Transcription factors are central to neuronal development, yet their functions beyond the cells in which they are expressed remain poorly understood. Here, we uncover unexpected non-cell-autonomous roles for UNC-3, a terminal selector of cholinergic motor neuron identity in C. elegans, whose human ortholog (EBF3) is linked to a neurodevelopmental syndrome. Single-cell RNA-sequencing reveals unc-3 loss in cholinergic motor neurons elicits pronounced transcriptional changes in GABAergic motor neurons that do not express unc-3, which can be rescued by cholinergic-specific UNC-3 restoration. Mechanistically, gene network analysis identifies the pro-regenerative bZIP factor CEBP-1/CEBPB as a key driver of these transcriptional changes. At the circuit level, unc-3 loss causes synaptic and axon pathfinding defects in GABA motor neurons alongside misregulation of neurite development genes. Finally, UNC-3 not only acts as a direct transcriptional activator but also suppresses inappropriate gene expression through indirect mechanisms. Together, these findings broaden terminal selectors as both intrinsic and extrinsic regulators of neuronal identity and circuit assembly, providing a mechanistic framework for understanding EBF3-associated neurodevelopmental disease.

Mutations in two highly related chromatin-remodeling complexes, BRG1/BRM-associated factor (BAF) and Polybromo-associated BAF (PBAF), cause various neurodevelopmental conditions. Although BAF activity is required at multiple steps of nervous system development, the specific neural functions of PBAF remain largely unexplored. Through an unbiased genetic screen in C. elegans, we identify here critical roles for PBAF in postmitotic neuronal identity. In nerve cord motor neurons, PBAF represses gene expression by antagonizing the terminal selector-type transcription factor UNC-3 (EBF1-4). In contrast, PBAF activates gene expression in caudal motor neurons. This dichotomy in PBAF function generates distinct motor neuron identities. PBAF disruption leads to locomotion defects. Genetic, genomic, and biochemical data suggest that the cell type-specific functions of PBAF in different motor neuron groups arise via its recruitment to specific gene loci by conserved transcription factors, such as MAB-9/TBX20. Altogether, our findings provide a conceptual framework to understand specific neuronal defects that arise in neurological conditions caused by mutations in broadly expressed chromatin-remodeling complexes.

Transcription factors (TFs) are essential for neuronal identity, yet their potential non-cell-autonomous functions remain largely unexplored. Here, we uncover both cell- and non-cell-autonomous roles for the conserved terminal selector UNC-3 in C. elegans motor neurons (MNs). UNC-3 is an ortholog of human EBF3, mutations in which cause a severe neurodevelopmental syndrome. Single-cell RNA sequencing of cholinergic MNs, which express unc-3, and downstream GABA MNs, which do not, revealed that unc-3 loss disrupts neuronal identity in distinct ways across MN classes. Four cholinergic MN classes lose their molecular identity entirely, whereas the AS class retains it partially, illuminating terminal selector-driven neuronal diversification processes. Integrated transcriptomic and genomic analyses uncovered a dual cell-autonomous role for UNC-3 as both a direct activator and repressor of neuron-type-specific genes in cholinergic MNs, including repression of alternate neurotransmitter programs. Unexpectedly, unc-3 loss also caused widespread transcriptional, morphological, and connectivity defects in downstream GABA MNs. Mechanistically, these non-cell-autonomous effects are mediated by cholinergic neurotransmission and include activation of the pro-regenerative bZIP TF CEBP-1 (C/EBP) and dysregulation of UNC-6/Netrin signaling. These findings redefine terminal selectors as both intrinsic and extrinsic regulators of neuronal identity and circuit assembly, offering a mechanistic framework for understanding EBF3 syndrome pathogenesis.

Latrophilins are conserved adhesion-type G-protein-coupled receptors associated with embryonic defects and lethality. However, their mechanistic roles and ligands in embryogenesis remain unknown. Here, we identified TOL-1, the sole Toll-like receptor in Caenorhabditis elegans, as a ligand for the C. elegans latrophilin, LAT-1. The extracellular lectin domain of LAT-1 directly binds to the second leucine-rich repeat domain of TOL-1. The crystal structure and cryo-electron microscopy density map of the LAT-1-TOL-1 extracellular region complex reveal a one-to-one lectin domain interaction with the convex face of a leucine-rich repeat domain. In C. elegans, endogenous mRNA and protein localization analyses showed mutually exclusive sites of expression, suggesting that in vivo LAT-1-TOL-1 interactions mostly occur in trans. Mutagenesis of key interface residues that disrupt the LAT-1-TOL-1 interaction led to partial lethality and malformed embryos. Thus, TOL-1 binding to LAT-1 represents a receptor-ligand axis essential for animal development.

Motor neurons (MNs) constitute an ancient cell type targeted by multiple adult-onset diseases. It is therefore important to define the molecular makeup of adult MNs in animal models and extract organizing principles. Here, we generated a comprehensive molecular atlas of adult Caenorhabditis elegans MNs and a searchable database (http://celegans.spinalcordatlas.org). Single-cell RNA-sequencing of 13,200 cells revealed that ventral nerve cord MNs cluster into 29 molecularly distinct subclasses. All subclasses are delineated by unique expression codes of either neuropeptide or transcription factor gene families. Strikingly, we found that combinatorial codes of homeodomain transcription factor genes define adult MN diversity both in C. elegans and mice. Further, molecularly defined MN subclasses in C. elegans display distinct patterns of connectivity. Hence, our study couples the connectivity map of the C. elegans motor circuit with a molecular atlas of its constituent MNs, and uncovers organizing principles and conserved molecular codes of adult MN diversity.

AT-rich interaction domain 3 (ARID3) transcription factors are expressed in the nervous system, but their mechanisms of action are largely unknown. Here, we provide, in vivo, a genome-wide binding map for CFI-1, the sole C. elegans ARID3 ortholog. We identify 6,396 protein-coding genes as putative direct targets of CFI-1, most of which encode neuronal terminal differentiation markers. In head sensory neurons, CFI-1 directly activates multiple terminal differentiation genes, thereby acting as a terminal selector. In motor neurons, however, CFI-1 acts as a direct repressor, continuously antagonizing three transcriptional activa-tors. By focusing on the glr-4/GRIK4 glutamate receptor locus, we identify proximal CFI-1 binding sites and histone methyltransferase activity as necessary for glr-4 repression. Rescue assays reveal functional redundancy between core and extended DNA-binding ARID domains and a strict requirement for REKLES, the ARID3 oligomerization domain. Altogether, this study uncovers cell-context-dependent mechanisms through which a single ARID3 protein controls the terminal differentiation of distinct neuron types.

Cellular sodium ion (Na+) homeostasis is integral to organism physiology. Our current understanding of Na+ homeostasis is largely limited to Na+ transport at the plasma membrane. Organelles may also contribute to Na+ homeostasis; however, the direction of Na+ flow across organelle membranes is unknown because organellar Na+ cannot be imaged. Here we report a pH-independent, organelle-targetable, ratiometric probe that reports lumenal Na+. It is a DNA nanodevice containing a Na+-sensitive fluorophore, a reference dye and an organelle-targeting domain. By measuring Na+ at single endosome resolution in mammalian cells and Caenorhabditis elegans, we discovered that lumenal Na+ levels in each stage of the endolysosomal pathway exceed cytosolic levels and decrease as endosomes mature. Further, we find that lysosomal Na+ levels in nematodes are modulated by the Na+/H+ exchanger NHX-5 in response to salt stress. The ability to image subcellular Na+ will unveil mechanisms of Na+ homeostasis at an increased level of cellular detail. A probe reports absolute sodium levels in acidic organelles with single-organelle resolution.

Latrophilins/ADGRLs are conserved adhesion-type G protein-coupled receptors associated with early embryonic morphogenesis defects, lethality, and sterility across multiple model organisms. However, their mechanistic roles in embryogenesis and the identity of their binding ligands remain unknown. Here, we identified a cell-surface receptor, TOL-1, the sole Toll-like receptor in C. elegans , as a novel ligand for the C. elegans Latrophilin, LAT-1. The extracellular lectin domain of LAT-1 directly binds to the second leucine-rich repeat domain of TOL-1. The highresolution crystal structure and the cryo-EM density map of the LAT-1–TOL-1 ectodomain complex reveal a previously-unobserved mode of one-to-one interaction enabled by a large interface. CRISPR/Cas9-mediated mutation of key interface residues selectively disrupted the endogenous LAT-1–TOL-1 interaction in C. elegans, leading to partial sterility, lethality, and malformed embryos. Thus, TOL-1 binding to LAT-1 represents a receptor-ligand axis essential for animal morphogenesis.

SUMMARY ARID3 transcription factors are expressed in the nervous system, but their functions and mechanisms of action are largely unknown. Here, we generated in vivo a genome-wide binding map for CFI-1, the sole C. elegans ARID3 ortholog. We identified 6,396 protein-coding genes as putative direct targets of CFI-1, most of which (77%) are expressed in post-mitotic neurons and encode terminal differentiation markers (e.g., neurotransmitter receptors, ion channels, neuropeptides). To gain mechanistic insights, we focused on two neuron types. In sensory neurons (IL2 class), CFI-1 exerts a dual role: it acts directly to activate, and indirectly to repress, distinct terminal differentiation genes. In motor neurons, however, CFI-1 acts directly as a repressor, continuously antagonizing three transcriptional activators (UNC-3/Ebf, LIN-39/Hox4-5, MAB-5/Hox6-8). By focusing on a glutamate receptor gene ( glr-4/GRIK1 ), we found CFI-1 exerts its repressive activity through proximal binding to the glr-4 locus. Further, the core DNA binding domain of CFI-1 is partially required for glr-4 repression in motor neurons. Altogether, this study uncovers cell context-dependent mechanisms through which a single ARID3 protein controls the terminal differentiation of distinct neuron types.

Hox transcription factors play fundamental roles during early patterning, but they are also expressed continuously, from embryonic stages through adulthood, in the nervous system. However, the functional significance of their sustained expression remains unclear. In C. elegans motor neurons (MNs), we find that LIN-39 (Scr/Dfd/Hox4-5) is continuously required during post-embryonic life to maintain neurotransmitter identity, a core element of neuronal function. LIN-39 acts directly to co-regulate genes that define cholinergic identity (e.g., unc-17/VAChT, cho-1/ChT). We further show that LIN-39, MAB-5 (Antp/Hox6-8) and the transcription factor UNC-3 (Collier/Ebf) operate in a positive feedforward loop to ensure continuous and robust expression of cholinergic identity genes. Finally, we identify a two-component design principle for homeostatic control of Hox gene expression in adult MNs: Hox transcriptional autoregulation is counterbalanced by negative UNC-3 feedback. These findings uncover a noncanonical role for Hox proteins during post-embryonic life, critically broadening their functional repertoire from early patterning to the control of neurotransmitter identity.

The nervous system emerges from a series of genetic programs that generate a remarkable array of neuronal cell types. Each cell type must acquire a distinct anatomical position, morphology, and function, enabling the gen-eration of specialized circuits that drive animal behavior. How are these diverse cell types and circuits patterned along the anterior-posterior (A-P) axis of the animal body? Hox genes encode transcription factors that regulate cell fate and patterning events along the A-P axis of the nervous system. While most of our understanding of Hox-mediated control of neuronal development stems from studies in segmented animals like flies, mice, and zebrafish, important new themes are emerging from work in a non-segmented animal: the nematode Caeno-rhabditis elegans. Studies in C. elegans support the idea that Hox genes are needed continuously and across different life stages in the nervous system; they are not only required in dividing progenitor cells, but also in post-mitotic neurons during development and adult life. In C. elegans embryos and young larvae, Hox genes control progenitor cell specification, cell survival, and neuronal migration, consistent with their neural patterning roles in other animals. In late larvae and adults, C. elegans Hox genes control neuron type-specific identity features critical for neuronal function, thereby extending the Hox functional repertoire beyond early patterning. Here, we provide a comprehensive review of Hox studies in the C. elegans nervous system. To relate to readers outside the C. elegans community, we highlight conserved roles of Hox genes in patterning the nervous system of invertebrate and vertebrate animals. We end by calling attention to new functions in adult post-mitotic neurons for these paradigmatic regulators of cell fate.

Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 14 homeobox genes in 24 distinct neuron classes that are mostly unrelated by location, function and lineage history. 12 of these 24 neuron classes had no homeobox gene function ascribed to them before, while in the other 12 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analyses, 113 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancestral feature of animal nervous systems.

Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and chromatin regulating factors that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.

4 5 Yinan Li , Jayson J. Smith, Filipe Marques, Anthony Osuma, Hsin-Chiao Huang, 6 Paschalis Kratsios 7 8 1 Department of Neurobiology, University of Chicago, Chicago, IL, USA 9 2 Committee on Neurobiology, University of Chicago, Chicago, IL, USA 10 3 Committee on Development, Regeneration and Stem Cell Biology, University of Chicago, 11 Chicago, IL, USA 12 4 University of Chicago Neuroscience Institute, Chicago, IL, USA 13 14

Light sheet fluorescence microscopy (LSFM) has become a method of choice for live imaging because of its fast acquisition and reduced photobleaching and phototoxicity. Despite the strengths and growing availability of LSFM systems, no generalized LSFM mounting protocol has been adapted for live imaging of post-embryonic stages of C. elegans. A major challenge has been to develop methods to limit animal movement using a mounting media that matches the refractive index of the optical system. Here, we describe a simple mounting and immobilization protocol using a refractive-index matched UV-curable hydrogel within fluorinated ethylene propylene (FEP) tubes for efficient and reliable imaging of larval and adult C. elegans stages.

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; Sulston and Horvitz, 1977). Quantification of DHB::GFP in this lineage revealed that shortly after the first and second divisions, CDK activity increases immediately after mitotic exit from an intermediate level, which we designate as a CDKinc state (Figure 2B and C; Figure 2—video 1), Conversely, CDK activity following the third and terminal division remains low, which we designate as a CDKlow state. Bootstrap analyses support a significant difference in DHB::GFP ratios between pre-terminal (CDKinc) and terminal divisions (CDKlow), but not among pre-terminal divisions (Figure 2—figure supplement 1A–C). We then quantified DHB::GFP ratios during the division of two somatic gonad lineages, the VU and SS cells. VU and SS cells undergo several rounds of division during the L3 larval stage and exit the cell cycle in the early L4 stage (Figure 2D; Sulston and Horvitz, 1977). We quantified a pre-terminal division and the subsequent division that leads to quiescence. Similar to the SM lineage, both somatic gonad lineages exit the round of cell division prior to their final division into a CDKinc state and then exit into a CDKlow state following their terminal division (Figure 2E and F, Figure 2—figure supplement 1D–F; Figure 2—video 2). Bootstrap analyses also support a significant difference between DHB::GFP ratios in pre-terminal versus terminal divisions in the developing somatic gonad (Figure 2—figure supplement 1D). Figure 2 with 3 supplements see all Download asset Open asset Sex myoblasts and somatic gonad cells exit terminal divisions into a CDKlow state. (A) SM lineage schematic. (B) Micrographs of a time-lapse showing SM cells at G1 and G0. (C) Quantification of CDK activity in SM cells (n ≥ 10). (D) Uterine lineage schematic. (E) Micrographs of a time-lapse showing uterine cells at G1 and G0. (F) Quantification of CDK activity in uterine cells (n ≥ 13). (G) Quantification of CDK activity in SM cells and uterine cells following ectopic expression of CKI-1 (hsp>CKI-1:: mTagBFP2) compared to non-heat shock controls and heat shock animals without the inducible transgene (n ≥ 36 cells per treatment). Pseudo-colored nuclei magenta, B; cyan, (E) indicate cells of interest. Scale bars = 10 μm. Dotted line in C and F indicates time of anaphase. Line and shaded error bands depict mean ± SD. Time series measured every 5 min. ns, not significant, ****p≤0.0001. Significance determined by statistical simulations; p-values in Supplementary file 1. Figure 2—source data 1 Source data for Figure 2. Raw data of DHB ratios used to generate Figure 2C, F and G in Microsoft Excel format. https://cdn.elifesciences.org/articles/63265/elife-63265-fig2-data1-v2.xlsx Download elife-63265-fig2-data1-v2.xlsx Figure 2—source data 2 Source date for Figure 2—figure supplement 1. Raw data of DHB ratios used to generate Figure 2—figure supplement 1A–D in Microsoft Excel format. https://cdn.elifesciences.org/articles/63265/elife-63265-fig2-data2-v2.xlsx Download elife-63265-fig2-data2-v2.xlsx Next, we sought to determine how the CDK sensor behaves under conditions in which cells are experimentally forced into G0. To accomplish this, we generated a single copy transgenic line of mTagBFP2-tagged CKI-1, the C. elegans homolog of p21Cip1/p27Kip1, under an inducible heat shock promoter (hsp), paired with a rps-0>DHB::mKate2 variant of the CDK sensor. Induced expression of CKI-1 is expected to result in G0 arrest (Hong et al., 1998; Matus et al., 2014; van der Horst et al., 2019). Indeed, in the SM and uterine lineages, induced expression of CKI-1 resulted in cells entering a CDKlow G0 state, with mean DHB ratios of 0.10 ± 0.05 and 0.12 ± 0.05, respectively (Figure 2G), as compared to control animals that lacked heat shock-induced expression (SM: 0.99 ± 0.82, uterine: 0.71 ± 0.35) or lacked the inducible transgene (SM: 0.96 ± 0.77, uterine: 1.00 ± 0.37). Thus, induced G0 arrest by ectopic expression of CKI-1 is functionally equivalent, by CDK-activity levels, to the G0 arrest that occurs following mitotic exit in an unperturbed cell destined to undergo quiescence. We next examined the divisions of the 1°- and 2°-fated VPC lineage. The C. elegans vulva is derived from three cells (P5.p–P7.p), which undergo three rounds of cell division during the L3 and early L4 larval stages (Figure 3A and B; Katz et al., 1995; Sternberg and Horvitz, 1986; Sulston and Horvitz, 1977). Rather than giving rise to 24 cells, the two D cells, the innermost granddaughters of the 2°-fated P5.p and P7.p, exit the cell cycle one round early. This results in a total of 22 cells, which comprise the adult vulva (Katz et al., 1995; Sulston and Horvitz, 1977). Quantification of DHB::GFP ratios during VPC divisions yielded the expected pattern. The daughters of P5.p–P7.p all exited their first division into a CDKinc state (Figure 3C and D). After the next division, the 12 granddaughters of P5.p–P7.p (named A–F symmetrically) are born, including the D cell (Katz et al., 1995; Sulston and Horvitz, 1977). At this division, the strong nuclear localization of DHB::GFP in the D cell was in stark contrast to the remaining proliferating VPCs. The D cell exited into and remained in a CDKlow state, while the remaining VPCs exited into a CDKinc state and continued to progress through the cell cycle (Figure 3C and D; Figure 3—video 1). All remaining VPCs exited into a CDKlow state at their terminal division. Consistent with these results, bootstrap analyses (Figure 3—figure supplement 1A–G) support our qualitative results, such that we can quantitatively distinguish between a cell that has completed mitosis and will continue to cycle (CDKinc) from a cell that exits mitosis and enters a G0 state (CDKlow). Figure 3 with 2 supplements see all Download asset Open asset Vulval precursor cells (VPCs) exit terminal divisions into a CDKlow state. (A) Schematic of primary (1°) and secondary (2°) fated VPCs. (B) All of the VPCs divide, with the exception of the D cells, to facilitate vulval morphogenesis. (C) Time series of CDK sensor localization in the 1° and 2° VPCs, as measured every 5 min. Note that the D cells are born into a CDKlow state (n ≥ 9 cells). Dotted line indicates time of anaphase. Shaded error bands depict mean ± SD. (D) Representative images of CDK sensor localization in the VPCs from the P6.p 2 cell stage to 8 cell stage. Nuclei (H2B) are highlighted in magenta for non-D cell 1° and 2° VPCs and green for the D cells. Scale bar = 10 μm. Figure 3—source data 1 Source data for Figure 3. Raw data of DHB ratios used to generate Figure 3C in Microsoft Excel format. https://cdn.elifesciences.org/articles/63265/elife-63265-fig3-data1-v2.xlsx Download elife-63265-fig3-data1-v2.xlsx Figure 3—source data 2 Source date for Figure 3—figure supplement 1. Raw data of DHB ratios used to generate Figure 3—figure supplement 1A–G in Microsoft Excel format. https://cdn.elifesciences.org/articles/63265/elife-63265-fig3-data2-v2.xlsx Download elife-63265-fig3-data2-v2.xlsx CKI-1 levels peak prior to cell cycle exit In mammalian cell culture, endogenous levels of p21Cip1 during G2 are predictive of whether a cell will go on to divide or enter quiescence, senescence, or terminal differentiation (Hsu et al., 2019; Moser et al., 2018; Overton et al., 2014; Spencer et al., 2013). This raises the intriguing possibility that endogenous levels of CKI-1 in C. elegans correlate with CDKlow or CDKinc activity. To co-visualize CKI-1 dynamics with our CDK sensor, we inserted a N-terminal GFP tag into the endogenous locus of cki-1 via CRISPR/Cas9 and introduced a DHB::2xmKate2 variant of the sensor (devoid of histone H2B) into this genetic background. Since endogenous levels of GFP::CKI-1 were too dim for time-lapse microscopy, likely due to its short half-life (Yang et al., 2017), we collected a developmental time series of static images over the L3 and L4 larval stages to characterize GFP::CKI-1 levels during pre-terminal and terminal divisions in the VPC lineage. We detected generally low levels of GFP::CKI-1 at the Pn.p 2 cell stage (Figure 4A–C, Figure 4—figure supplement 1A–C). In their daughter cells, at the Pn.p 4 cell stage, we detected an increase in GFP::CKI-1 levels in cycling cells prior to their next cell division, peaking in G2 (Figure 4A, B and D, Figure 4—figure supplement 1A–C). Notably, the D cell, which becomes post-mitotic after this cell division, exits mitosis with higher levels of GFP::CKI-1 than its CD mother (Figure 4B, Figure 4—figure supplement 1B). This trend holds true for the remaining VPCs at the Pn.p 6 cell and 8 cell stage, which show high levels of GFP::CKI-1 that peak immediately after mitotic exit and remain high during the post-mitotic L4 stage (Figure 4A, B, E and F, Figure 4—figure supplement 1A–C). We also observed increasing levels of GFP::CKI-1 in the G2 phase of mother cells that peak in their quiescent daughter cells in the uterine (Figure 4—figure supplement 1D) and SM cell lineages (Figure 4—figure supplement 1E). Thus, levels of GFP::CKI-1 increase in mother cells and remain high upon mitotic exit in daughter cells with CDKlow activity. These results suggest that the proliferation-quiescence decision is already underway in the G2 phase of the previous cell cycle and correlates with CKI-1 levels in the mother cell. Figure 4 with 1 supplement see all Download asset Open asset CKI-1 levels peak prior to cell cycle exit. (A) Schematic of VPC divisions in the L3-L4 larval stage. (B) CDK activity and CKI-1 levels across pseudo-time and DHB ratios for all VPCs (black line) and D cells (dark green line). GFP::CKI-1 fluorescence in VPCs (gray line) and D cell (light green line); n ≥ 93 cells per lineage. (C) Representative images of VPCs at the Pn.p 2-cell stage at G1 and G2 (white asterisk). (D) Representative images of VPCs at the Pn.p 4-cell stage at G1 and G2; early quiescent C cells (cyan arrows) with low levels of GFP::CKI-1. (E, F) Representative images of VPCs at the Pn.p 6- cell stage (E) and 8-cell stage (F); arrows show early quiescent C (cyan) and B cell (dark blue), F cell (magenta), and A cell (purple). (G) GFP::CKI-1 fluorescence in each cell of the VPC lineage (n ≥ 16, except C normal and A early n = 2, E early n = 3). (H) Percentage of cells of each lineage that showed signs of early quiescence and did not undergo their final division. (I) Overexpression of CKI-1 via heat shock causes cells to pre-maturely enter and remain in G0 (n ≥ 36 cells per treatment). Scale bar = 10 μm. ns, not significant, *p≤0.05, ****p≤0.0001. Significance determined by statistical simulations; p-values in Supplementary file 1. Figure 4—source data 1 Source data for Figure 4. Raw data of DHB ratios and fluorescence intensity measurements of GFP::CKI-1 used to generate Figure 4B, and raw data used to generate Figure 4G–I in Microsoft Excel format. Data also corresponds to lineage specific data used to generate plots in Figure 4—figure supplement 1A–C. https://cdn.elifesciences.org/articles/63265/elife-63265-fig4-data1-v2.xlsx Download elife-63265-fig4-data1-v2.xlsx During our collection of static images of GFP::CKI-1 animals, we observed significant deviations in the expected VPC lineage pattern in the early L4 larval stage. In particular, we noted that many cells appeared to bypass their final division and undergo early cell-cycle quiescence with coincident high levels of GFP::CKI-1 and low DHB ratios. We hypothesized that the line we generated could be behaving as a gain-of-function mutant, as GFP insertions at the N-terminus could interfere with proteasome-mediated protein degradation of CKI

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.

Cellular invasion is a key part of development, immunity and disease. Using an in vivo model of Caenorhabditis elegans anchor cell invasion, we characterize the gene regulatory network that promotes cell invasion. The anchor cell is initially specified in a stochastic cell fate decision mediated by Notch signaling. Previous research has identified four conserved transcription factors, fos-1 (Fos), egl-43 (EVI1/MEL), hlh-2 (E/Daughterless) and nhr-67 (NR2E1/TLX), that mediate anchor cell specification and/or invasive behavior. Connections between these transcription factors and the underlying cell biology that they regulate are poorly understood. Here, using genome editing and RNA interference, we examine transcription factor interactions before and after anchor cell specification. Initially, these transcription factors function independently of one another to regulate LIN-12 (Notch) activity. Following anchor cell specification, egl-43, hlh-2 and nhr-67 function largely parallel to fos-1 in a type I coherent feed-forward loop with positive feedback to promote invasion. Together, these results demonstrate that the same transcription factors can function in cell fate specification and differentiated cell behavior, and that a gene regulatory network can be rapidly assembled to reinforce a post-mitotic, pro-invasive state.

Cell proliferation and terminal differentiation are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these cell cycle-associated events live in C. elegans and zebrafish. 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 terminally differentiated cells in G0, revealing a commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. We also derive a predictive model of future proliferation behavior in C. elegans and zebrafish 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.### Competing Interest StatementThe authors have declared no competing interest.

Cellular invasion is a key part of development, immunity, and disease. Using thein vivomodel ofC. elegansanchor cell invasion, we characterize the gene regulatory network that promotes invasive differentiation. The anchor cell is initially specified in a stochastic cell fate decision mediated by Notch signaling. Previous research has identified four conserved transcription factors,fos-1a(Fos),egl-43(EVI1/MEL),hlh-2(E/Daughterless) andnhr-67(NR2E1/TLX), that mediate anchor cell specification and/or invasive differentiation. Connections between these transcription factors and the underlying cell biology that they regulate is poorly understood. Here, using genome editing and RNA interference, we examine transcription factor interactions prior to and after anchor cell specification. During invasion we identify thategl-43,hlh-2, andnhr-67function together in a type I coherent feed-forward loop with positive feedback. Conversely, prior to specification, these transcription factors function independent of one another to regulate LIN-12 (Notch) activity. Together, these results demonstrate that, although the same transcription factors can function in fate specification and differentiated cell behavior, a gene regulatory network can be rapidly re-wired to reinforce a post-mitotic, pro-invasive state.SUMMARY STATEMENTBasement membrane invasion by theC. elegansanchor cell is coordinated by a dynamic gene regulatory network encompassing cell cycle dependent and independent sub-circuits.