horvitz, 1977). We quantified a division and the division that to quiescence. to the SM lineage, both somatic lineages exit the round of cell division to their final division into a CDKinc state and then exit into a CDKlow state following their terminal division (Figure and F, Figure supplement Figure also a between DHB::GFP ratios in terminal divisions in the somatic (Figure supplement Figure 2 with 3 supplements see all Download asset Open asset myoblasts and somatic cells exit terminal divisions into a CDKlow state. (A) SM lineage (B) of a time-lapse showing SM cells at G1 and (C) Quantification of CDK activity in SM cells (n ≥ (D) lineage (E) of a time-lapse showing uterine cells at G1 and (F) Quantification of CDK activity in uterine cells (n ≥ (G) Quantification of CDK activity in SM cells and uterine cells following expression of CKI-1 compared to and the (n ≥ cells per nuclei (E) indicate cells of interest. Scale bars = 10 Dotted line in and F indicates time of anaphase. and shaded error bands depict mean ± SD. series measured every 5 not ****p≤0.0001. Significance determined by statistical p-values in Supplementary file 1. Figure data 1 Source data for Figure Raw data of DHB ratios used to generate Figure F and in Microsoft Excel format. Download Figure data 2 Source for Figure supplement 1. Raw data of DHB ratios used to generate Figure supplement in Microsoft Excel format. Download Next, we to determine the CDK sensor under in which cells are into To accomplish this, we generated a single transgenic line of CKI-1, the C. elegans of under an promoter paired with a of the CDK expression of CKI-1 is to result in G0 arrest et al., et al., van et al., in the SM and uterine expression of CKI-1 in cells a CDKlow G0 state, with mean DHB ratios of 0.10 ± and ± respectively (Figure as compared to control that expression ± ± or the ± 1.00 ± Thus, G0 arrest by expression of CKI-1 is by CDK-activity levels, to the G0 arrest that occurs following mitotic exit in an cell to undergo quiescence. We next examined the divisions of the and VPC lineage. The C. elegans is derived from three cells which undergo three rounds of cell division during the L3 and early L4 larval stages (Figure and et al., and Horvitz, and Horvitz, 1977). than rise to the two the of the and exit the cell cycle one round This results in a of which the adult et al., and Horvitz, 1977). Quantification of DHB::GFP ratios during VPC divisions the The of all exited their first division into a CDKinc state (Figure and After the next division, the 12 of are the cell et al., and Horvitz, 1977). this division, the nuclear localization of DHB::GFP in the cell was in to the proliferating VPCs. The cell exited into and in a CDKlow state, while the VPCs exited into a CDKinc state and to through the cell cycle (Figure and Figure 1). All VPCs exited into a CDKlow state at their terminal Consistent with these (Figure supplement our such that we can quantitatively distinguish between a cell that has completed mitosis and will continue to cycle (CDKinc) from a cell that mitosis and a G0 state Figure 3 with 2 supplements see all Download asset Open asset precursor cells (VPCs) exit terminal divisions into a CDKlow state. (A) Schematic of and VPCs. (B) All of the VPCs with the of the to facilitate vulval (C) series of CDK sensor localization in the and VPCs, as measured every 5 that the cells are born into a CDKlow state (n ≥ Dotted line indicates time of anaphase. error bands depict mean ± SD. (D) Representative images of CDK sensor localization in the VPCs from the 2 cell stage to cell are highlighted in for cell and VPCs and green for the cells. Scale bar = 10 Figure data 1 Source data for Figure Raw data of DHB ratios used to generate Figure in Microsoft Excel format. Download Figure data 2 Source for Figure supplement 1. Raw data of DHB ratios used to generate Figure supplement in Microsoft Excel format. Download CKI-1 levels peak to cell cycle exit In mammalian cell culture, endogenous levels of during G2 are predictive of whether a cell will on to divide or enter or terminal differentiation et al., 2019; et al., 2018; et al., Spencer et al., 2013). This the that endogenous levels of CKI-1 in C. elegans correlate with CDKlow or CDKinc activity. To CKI-1 dynamics with our CDK sensor, we inserted a GFP into the endogenous of via and a DHB::2xmKate2 of the sensor of into this endogenous levels of were for time-lapse to its et al., we a developmental time series of static images over the L3 and L4 larval stages to levels during and terminal divisions in the VPC lineage. We detected generally low levels of at the 2 cell stage (Figure Figure supplement In their at the 4 cell we detected an in levels in cycling cells to their next cell division, in G2 (Figure B and Figure supplement Notably, the which after this cell division, mitosis with higher levels of than its mother (Figure Figure supplement 1B). This for the VPCs at the cell and cell which show high levels of that peak after mitotic exit and high during the L4 stage (Figure E and F, Figure supplement We also observed increasing levels of in the G2 phase of mother cells that peak in their quiescent cells in the uterine (Figure supplement and SM cell lineages (Figure supplement Thus, levels of in mother cells and high upon mitotic exit in cells with CDKlow activity. These results suggest that the proliferation-quiescence decision is in the G2 phase of the cell cycle and with CKI-1 levels in the mother Figure 4 with 1 supplement see all Download asset Open asset CKI-1 levels peak to cell cycle exit. (A) Schematic of VPC divisions in the larval (B) CDK activity and CKI-1 levels across and DHB ratios for all VPCs and cells green fluorescence in VPCs and cell green ≥ cells per lineage. (C) Representative images of VPCs at the stage at G1 and G2 (D) Representative images of VPCs at the stage at G1 and early quiescent cells with low levels of Representative images of VPCs at the cell stage (E) and stage show early quiescent and B cell F cell and A cell (G) fluorescence in each cell of the VPC lineage (n ≥ and A early = E early = (H) of cells of each lineage that showed of early quiescence and not undergo their final (I) of CKI-1 via cells to enter and in G0 (n ≥ cells per Scale bar = 10 not ****p≤0.0001. Significance determined by statistical p-values in Supplementary file 1. Figure data 1 Source data for Figure Raw data of DHB ratios and fluorescence measurements of used to generate Figure and data used to generate Figure in Microsoft Excel format. Data also to lineage data used to generate in Figure supplement Download our of static images of we observed in the VPC lineage pattern in the early L4 larval In we that many cells to their final division and undergo early cell-cycle quiescence with coincident high levels of and low DHB We that the line we generated could be as a as GFP at the could with protein of