Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch in C. elegans.

A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of NHR-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which the association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.

A simple method to dramatically increase C. elegans germline microinjection efficiency.

Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans. While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.

Architecture of the cortical actomyosin network driving apical constriction in C. elegans.

Apical constriction is a cell shape change that drives key morphogenetic events during development, including gastrulation and neural tube formation. The forces driving apical constriction are primarily generated through the contraction of apicolateral and/or medioapical actomyosin networks. In the Drosophila ventral furrow, the medioapical actomyosin network has a sarcomere-like architecture, with radially polarized actin filaments and centrally enriched non-muscle myosin II and myosin activating kinase. To determine if this is a broadly conserved actin architecture driving apical constriction, we examined actomyosin architecture during C. elegans gastrulation, in which two endodermal precursor cells internalize from the surface of the embryo. Quantification of protein localization showed that neither the non-muscle myosin II NMY-2 nor the myosin-activating kinase MRCK-1 is enriched at the center of the apex. Further, visualization of barbed- and pointed-end capping proteins revealed that actin filaments do not exhibit radial polarization at the apex. Our results demonstrate that C. elegans endodermal precursor cells apically constrict using a mixed-polarity actin filament network and with myosin and a myosin activator distributed throughout the network. Taken together with observations made in other organisms, our results demonstrate that diverse actomyosin architectures are used in animal cells to accomplish apical constriction.

A simple method to dramatically increase C. elegans germline microinjection efficiency.

Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans . While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.

Architecture of the cortical actomyosin network driving apical constriction in C. elegans.

Apical constriction is a cell shape change that drives key morphogenetic events during development, including gastrulation and neural tube formation. The forces driving apical constriction are primarily generated through the contraction of apicolateral and/or medioapical actomyosin networks. In the Drosophila ventral furrow, the medioapical actomyosin network has a sarcomere-like architecture, with radially polarized actin filaments and centrally enriched non-muscle myosin II and myosin activating kinase. To determine if this is a broadly conserved actin architecture driving apical constriction, we examined actomyosin architecture during C. elegans gastrulation, in which two endodermal precursor cells internalize from the surface of the embryo. Quantification of protein localization showed that neither the non-muscle myosin II NMY-2 nor the myosin-activating kinase MRCK-1 is enriched at the center of the apex. Further, visualization of barbed- and pointed-end capping proteins revealed that actin filaments do not exhibit radial polarization at the apex. Taken together with observations made in other organisms, our results demonstrate that diverse actomyosin architectures are used in animal cells to accomplish apical constriction. Summary: Through live-cell imaging of endogenously-tagged proteins, Zhang, Medwig-Kinney, and Goldstein show that the medioapical actomyosin network driving apical constriction during C. elegans gastrulation is organized diffusely, in contrast to the sarcomere-like architecture previously observed in the Drosophila ventral furrow.

A new toolkit to visualize and perturb endogenous LIN-12/Notch signaling in C. elegans.

Notch signaling mediates cell-cell interactions during development and homeostasis. Methods for visualizing and manipulating Notch activity in vivo are essential to elucidate how the Notch pathway functions. Here, we provide new tools for use in C. elegans to visualize and perturb Notch signaling in vivo using endogenously tagged alleles of the Notch receptor lin-12 . Tagging the endogenous LIN-12 intracellular domain with the fluorescent protein mNeonGreen (mNG) allowed for visualization of both its membrane-localized state and translocation of the Notch intracellular domain into the nucleus upon ligand activation. LIN-12::mNG localized to the nucleus in cells where and when Notch signaling is known to be active and provided a real-time readout of Notch activity in vivo that complements existing biosensors and transcriptional reporters. We also report an allele of endogenous lin-12 that we tagged with both mNG and an auxin-inducible degron, to facilitate conditional LIN-12 protein degradation. This toolkit provides novel reagents for the C. elegans research community to investigate mechanisms of Notch signaling and its functions in vivo .

An in vivo toolkit to visualize endogenous LAG-2/Delta and LIN-12/Notch signaling in C. elegans.

Notch/Delta signaling regulates numerous cell-cell interactions that occur during development, homeostasis, and in disease states. In many cases, the Notch/Delta pathway mediates lateral inhibition between cells to specify alternative fates. Here, we provide new tools for use in C. elegans to investigate feedback between the Notch receptor LIN-12 and the ligand LAG-2 (Delta) in vivo . We report new, endogenously tagged strains to visualize LAG-2 protein and lag-2 transcription, which we combined with endogenously tagged LIN-12 to visualize Notch and Delta dynamics over the course of a stochastic Notch-mediated cell fate decision. To validate these tools in a functional context, we demonstrated that our endogenous lag-2 transcriptional reporter was expressed in ectopic anchor and primary vulval precursor cells after auxin-mediated depletion of LIN-12. This toolkit provides new reagents for the C. elegans research community to further investigate Notch/Delta signaling mechanisms and functions for this pathway in vivo .

The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo.

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.

An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans.

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.

Deletion of a putative HDA-1 binding site in the hlh-2 promoter eliminates expression in C. elegans dorsal uterine cells.

The helix-loop-helix transcription factor hlh-2 (E/Daughterless) has been shown to play an important role in regulating cell fate patterning, cell cycle, and basement membrane invasion in the context of the development of the C. elegans somatic gonad. Here, using CRISPR/Cas9 genome engineering, we generated a new hlh-2 allele (hlh-2(Δ-1303-702)) in the endogenous, GFP-tagged hlh-2 locus. This allele represents a deletion of a 601 bp region in the hlh-2 promoter that contains a putative binding site of the histone deacetylase hda-1 (HDAC) according to publicly available ChIP-sequencing data. Strikingly, we find that HLH-2 expression is virtually absent in the dorsal uterine cells of hlh-2(Δ-1303-702) animals compared to wild type controls. Levels of HLH-2 in the anchor cell and ventral uterine cells are only modestly reduced in the mutant; however, this does not seem to be functionally significant based on the lack of relevant phenotypes and expression levels of a downstream gene, NHR-67 (TLX/Tailless/NR2E1), in these cells. Taken together, these results support growing evidence that HDACs can potentially positively regulate transcription and provide a new reagent for studying hlh-2 regulation.

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans.

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

Visualizing the metazoan proliferation-quiescence decision in vivo.

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.

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.

A developmental gene regulatory network for C. elegans anchor cell invasion.

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.

Rapid Degradation of Caenorhabditis elegans Proteins at Single-Cell Resolution with a Synthetic Auxin.

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system allows for spatial and temporal control of protein degradation via a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate-recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID system by utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 min of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 depends on C. elegans SKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the FTZ-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work improves our use and understanding of the AID system for dissecting gene function at the single-cell level during C. elegans development.