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Employing molecular genetic analysis of a G protein–coupled receptor and its cognate ligands, Ringstad and Horvitz describe a neuropeptide pathway that modulates egg-laying behavior in C. elegans . This signaling pathway is shown to act in a collaborative fashion with cholinergic signaling to inhibit this behavior. Egg-laying behavior of the Caenorhabditis elegans hermaphrodite is regulated by G protein signaling pathways. Here we show that the egg laying–defective mutant egl-6(n592) carries an activating mutation in a G protein–coupled receptor that inhibits C. elegans egg-laying motor neurons in a G o -dependent manner. Ligands for EGL-6 are Phe-Met-Arg-Phe-NH 2 (FMRFamide)-related peptides encoded by the genes flp-10 and flp-17 . flp-10 is expressed in both neurons and non-neuronal cells. The major source of flp-17 peptides is a pair of presumptive sensory neurons, the BAG neurons. Genetic analysis of the egl-6 pathway revealed that the EGL-6 neuropeptide signaling pathway functions redundantly with acetylcholine to inhibit egg-laying. The retention of embryos in the uterus of the C. elegans hermaphrodite is therefore under the control of a presumptive sensory system and is inhibited by the convergence of signals from neuropeptides and the small-molecule neurotransmitter acetylcholine.

How cells regulate gene expression in a precise spatiotemporal manner during organismal development is a fundamental question in biology. Although the role of transcriptional condensates in gene regulation has been established, little is known about the function and regulation of these molecular assemblies in the context of animal development and physiology. Here we show that the evolutionarily conserved DEAD-box helicase DDX-23 controls cell fate in Caenorhabditis elegans by binding to and facilitating the condensation of MAB-10, the C. elegans homolog of mammalian NGFI-A-binding (NAB) protein. MAB-10 is a transcriptional cofactor that functions with the early growth response (EGR) protein LIN-29 to regulate the transcription of genes required for exiting the cell cycle, terminal differentiation, and the larval-to-adult transition. We suggest that DEAD-box helicase proteins function more generally during animal development to control the condensation of NAB proteins important in cell identity and that this mechanism is evolutionarily conserved. In mammals, such a mechanism might underlie terminal cell differentiation and when dysregulated might promote cancerous growth. The mechanism of spatiotemporal gene regulation during animal development is a fundamental question in biology. Here the authors show that the DEAD-box helicase DDX-23 controls stem cell fate by driving the formation of NAB repressive transcriptional condensates.

Cells programmed to die during C. elegans embryogenesis can be eliminated from the embryo and undergo apoptosis in the absence of caspase activity via an extrusion mechanism that depends on activation of the AMPK-related kinase PIG-1 by an LKB1-like kinase complex. Apoptosis without caspases Programmed cell death is an important part of normal development, used to eliminate defective or overabundant cells and in response to infection, for instance. Most such cell deaths are a result of mechanisms dependent on a family of proteases known as caspases. Caspase-independent cell death does occur, but the mechanisms involved are not well understood. Here, Robert Horvitz and colleagues describe how cells that are programmed to die during the development of the nematode Caenorhabditis elegans can be eliminated in a caspase-independent manner. These cells are extruded from the developing embryo even in the absence of caspase activity, and appear almost identical to apoptotic cells. The authors identify an AMPK-related kinase pathway required for this form of apoptosis. The elimination of unnecessary or defective cells from metazoans occurs during normal development and tissue homeostasis, as well as in response to infection or cellular damage 1 . Although many cells are removed through caspase-mediated apoptosis followed by phagocytosis by engulfing cells 2 , other mechanisms of cell elimination occur 3 , including the extrusion of cells from epithelia through a poorly understood, possibly caspase-independent, process 4 . Here we identify a mechanism of cell extrusion that is caspase independent and that can eliminate a subset of the Caenorhabditis elegans cells programmed to die during embryonic development. In wild-type animals, these cells die soon after their generation through caspase-mediated apoptosis. However, in mutants lacking all four C. elegans caspase genes, these cells are eliminated by being extruded from the developing embryo into the extra-embryonic space of the egg. The shed cells show apoptosis-like cytological and morphological characteristics, indicating that apoptosis can occur in the absence of caspases in C. elegans . We describe a kinase pathway required for cell extrusion involving PAR-4, STRD-1 and MOP-25.1/-25.2, the C. elegans homologues of the mammalian tumour-suppressor kinase LKB1 and its binding partners STRADα and MO25α. The AMPK-related kinase PIG-1, a possible target of the PAR-4–STRD-1–MOP-25 kinase complex, is also required for cell shedding. PIG-1 promotes shed-cell detachment by preventing the cell-surface expression of cell-adhesion molecules. Our findings reveal a mechanism for apoptotic cell elimination that is fundamentally distinct from that of canonical programmed cell death.

Removal of cells during development in Caenorhabditis elegans requires the precise execution of cell-death programs, which can include both caspase-dependent and -independent pathways; here it is shown that a single upstream transcription factor can drive both, in parallel, to destroy a single cell. Two roads to programmed cell death The removal of cells during development requires the precise execution of cell-death programs, which can include both caspase-dependent and caspase-independent pathways. Takashi Hirose and H. Robert Horvitz report that a single upstream factor can drive both, in parallel, to destroy a single cell. They show in C. elegans that the transcription factor SPTF-3 not only drives the transcription of the egl-1 gene, which promotes apoptosis through the activation of caspases, but also the transcription of pig-1 , which codes for a protein kinase and kills cells in a caspase-independent manner. Thus different cell-killing pathways can be coordinated by a single transcription factor. The authors suggest that such 'cell-death nodes' might be important therapeutic targets for diseases caused by excessive cell death. During animal development, the proper regulation of apoptosis requires the precise spatial and temporal execution of cell-death programs, which can include both caspase-dependent and caspase-independent pathways 1 , 2 . Although the mechanisms of caspase-dependent and -independent cell killing have been examined extensively, how these pathways are coordinated within a single cell that is fated to die is unknown. Here we show that the Caenorhabditis elegans Sp1 transcription factor SPTF-3 specifies the programmed cell deaths of at least two cells—the sisters of the pharyngeal M4 motor neuron and the AQR sensory neuron—by transcriptionally activating both caspase-dependent and -independent apoptotic pathways. SPTF-3 directly drives the transcription of the gene egl-1 , which encodes a BH3-only protein that promotes apoptosis through the activation of the CED-3 caspase 3 . In addition, SPTF-3 directly drives the transcription of the AMP-activated protein kinase-related gene pig-1 , which encodes a protein kinase and functions in apoptosis of the M4 sister and AQR sister independently of the pathway that activates CED-3 (refs 4 , 5 ). Thus, a single transcription factor controls two distinct cell-killing programs that act in parallel to drive apoptosis. Our findings reveal a bivalent regulatory node for caspase-dependent and -independent pathways in the regulation of cell-type-specific apoptosis. We propose that such nodes might act as features of a general mechanism for regulating cell-type-specific apoptosis and could be therapeutic targets for diseases involving the dysregulation of apoptosis through multiple cell-killing mechanisms.

The proper regulation of apoptosis requires precise spatial and temporal control of gene expression. While the transcriptional and translational activation of pro-apoptotic genes is known to be crucial to triggering apoptosis, how different mechanisms cooperate to drive apoptosis is largely unexplored. Here we report that pro-apoptotic transcriptional and translational regulators act in distinct pathways to promote programmed cell death. We show that the evolutionarily conserved C. elegans translational regulators GCN-1 and ABCF-3 contribute to promoting the deaths of most somatic cells during development. GCN-1 and ABCF-3 are not obviously involved in the physiological germ-cell deaths that occur during oocyte maturation. By striking contrast, these proteins play an essential role in the deaths of germ cells in response to ionizing irradiation. GCN-1 and ABCF-3 are similarly co-expressed in many somatic and germ cells and physically interact in vivo, suggesting that GCN-1 and ABCF-3 function as members of a protein complex. GCN-1 and ABCF-3 are required for the basal level of phosphorylation of eukaryotic initiation factor 2α (eIF2α), an evolutionarily conserved regulator of mRNA translation. The S. cerevisiae homologs of GCN-1 and ABCF-3, which are known to control eIF2α phosphorylation, can substitute for the worm proteins in promoting somatic cell deaths in C. elegans. We conclude that GCN-1 and ABCF-3 likely control translational initiation in C. elegans. GCN-1 and ABCF-3 act independently of the anti-apoptotic BCL-2 homolog CED-9 and of transcriptional regulators that upregulate the pro-apoptotic BH3-only gene egl-1. Our results suggest that GCN-1 and ABCF-3 function in a pathway distinct from the canonical CED-9-regulated cell-death execution pathway. We propose that the translational regulators GCN-1 and ABCF-3 maternally contribute to general apoptosis in C. elegans via a novel pathway and that the function of GCN-1 and ABCF-3 in apoptosis might be evolutionarily conserved.

Cell identity is characterized by a distinct combination of gene expression, cell morphology, and cellular function established as progenitor cells divide and differentiate. Following establishment, cell identities can be unstable and require active and continuous maintenance throughout the remaining life of a cell. Mechanisms underlying the maintenance of cell identities are incompletely understood. Here, we show that the gene ctbp-1, which encodes the transcriptional corepressor C-t erminal b inding p rotein-1 (CTBP-1), is essential for the maintenance of the identities of the two AIA interneurons in the nematode Caenorhabditis elegans. ctbp-1 is not required for the establishment of the AIA cell fate but rather functions cell-autonomously and can act in later larval stage and adult worms to maintain proper AIA gene expression, morphology and function. From a screen for suppressors of the ctbp-1 mutant phenotype, we identified the gene egl-13, which encodes a SOX family transcription factor. We found that egl-13 regulates AIA function and aspects of AIA gene expression, but not AIA morphology. We conclude that the CTBP-1 protein maintains AIA cell identity in part by utilizing EGL-13 to repress transcriptional activity in the AIAs. More generally, we propose that transcriptional corepressors like CTBP-1 might be critical factors in the maintenance of cell identities, harnessing the DNA-binding specificity of transcription factors like EGL-13 to selectively regulate gene expression in a cell-specific manner.

Neural control of muscle function is fundamental to animal behavior. Many muscles can generate multiple distinct behaviors. Nonetheless, individual muscle cells are generally regarded as the smallest units of motor control. We report that muscle cells can alter behavior by contracting subcellularly. We previously discovered that noxious tastes reverse the net flow of particles through the C. elegans pharynx, a neuromuscular pump, resulting in spitting. We now show that spitting results from the subcellular contraction of the anterior region of the pm3 muscle cell. Subcellularly localized calcium increases accompany this contraction. Spitting is controlled by an ‘hourglass’ circuit motif: parallel neural pathways converge onto a single motor neuron that differentially controls multiple muscles and the critical subcellular muscle compartment. We conclude that subcellular muscle units enable modulatory motor control and propose that subcellular muscle contraction is a fundamental mechanism by which neurons can reshape behavior.

Cell extrusion is a mechanism of cell elimination that is used by organisms as diverse as sponges, nematodes, insects and mammals 1 – 3 . During extrusion, a cell detaches from a layer of surrounding cells while maintaining the continuity of that layer 4 . Vertebrate epithelial tissues primarily eliminate cells by extrusion, and the dysregulation of cell extrusion has been linked to epithelial diseases, including cancer 1 , 5 . The mechanisms that drive cell extrusion remain incompletely understood. Here, to analyse cell extrusion by Caenorhabditis elegans embryos 3 , we conducted a genome-wide RNA interference screen, identified multiple cell-cycle genes with S-phase-specific function, and performed live-imaging experiments to establish how those genes control extrusion. Extruding cells experience replication stress during S phase and activate a replication-stress response via homologues of ATR and CHK1. Preventing S-phase entry, inhibiting the replication-stress response, or allowing completion of the cell cycle blocked cell extrusion. Hydroxyurea-induced replication stress 6 , 7 triggered ATR–CHK1- and p53-dependent cell extrusion from a mammalian epithelial monolayer. We conclude that cell extrusion induced by replication stress is conserved among animals and propose that this extrusion process is a primordial mechanism of cell elimination with a tumour-suppressive function in mammals. A cell-cycle checkpoint triggers the extrusion of both nematode and mammalian cells experiencing replication stress.

MicroRNAs (miRNAs) are small noncoding RNAs, about 21 nucleotides in length, that can regulate gene expression by base-pairing to partially complementary mRNAs. Regulation by miRNAs can play essential roles in embryonic development. We determined the temporal and spatial expression patterns of 115 conserved vertebrate miRNAs in zebrafish embryos by microarrays and by in situ hybridizations, using locked-nucleic acid-modified oligonucleotide probes. Most miRNAs were expressed in a highly tissue-specific manner during segmentation and later stages, but not early in development, which suggests that their role is not in tissue fate establishment but in differentiation or maintenance of tissue identity.

The HIF (hypoxia-inducible factor) transcription factor is the master regulator of the metazoan response to chronic hypoxia. In addition to promoting adaptations to low oxygen, HIF drives cytoprotective mechanisms in response to stresses and modulates neural circuit function. How most HIF targets act in the control of the diverse aspects of HIF-regulated biology remains unknown. We discovered that a HIF target, the C. elegans gene cyp-36A1, is required for numerous HIF-dependent processes, including modulation of gene expression, stress resistance, and behavior. cyp-36A1 encodes a cytochrome P450 enzyme that we show controls expression of more than a third of HIF-induced genes. CYP-36A1 acts cell non-autonomously by regulating the activity of the nuclear hormone receptor NHR-46, suggesting that CYP-36A1 functions as a biosynthetic enzyme for a hormone ligand of this receptor. We propose that regulation of HIF effectors through activation of cytochrome P450 enzyme/nuclear receptor signaling pathways could similarly occur in humans.