Liu, Z. et al. (2015) International Worm Meeting "Existence of a nematode alarm pheromone."

Zhong, W., Nguyen, J., Aleman-Meza, B., Liu, Z., Jung, S.

[International Worm Meeting, 2015]

It is crucial for animal survival to detect dangers such as predators. A good indicator of dangers is injury of conspecifics. It was reported that in C. elegans when one worm exploded upon a glass needle, nearby worms would move away from the victim (Thomas and Horvitz, 1988). Using three newly-designed quantitative methods, we confirmed the existence of an alarm pheromone released by injured nematodes. C. elegans avoided the alarm pheromone in a dose-dependent pattern. We then explored the molecular properties of the alarm pheromone and found that it was not any of the known avoidance chemicals. The alarm pheromone appeared to be ascaroside-independent because ascaroside-synthesis mutants still generated the alarm pheromone and ascaroside-receptor mutants still detected the alarm pheromone. The alarm pheromone was an endogenous factor present in all developmental stages, including embryos, larvae, adults and dauers, and only released to the environment upon injury. The alarm pheromone appeared to be nematode specific, because C. elegans did not avoid injured fruit fly larvae, but avoided alarm pheromones from multiple nematode species, including the insect parasite Steinernema. Interestingly, Steinernema only avoided the alarm pheromone from Steinernema but not C. elegans. We further examined the effects of the alarm pheromone on C. elegans. Constant exposure to the alarm pheromone did not reduce the lifespan of C. elegans, suggesting that it imposed no physical damage to the worms. Avoidance of the alarm pheromone was not affected by gender, developmental stage or feeding status. Males, hermaphrodites, dauers, and starved worms showed equal avoidance of this alarm pheromone, suggesting that avoidance of the alarm pheromone was a basic response important for animal survival in the wild environment. Multiple neurotransmitters were required for the alarm response. Exposure to the anti-anxiety drug Fluoxetine (Prozac) abolished C. elegans avoidance to the alarm pheromone. Finally, we investigated the molecular and cellular pathways used by C. elegans to detect the alarm pheromone. Mutant analysis showed that detection of the alarm pheromone was modulated through the cGMP-gated ion channels TAX-2 and TAX-4, guanylyl cyclases DAF-11, ODR-1, GCY-5, GCY-9 and GCY-22, and G proteins GOA-1, GPA-3, GPA-7 and GPA-14. Genetic and laser ablation of neurons showed that the amphid neurons AWC, ASI, and ASK were required for the detection of the alarm pheromone. .

Liu, Z. et al. (2017) International Worm Meeting "Sulfolipids drive C. elegans defensive responses to a predator."

Pribadi, A., Srinivasan, J., Liu, Z., Curran, K., Leinwand, S., Schroeder, F., Bose, N., Chalasani, S., Tong, A., Chute, C., Kariya, M.

[International Worm Meeting, 2017]

Animals respond to predator threats by implementing characteristic behavioural and physiological changes, but it is not known how the underlying neuronal ensembles process these threats. The simple, well-defined nervous system of the nematode, Caenorhabditis elegans facilitates uncovering conserved principles in neuronal processing. Here we show that C. elegans responds to excretions from a nematode predator, Pristionchus pacificus with instantaneous escape behaviour and a prolonged reduction of oviposition. Chemical analysis of the P. pacificus exo-metabolome revealed a series of novel sulfated lipids that instruct these C. elegans responses. These sulfolipids are specific to the predator and originate from a biosynthetic pathway that ties into endocrine signalling required for predacious behavior.

Liu, Z. et al. (2019) International Worm Meeting "Tetraspanins TSP-12 and TSP-14 function redundantly to promote the trafficking of the type II BMP receptor in Caenorhabditis elegans."

Liu, Z., Nzessi, A., Grant, B., Liu, J., Shi, H.

[International Worm Meeting, 2019]

Tetraspanins are a unique family of four-pass transmembrane proteins that play important roles in a variety of cell biological processes. One of their roles is to regulate the trafficking of their associated proteins. We have previously shown that two paralogous tetraspanins in C. elegans, TSP-12 and TSP-14, function redundantly to promote bone morphogenetic protein (BMP) signaling [1]. To dissect the functions of TSP-12 and TSP-14 in BMP signaling, we examined the expression and subcellular localization patterns of endogenously tagged TSP-12 and TSP-14 proteins. We found that TSP-12 and TSP-14 share overlapping expression patterns in multiple tissues, including the hypodermis, where the BMP receptors function to regulate body size, one of the phenotypic outputs of the BMP pathway. TSP-12 and TSP-14 also share overlapping localization patterns on the cell surface and partial co-localization in intracellular vesicles. Using markers for various intracellular vesicles in hypodermal cells, we found that TSP-12 and TSP-14 are primarily localized to the early endosomes, late endosomes and recycling endosomes. In tsp-12(0); tsp-14(0)double mutants, the morphology of the endosomes is impaired and an intestinally expressed DAF-4::GFP is mis-localized to lysosomes or lysosome-related organelles, indicating that TSP-12 and TSP-14 are required for the intracellular trafficking of the type II BMP receptor DAF-4. Together with previous findings by Gleason et al. showing that the type I receptor SMA-6 is recycled via the retromer complex [2], our work demonstrates the involvement of distinct recycling pathways for the type I and type II BMP receptors, and highlights the importance of intracellular trafficking in the regulation of BMP signaling in vivo. 1. Wang, L. et al. (2017) Two paralogous tetraspanins TSP-12 and TSP-14 function with the ADAM10 metalloprotease SUP-17 to promote BMP signaling in C. elegans.PLoS GeneticsDOI:10.1371/journal.pgen.1006568. 2. Gleason, R. J. et al. (2014) BMP signaling requires retromer-dependent recycling of the type I receptor. PNASDOI:10.1073/pnas.1319947111

Khan, Munzareen et al. (2021) International Worm Meeting "

Khan, Munzareen, Sengupta, Piali, Dwyer, Noelle, Hartmann, Anna, Bargmann, Cori, Piccione, Madeline

[International Worm Meeting, 2021]

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Wu S-L et al. (1996) East Coast Worm Meeting "CHARACTERIZATION OF AN ATYPICAL PROTEIN KINASE C IN C. Elegans."

Rubin CS, Wu S-L

[East Coast Worm Meeting, 1996]

Protein kinase Cz (PKCz) is an atypical member of the PKC superfamily. PKCz contains Cys-rich zinc binding and catalytic domains that are homologous with corresponding regions in other PKCs. Unlike other PKCs, the zeta isoform in not activated by diacyl glycerol, TPA or calcium. However, PKCz appears to be a target for activation by alternative lipid second messengers (3,4,5-phosphoinositol, ceramide) generated by PI-3-kinase and/or sphingomyelinase. Activated PKCz has been implicated in the transmission of regulatory signals from the cytoplasm to the nucleus, but its precise physiological roles remain to be rigorously determined. We have initiated studies on the structure, function and regulation of an atypical C. elegans PKC (designated PKC-Z) that is related to mammalian PKCz. A cDNA that encodes full-length PKC-Z was cloned and sequenced. The predicted PKC-Z protein (598 amino acid residues, molecular weight = 68,000) is only ~55% identical with mammalian PKCz. N-terminal and central portions of the two proteins are highly divergent. However, the catalytic, psuedosubstrate and Cys-rich domains are highly conserved. Comparison of the cDNA sequence with data from the C. elegans genome project indicates that the PKC-Z gene (LGII) encompasses 3.6 kbp of DNA and contains 9 exons. A C terminal fragment (120 amino acids) of PKC-Z was expressed as a His-tagged fusion protein in E. coli, purified and injected into rabbits to produce antiserum. Western immunoblot analysis revealed that PKC-Z is most abundantly expressed in the soluble fraction of embryos. The kinase is also evident in L1-L4 larvae and adult nematodes, but the bulk of the enzyme is tightly associated with the particulate fractions of post-embryonic animals. Consistent with the latter observation, immunostaining and confocal microscopy of permeabilized C. elegans revealed that PKC-Z is localized and concentrated in discrete intracellular clusters. The nature of the intracellular structure at which PKC-Z accumulates is not yet known. PKC-Z is being expressed in baculovirus/Sf9 cells and in mammalian cells for enzymic characterization. In addition, injected anti-sense oligonucleotides are being used to probe the function(s) of PKC-Z in embryogenesis.

Hristova M et al. (2000) East Coast Worm Meeting "Identifying lin-14 targets with the help of a DNA microarray"

Ambros VR, Hristova M

[East Coast Worm Meeting, 2000]

The heterochronic regulator lin-14 affects certain somatic cell-fate choices during post-embryonic development in C. elegans. In lin-14 mutants, cells express stage-specific developmental programs, such as cell divisions or dauer entry, at inappropriate times in development.1,2 Loss-of-function mutations in lin-14 result in the precocious execution of L2-type programs during the first larval stage and the omission of L1-type programs, while gain-of-function mutations retard the execution of L2-specific programs during the second larval stage and instead cause the re-iteration of L1-specific programs. While the biochemical activity of lin-14 remains unknown, we hypothesize that lin-14 functions to either repress the expression/execution of L2-type fates during the first larval stage, and/or to promote the expression of L1-type fates during that stage. Since LIN-14 is a nuclear protein, it is likely that it controls state-specific fates by regulating gene expression at the level of mRNA abundance. We chose to look for downstream targets of lin-14 using a microarray of 12,000 ORFs prepared by the Kim lab, at Stanford University. The two synchronized mid-L1 populations compared were N2 and lin-14 (n179ts) grown at the non-premissive temperature. We chose 4 genes for follow-up by Northern and GFP-fusion analysis of stage-specific and cell-type specific expression. Three of these four genes show mRNA misexpression in the L1 in the absense of lin-14 function. The expression of promoter fusions to GFP suggests that at least one of the genes, ins-33, appears to be developmentally regulated by lin-14 at the transcriptional level. 1. Ambros, V., and H.R. Horvitz, Genes and Development (1987) 1:398 2. Liu, Z., and V. Ambros, Genes and Development (1989) 3:2039

Jeon M et al. (1996) Midwest Worm Meeting "CHARACTERIZATION OF HETEROCHRONIC MUTANTS"

Abrahante JE, Rougvie AE, Miller EA, Jeon M

[Midwest Worm Meeting, 1996]

In C. elegans, the heterochronic genes lin-4, lin-14, lin-28 and lin-29, control the relative timing and sequence of many events during post-embryonic development, including the terminal differentiation of lateral hypodermal seam cells. We have been performing a variety of genetic screens aimed at identifying additional members of the heterochronic gene pathway (see related abstract, Abrahante et al.). We will present a summary of our phenotypic and genetic analyses of the mutants isolated from these screens. One additional member of the heterochronic gene pathway is lin-42, a gene for which a single mutant allele has been reported (Liu, 1990). lin-42(n1089) mutants execute seam cell terminal differentiation precociously, during the L3-to-L4 molt. We have identified a number of new lin-42 alleles in our screens and are investigating lin-42's role in the timing of seam cell terminal differentiation. lin-29 is epistatic to lin-42(n1089) (Liu, 1990) and to our new alleles. All of the alleles we have isolated are partially epistatic to lin-4. That is, some, but not all, of the seam cells in lin-42 lin-4 double mutants terminally differentiate during the L3-to-L4 molt, resulting in animals with gaps in their alae. These alleles exhibit variable penetrance of the mutant phenotypes and we are ordering them in an allelic series with respect to alae synthesis and suppression of lin-4 defects. We are in the process of isolating g-ray induced alleles to describe the lin-42 null phenotype and to aid in the cloning of the gene. We have initiated a positional cloning strategy and will present our progress toward localizing the gene on the left arm of Linkage Group II. Liu, Z. (1990). Ph.D. Thesis, Harvard University, Cambridge, MA.

Jeon M et al. (1997) International C. elegans Meeting "Characterization and Mapping of the Heterochronic Gene lin-42"

Miller EA, Jeon M, Rougvie AE

[International C. elegans Meeting, 1997]

lin-42 is a heterochronic gene for which a single mutant allele has been reported (Liu, 1990). lin-42(n1089) mutants execute seam cell terminal differentiation one stage earlier than wild type animals, during the L3-to-L4 molt. To better characterize the function of lin-42 and understand its role in the heterochronic pathway, we have isolated and characterized seven new lin-42 alleles and are working toward cloning the lin-42 locus. One allele, ve16, is cold sensitive: when raised at 15 C, all animals (n=26) synthesize adult alae during the L3 molt, while at 25 C, no (n=24) L3 molt animals have alae. We are using this allele in temperature-shift experiments to determine the time of lin-42 action with respect to seam cell terminal differentiation. Preliminary results indicate a requirement for lin-42 late during embryogenesis, between egg-laying and hatching, a time prior to the lin-14 temperature sensitive period (Ambros and Horvitz, 1987). In our efforts to clone lin-42, we have isolated two Tc1-tagged lin-42 alleles. We have outcrossed these alleles and have performed Southern blot analysis to identify closely linked Tc1 elements. At present, we have identified five possible linked elements and are assessing the segregation of these elements relative to the lin-42 phenotype. We have also mapped lin-42 between two polymorphic markers, nP48 and veP2. We will perform transformation rescue experiments using YAC clones spanning this region and search for allele-specific RFLPs in DNA isolated from two gamma-ray induced alleles. Our progress in cloning lin-42 using these combined approaches will be reported. Ambros, V. and H.R. Horvitz. (1987). Genes & Dev. 1: 398-414. Liu, Z. (1990). Ph.D. Thesis, Harvard University, Cambridge, MA.

Marta Hristova et al. (2001) International C. elegans Meeting "Control of ins-33 transcription by lin-14 in the C. elegans hypodermis"

Marta Hristova, Victor Ambros

[International C. elegans Meeting, 2001]

The heterochronic regulator lin-14 affects certain somatic cell-fate choices during post-embryonic development in C. elegans . In lin-14 mutants, cells express stage-specific developmental programs, such as cell divisions or dauer entry, at inappropriate times in development. 1,2 Loss-of-function mutations in lin-14 result in the precocious execution of L2-specific programs during the first larval stage and the omission of L1-specific programs, while gain-of-function mutations retard the execution of L2-specific programs during the second larval stage and instead cause the re-iteration of L1-specific programs. We chose to study the molecular mechanisms of lin-14 function using two complementary approaches. First, we characterized lin-14 biochemically by analyzing its association with nuclei in vivo and by performing in vitro DNA binding assays. We also performed microarray analysis to identify genes misregulated under lin-14 loss-of-function or gain-of-function conditions. We identified a number of genes whose transcript levels are regulated either positively or negatively by lin-14 , and which could be among lin-14 's in vivo targets. Using Northern blot and GFP-reporter fusion analysis we have begun to investigate whether these putatuve targets are transcriptionally controlled by lin-14 , and whether this control is direct or indirect. The expression of one of these targets, ins-33 , a C. elegans insulin homolog, appears to be controlled by lin-14 at the level of transcription in vivo . LIN-14 protein can bind the ins-33 promoter in vitro at at least two sites. We are currently characterizing the in vivo significance of these sites to lin-14 dependent ins-33 transcription. 1. Ambros, V., and H.R. Horvitz, Genes and Development (1987) 1:398 2. Liu, Z., and V. Ambros, Genes and Development (1989) 3:2039

Gilleard JS et al. (1995) International C. elegans Meeting "REGULATION OF THE CUTICULAR COLLAGEN GENE dpy-7"

Barry JD, Gilleard JS, Johnstone IL

[International C. elegans Meeting, 1995]

We are interested in identifying trans acting factors which control cuticular collagen gene expression. Sequences sufficient for regulated expression of dpy-7 appear to reside relatively close to the initiator ATG since the wild type gene only requires 310bp of 5' flanking sequence for transformation repair of phenotype. We have analysed sequences necessary and sufficient for dpy-7 expression using lac-Z fusions and we have identified a 95bp fragment, encompassing the transcritional start sites, which is sufficient to produce lac-Z expression predominantly, or possibly exclusively, in hypodermal cells. This fragment includes a region in which 65 out of 72bp are conserved in the 5' flank of the C.briggsae dpy-7 homolog and interspecies transformation rescue and lac-Z fusion experiments have shown that the dpy-7 cis regulatory elements are functionally conserved. A 15bp deletion at the 5' end of this homology which removes a AGATAA motif, also present in C.briggsae, completely ablates lac-Z expression . We are attempting to identify transcription factors which bind to this motif by South Western expression library screening. We are also searching for new C.elegans GATA factor homologs with a particular interest in any which are expressed in hypodermal cells postembryonically.