The PHA-4 Gene is Required to Generate the Pharyngeal Primordium of Caenorhabditis-Elegans

In the 4-cell Caenorhabditis elegans embryo, two blastomeres are destined to generate pharyngeal cells, each by a distinct developmental strategy: one pathway is inductive, while the other is autonomous. Here, we identify the pha-4 locus. In animals lacking pha-4 activity, an early step in pharyngeal organogenesis is blocked: no pharyngeal primordium is formed and differentiated pharyngeal cells are absent. Most other tissues are generated normally in pha-4 mutants, including cells related to pharyngeal cells by cell lineage and position. Thus, pha-4 activity is required to form the pharyngeal primordium. We propose that pha-4 marks a convergence of the inductive and autonomous pathways of pharyngeal development and suggest that establishment of pharyngeal organ identity is a crucial step for pharyngeal organogenesis

LAG-2 may Encode a Signaling Ligand for the GLP-1 and LIN-12 Receptors of C-Elegans

The C. elegans lag-2 gene is required for several cell-cell interactions that rely on the receptors GLP-1 and LIN-12. In this paper, we report that lag-2 encodes a putative membrane protein with sequence similarity to Drosophila Delta, a proposed ligand for the Notch receptor. Furthermore, we show that the lag-2 promoter drives expression of a reporter protein in the signaling distal tip cell (DTC) of the DTC/germline interaction. By in situ hybridization, we have found that endogenous lag-2 mRNA is present in the DTC but not the germ line. One fusion protein, called LAG-2::beta-gal(intra), rescues a lag-2 null mutant and can be detected in both DTC and germ line. Taking these results together, we propose that lag-2 may encode a signaling ligand for GLP-1/LIN-12 and that the entire LAG-2 protein may be taken up into the receiving cell during induction by GLP-1 and lateral signaling by LIN-12

In vivo labeling of endogenous genomic loci in C. elegans using CRISPR/dCas9

Visualization of genomic loci with open chromatin state has been reported in mammalian tissue culture cells using a CRISPR/Cas9-based system that utilizes an EGFP-tagged endonuclease-deficient Cas9 protein (dCas9::EGFP) (Chen et al. 2013). Here, we adapted this approach for use in Caenorhabditis elegans . We generated a C. elegans strain that expresses the dCas9 protein fused to two nuclear-localized EGFP molecules (dCas9::NLS::2xEGFP::NLS) in an inducible manner. Using this strain, we report the visualization in live C. elegans embryos of two endogenous repetitive loci, rrn-4 and rrn-1 , from which 5S and 18S ribosomal RNAs are constitutively generated

Novel Alleles of gon-2, a C-elegans Ortholog of Mammalian TRPM6 and TRPM7, Obtained by Genetic Reversion Screens

TRP (Transient Receptor Potential) cation channels of the TRPM subfamily have been found to be critically important for the regulation of Mg2+ homeostasis in both protostomes (e.g.,the nematode, C. elegans, and the insect, D. melanogaster) and deuterostomes (e.g.,humans). Although significant progress has been made toward understanding how the activities of these channels are regulated, there are still major gaps in our understanding of the potential regulatory roles of extensive, evolutionarily conserved, regions of these proteins. The C. elegans genes, gon-2, gtl-1 and gtl-2, encode paralogous TRP cation channel proteins that are similar in sequence and function to human TRPM6 and TRPM7. We isolated fourteen revertants of the missense mutant, gon-2(q338),and these mutations affect nine different residues within GON-2. Since eight of the nine affected residues are situated within regions that have high similarity to human TRPM1, 3, 6 and 7, these mutations identify sections of these channels that are potentially critical for channel regulation. We also isolated a single mutant allele of gon-2 during a screen for revertants of the Mg2+-hypersensitive phenotype of gtl-2(-) mutants. This allele of gon-2 converts a serine to phenylalanine within the highly conserved TRP domain, and is antimorphic against both gon-2(+) and gtl-1 (+). Interestingly, others have reported that mutation of the corresponding residue in TRPM7 to glutamate results in deregulated channel activity

Engulfment pathways promote programmed cell death by enhancing the unequal segregation of apoptotic potential

Components of the conserved engulfment pathways promote programmed cell death in Caenorhabditis elegans (C. elegans) through an unknown mechanism. Here we report that the phagocytic receptor CED-1 mEGF10 is required for the formation of a dorsal-ventral gradient of CED-3 caspase activity within the mother of a cell programmed to die and an increase in the level of CED-3 protein within its dying daughter. Furthermore, CED-1 becomes enriched on plasma membrane regions of neighbouring cells that appose the dorsal side of the mother, which later forms the dying daughter. Therefore, we propose that components of the engulfment pathways promote programmed cell death by enhancing the polar localization of apoptotic factors in mothers of cells programmed to die and the unequal segregation of apoptotic potential into dying and surviving daughters. Our findings reveal a novel function of the engulfment pathways and provide a better understanding of how apoptosis is initiated during C. elegans development

PIG-1 MELK-dependent phosphorylation of nonmuscle myosin II promotes apoptosis through CES-1 Snail partitioning

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that theC.elegansorthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. TheC.elegansNSM neuroblast divides to produce a larger cell that differentiates into a neuron and a smaller cell that dies. We find that in this context, PIG-1 is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic geneegl-1BH3-only.pig-1MELK is controlled by both aces-1Snail- andpar-4LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose thatpig-1MELK-induced local contractility of the actomyosin network plays a conserved role in the acquisition of the apoptotic fate. Our work also uncovers an auto-regulatory loop through whichces-1Snail controls its own activity through the formation of a gradient of CES-1 Snail protein. Author summary Apoptosis is critical for the elimination of 'unwanted' cells. What distinguishes wanted from unwanted cells in developing animals is poorly understood. We report that in theC.elegansNSM neuroblast lineage, the level of CES-1, a Snail-family member and transcriptional repressor of the pro-apoptotic geneegl-1, contributes to this process. In addition, we demonstrate thatC.elegansPIG-1, the orthologue of mammalian proto-oncoprotein MELK, plays a critical role in controlling CES-1(Snail)levels. Specifically, during NSM neuroblast division, PIG-1(MELK)controls partitioning of CES-1(Snail)into one but not the other daughter cell thereby promoting the making of one wanted and one unwanted cell. Furthermore, we present evidence that PIG-1(MELK)acts prior to NSM neuroblast division by locally activating the actomyosin network

A caspase-RhoGEF axis contributes to the cell size threshold for apoptotic death in developing Caenorhabditis elegans

A cell's size affects the likelihood that it will die. But how is cell size controlled in this context and how does cell size impact commitment to the cell death fate? We present evidence that the caspase CED-3 interacts with the RhoGEF ECT-2 in Caenorhabditis elegans neuroblasts that generate "unwanted" cells. We propose that this interaction promotes polar actomyosin contractility, which leads to unequal neuroblast division and the generation of a daughter cell that is below the critical "lethal" size threshold. Furthermore, we find that hyperactivation of ECT-2 RhoGEF reduces the sizes of unwanted cells. Importantly, this suppresses the "cell death abnormal" phenotype caused by the partial loss of ced-3 caspase and therefore increases the likelihood that unwanted cells die. A putative null mutation of ced-3 caspase, however, is not suppressed, which indicates that cell size affects CED-3 caspase activation and/or activity. Therefore, we have uncovered novel sequential and reciprocal interactions between the apoptosis pathway and cell size that impact a cell's commitment to the cell death fate

In vivo construction of recombinant molecules within the Caenorhabditis elegans germ line using short regions of terminal homology

Homologous recombination provides a means for the in vivo construction of recombinant DNA molecules that may be problematic to assemble in vitro. We have investigated the efficiency of recombination within the Caenorhabditis elegans germ line as a function of the length of homology between recombining molecules. Our findings indicate that recombination can occur between molecules that share only 10 bp of terminal homology, and that 25 bp is sufficient to mediate relatively high levels of recombination. Recombination occurs with lower efficiency when the location of the homologous segment is subterminal or internal. As in yeast, recombination can also be mediated by either single- or double-stranded bridging oligonucleotides. We find that ligation between cohesive ends is highly efficient and does not require that the ends be phosphorylated; furthermore, precise intermolecular ligation between injected molecules that have blunt ends can also occur within the germ line

Magnesium Excretion in C. elegans Requires the Activity of the GTL-2 TRPM Channel

Systemic magnesium homeostasis in mammals is primarily governed by the activities of the TRPM6 and TRPM7 cation channels, which mediate both uptake by the intestinal epithelial cells and reabsorption by the distal convoluted tubule cells in the kidney. In the nematode, C. elegans, intestinal magnesium uptake is dependent on the activities of the TRPM channel proteins, GON-2 and GTL-1. In this paper we provide evidence that another member of the TRPM protein family, GTL-2, acts within the C. elegans excretory cell to mediate the excretion of excess magnesium. Thus, the activity of GTL-2 balances the activities of the paralogous TRPM channel proteins, GON-2 and GTL-1

Overlapping expression patterns and functions of three paralogous P5B ATPases in Caenorhabditis elegans

P5B ATPases are present in the genomes of diverse unicellular and multicellular eukaryotes, indicating that they have an ancient origin, and that they are important for cellular fitness. Inactivation of ATP13A2, one of the four human P5B ATPases, leads to early-onset Parkinson's disease (Kufor-Rakeb Syndrome). The presence of an invariant PPALP motif within the putative substrate interaction pocket of transmembrane segment M4 suggests that all P5B ATPases might have similar transport specificity;however, the identity of the transport substrate(s) remains unknown. Nematodes of the genus Caenorhabditis possess three paralogous P5B ATPase genes, catp-5, catp-6 and catp-7, which probably originated from a single ancestral gene around the time of origin of the Caenorhabditid clade. By using CRISPR/Cas9, we have systematically investigated the expression patterns, subcellular localization and biological functions of each of the P5B ATPases of C. elegans. We find that each gene has a unique expression pattern, and that some tissues express more than one P5B. In some tissues where their expression patterns overlap, different P5Bs are targeted to different subcellular compartments (e.g., early endosomes vs. plasma membrane), whereas in other tissues they localize to the same compartment (plasma membrane). We observed lysosomal co-localization between CATP-6::GFP and LMP-1::RFP in transgenic animals;however, this was an artifact of the tagged LMP-1 protein, since anti-LMP-1 antibody staining of native protein revealed that LMP-1 and CATP-6::GFP occupy different compartments. The nematode P5Bs are at least partially redundant, since we observed synthetic sterility in catp-5(0);catp-6(0) and catp-6(0) catp-7(0) double mutants. The double mutants exhibit defects in distal tip cell migration that resemble those of ina-1 (alpha integrin ortholog) and vab-3 (Pax6 ortholog) mutants, suggesting that the nematode P5Bs are required for ina-1and/or vab-3 function. This is potentially a conserved regulatory interaction, since mammalian ATP13A2, alpha integrin and Pax6 are all required for proper dopaminergic neuron function