Nicholas J D Gower et al. (2001) International C. elegans Meeting "Dissection of the upstream regulatory elements of the Inositol 1,4,5-trisphosphate receptor gene itr-1"

Gillian R Temple, Marco Marra, Nicholas J D Gower, Denise S Walker, Jacqueline E Schein, Howard A Baylis

[International C. elegans Meeting, 2001]

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a central role in the organisation of calcium signals by regulating intracellular calcium flux. IP3Rs in C. elegans are encoded by a single gene, itr-1 (also called lfe-1 (1) and dec-4 (2) ). In previous work (3), we characterised the gene structure in detail and identified three putative transcriptional start sites and three alternative splice sites. Sequence comparisons to the C. briggsae itr-1 homologue support the hypothesis that the itr-1 gene has three promoters (pA, pB and pC), each of which gives rise to an alternative mRNA and hence unique protein. The pattern of expression directed by each promoter was determined using GFP reporter gene fusions. pA directs expression in the pharyngeal terminal bulb, the rectal epithelial cells and vulva; pB directs expression in the amphid socket cells, the PDA motor neuron and the spermatheca; pC directs expression in the spermathecal valve, uterine sheath cells, pharyngeal isthmus and intestine (4). Thus tissue-specific expression of itr-1 variants is directed by three promoters and this results in adjacent cells in the same tissue containing different IP3R isoforms. Analysis of the sequence conversation between promoter A of C. elegans and C. briggsae identified four short regions (pA-A to pA-D) of conservation. Deletion analysis of pA demonstrated that the region containing pA-C is required for expression in the terminal bulb and rectal epithelial cells and the region containing pA-D is required for expression in the vulva. pA-C contains sequences which show homology to binding sites for known C. elegans transcription factors. We are currently identifying factors that regulate the promoter using a yeast one-hybrid screen. 1. Clandinin et al (1998) Cell: 92, 523-533. 2. Dal Santo et al (1999) Cell: 98 757-767. 3. Baylis et al (1999) J. Mol Biol: 294, 467-476. 4. Gower et al (2001) J. Mol Biol: 306, 145-157.

Johnsen, Holly et al. (2013) International Worm Meeting "Assisted Suicide: a Caspase- and Engulfment-Dependent Cell Death."

Horvitz, Bob, Johnsen, Holly

[International Worm Meeting, 2013]

Programmed cell death occurs during the development of many organisms. The C. elegans cell-death pathway has been extensively studied and is evolutionarily conserved. During programmed cell death, caspases are activated in the dying cell. The cell corpse is engulfed by a neighboring cell and degraded. Almost all cell deaths are "suicides"-they are cell-autonomous, caspase-dependent and can occur even in engulfment-defective animals. In the C. elegans male, the cells B.alapaav and B.arapaav are generated during the late L3 stage. During the early L4 stage one of these cells dies, and the other survives and adopts an epithelial fate. These two cells form an equivalence group; the decision of which cell dies and which survives is stochastic and takes place during the L3/L4 lethargus. The cell that dies is engulfed by the neighboring cell P12.pa. In contrast to most C. elegans cell deaths, the B.al/rapaav death is engulfment-dependent; if engulfment is blocked by a mutation in one of the genes in the engulfment pathway, both B.alapaav and B.arapaav survive. Furthermore, we have found that if the engulfing cell P12.pa is ablated, the B.al/rapaav death fails to occur in approximately 60% of animals. These observations suggest that cell interactions between B.alapaav and B.arapaav as well as between B.al/rapaav and P12.pa are involved in this cell death, leading some to suggest that P12.pa "murders" B.al/rapaav. We are investigating the control and execution of the B.al/rapaav cell death. When the B.al/rapaav cell death is blocked by engulfment defects or P12.pa ablation, the undead cell still initiates the cell-death pathway. Similar to other dying cells, the undead cell looks round by Nomarski and EM and exposes phosphatidylserine on its surface. egl-1 and ced-3 are required for the B.al/rapaav cell death and are expressed in the undead cell, suggesting that the core cell-death pathway is required but not sufficient for this cell death, i.e. that this death is an assisted suicide. We hope our studies will provide insight into new mechanisms of programmed cell death, cell-cell signaling, and fate determination within equivalence groups.

Qiang Liu et al. (2004) East Coast Worm Meeting "CaM KII Regulates Neurotransmitter Release at the C. elegans Neuromuscular Junction"

Zhao-Wen Wang, Qiang Liu

[East Coast Worm Meeting, 2004]

Ca 2+ /calmodulin-dependent kinase II (CaM KII) plays an important role in synaptic plasticity and development of the nervous system. Malfunction of CaM KII is associated with a variety of diseases including epilepsy. In the nervous system, CaM KII is expressed at both pre- and post-synaptic sites. Little is known about the potential role of CaM KII in neurotransmitter release. We have embarked on a project to study the function of CaM KII in neurotransmitter release using C. elegans as model system. C. elegans has a single CaM KII gene, unc-43 . Both gain-of-function and loss-of-function mutants are available for analysis. To evaluate the function of CaM KII in neurotransmitter release, miniature and evoked postsynaptic currents (mPSCs and ePSCs) were recorded at the neuromuscular junction with the postsynaptic body-wall muscle cell clamped at -60 mV. We initially analyzed mPSCs and ePSCs in unc-43(n498) , a gain-of-function mutant. Compared with the wild-type, the mutant showed a significant reduction in the amplitude of ePSCs (wild-type 1579 +/- 129 pA, n = 8; n-498 721 +/- 79 pA, n = 12) and in the frequency of mPSCs (wild-type 50.6 +/- 3.4 Hz, n = 22; n-498 22.6 +/- 3.4 Hz, n = 6). Postsynaptic receptor sensitivity appeared normal in the mutant since the amplitude of mPSCs did not change (wild-type 27.4 +/- 1.6 pA, n = 22; n-498 28.4 +/- 4.3 pA, n = 6). These results suggest that a normal function of CaM KII is to regulate neurotransmitter release. Experiments are under way to determine 1) whether unc-43 loss-of-function mutants show opposite changes of mPSCs and ePSCs compared with the gain-of-function mutant; 2) whether the apparent effect on neurotransmitter release is due to an action of presynaptic CaM KII, or a retrograde signal generated by postsynaptic CaM KII; and 3) what are the phosphorylation targets of CaM KII in regulating neurotransmitter release.

Yavorov, Deyan et al. (2021) International Worm Meeting "Pediococcus acidilactici CECT9879 reduces fat accumulation in C. elegans by affecting the insulin signaling pathway"

Aranaz, Paula, Ayo, Josune, Yavorov, Deyan, Milagro, Fermin I., Oneca, Maria

[International Worm Meeting, 2021]

The increasing prevalence of metabolic syndrome-related diseases, such as obesity and type-2 diabetes, makes urgent to develop alternative therapies to prevent or reduce the progression of these pathologies. On the other hand, during the last years it has been demonstrated the important role that gut microbiota plays on human health, and the influence that alterations in this bacterial balance plays in the development of metabolic diseases. Thus, different bacterial strains have emerged as potential probiotics for the prevention of the complications characteristic of obesity, such as the excess of adiposity or the dysregulation of glycemia. In this study, we have used Caenorhabditis elegans (C. elegans) as a model to examine the potential probiotic activities of the bacterium Pediococcus acidilactici (PA) CECT9879, together with the underlying mechanisms of action. Our work revealed that the treatment with PA reduced the fat accumulation of C. elegans in a 9% when grown in NGM medium, and a 16% in a glucose-loaded (10 mM) NGM medium, quantified by Nile Red staining, without affecting the development of the worm. The reduced fat content was also accompanied by an extension of the nematode median survival of two days in comparison with untreated control worms. Subsequent gene expression analyses demonstrated that the probiotic activities were mediated by modulation of the insulin/IGF-1 signaling (IIS) pathway through the overexpression of daf-16 and ins-6 mediators, but also by an increased expression of genes involved in the fatty acid peroxisomal beta-oxidation. Taken together, our data suggest that PA CECT9879 could be considered a potential probiotic strain for the prevention of the metabolic syndrome-related disturbances, and highlight the use of C. elegans as an appropriate in vivo model for the study of the mechanisms underlying these diseases.

Xin Li et al. (2006) Development & Evolution Meeting "Autoregulatory sequences influence egl-5 expression in the P12 cell lineage"

Xin Li, Russell Hill, Helen Chamberlin

[Development & Evolution Meeting, 2006]

In C. elegans, ventral ectoblasts called P cells divide postembryonically to produce ventral cord neurons and hypodermal cells. Initially, the two most posterior P ectoblasts have similar potential to undergo two patterns of development known as the P11 and P12 cell fates. In normal development, the WNT and EGF signaling pathways and the HOX gene egl-5 direct only the most posterior cell to adopt the P12 cell fate. Through epistasis analysis, we have found that egl-5 acts downstream of the WNT pathway, which combined with other studies, indicates EGL-5 is a functional target of both signaling pathways. Given the central role of egl-5 in P12 cell fate specification, we have sought to better understand the molecular mechanisms that regulate egl-5 expression. Previously, another group identified a 1.3 kb egl-5 enhancer fragment (present in pLG7) that can activate GFP expression in P12.pa, a cell type unique to the P12 cell fate (Teng et al., 2004, Dev Biol 276:476-492). We have found that pLG7 does not express in the P12 cell lineage in egl-5(-) and Wnt pathway mutants that lack P12 cell fates. Notably, we found that ectopic expression of EGL-5 protein can activate GFP expression from pLG7, even when the Wnt pathway is inactive. This result suggests that pLG7 contains an EGL-5 response element. By producing deletion variants of pLG7, we have identified a minimal 364bp region (pXL323) sufficient for expression in P12.pa. This minimal region contains a consensus sequence for a HOX binding site and this site is conserved in two other Caenorhabditis species. We have shown that mutating this HOX site in pLG7 or pXL323 causes a loss of GFP expression in P12.pa (but not in other cells). These results are consistent with the model that the pLG7 enhancer contains an EGL-5 autoregulatory site that maintains EGL-5 expression in P12.pa. To test whether the pLG7 enhancer is sufficient to activate expression in the P12 cell lineage, we used this enhancer to express EGL-5 protein in an egl-5 null mutant and found that this did not restore P12 cell fates. This result suggests that some of the cis regulatory sequences that normally activate egl-5 expression lay outside of pLG7. We propose that functionally separable regulatory elements initiate and maintain egl-5 expression in the P12 cell lineage. We are continuing experiments to characterize both types of elements.

Rashmi Deshpande et al. (2005) International Worm Meeting "Wnt signaling and P11/ P12 specification in C. elegans"

Russell J Hill, Rashmi Deshpande, Xin Li, John Tychonievich, Soumitra Sen

[International Worm Meeting, 2005]

Wnt signaling pathways control many aspects of animal development including cell type specification, cell migration and spindle orientation. In C. elegans, a Wnt pathway regulates the development of two neuroectodermal precursors called P11 and P12. In normal development, the posterior cell adopts the P12 cell fate in response to Wnt (and EGF signals) while the anterior cell does not and instead adopts the P11 cell fate. One difference between these two cell fates is that P11 produces a large hypodermal cell called P11.p whereas P12 produces a small hypodermal cell called P12.pa. These cell types P11.p and P12.pa are commonly used as markers for the P11 and P12 fates. We are investigating the mechanisms of Wnt signaling in P12 development. Prior studies have shown that lin-44/Wnt, lin-17/Frizzled and bar-1/beta-catenin function in P12 specification. We have found that a constitutively active form of bar-1 (deltaN-BAR-1) causes P11 to P12 transformations. Together, these results suggest bar-1 is necessary and sufficient for P12 specification. Because beta-catenins typically activate the transcription factor TCF-1, this predicts that pop-1, the sole TCF-1 homolog of C. elegans, will also act in P12 specification. Consistent with this prediction, we have found that the pop-1 hypomorph hu9 causes a P12 to P11 transformation. Surprisingly, however, we find that pop-1 RNAi causes the opposite effect resulting in a loss of P11.p and the presence of an extra P12.pa-like cell. This apparent P11 to P12 cell fate transformation suggests that POP-1 has an additional function in P11 specification. Epistasis experiments indicate that this latter function of pop-1 is independent of bar-1. We find that multiple Wnts act in P12 specification. lin-44/Wnt(n1792) and egl-20/Wnt(n585) double mutants display an enhanced rate of P12 to P11 transformations. Other possible roles for additional Wnts and Wnt receptors in P11/P12 development will be discussed.

Johnsen, Holly L. et al. (2011) International Worm Meeting "A Screen for Genes Involved in Engulfment-Dependent Cell Death."

Horvitz, H.R., Johnsen, Holly L.

[International Worm Meeting, 2011]

Programmed cell death occurs during the normal development of many organisms. The C. elegans cell-death pathway has been extensively studied for a variety of cells that are fated to die. During programmed cell death, caspases are activated in the dying cell. The cell corpse is then engulfed by a neighboring cell and degraded. In most cases, cell death is cell-autonomous and caspase-dependent, and can occur even in engulfment-defective animals. During development of the C. elegans male, the cells B.alapaav and B.arapaav are generated during the late L3 stage. During the early L4 stage one of these cells undergoes caspase-dependent programmed cell death and the other survives. The decision of which cell dies and which survives is stochastic. The cell that dies is engulfed by the neighboring cell P12.pa. If engulfment is blocked through a mutation in one of the genes in the engulfment pathway or by ablating P12.pa, both B.alapaav and B.arapaav survive. If one cell is ablated by laser microsurgery, then the other cell will invariably survive. These cells form an equivalence group: both have the potential to either survive and form part of the male tail or undergo programmed cell death. These observations suggest that cell interactions between B.alapaav and B.arapaav and between B.al/rapaav and P12.pa are involved in this cell death. We will investigate the regulation of this unusual programmed cell death. To this end, we will perform a genetic screen to identify genes that function in B.al/rapaav cell death. cog-1::GFP has been reported to be expressed in B.alapaav and B.arapaav. Using this reporter strain, we will screen for mutants in which neither or both B.alapaav and B.arapaav are present because of misregulation of fate determination or of engulfment-dependent cell death. This screen might identify new genes involved in fundamental mechanisms of programmed cell death, cell-cell signaling and fate determination within equivalence groups.

Eric Rutledge et al. (2001) International C. elegans Meeting "The ClC channel CLH-3 is activated during meiotic maturation in C. elegans oocytes"

Michael Miller, Christoph Boehmer, David Greenstein, Eric Rutledge, Kevin Strange

[International C. elegans Meeting, 2001]

C. elegans oocytes express an inwardly rectifying Cl - channel that is activated by cell swelling and is encoded by the ClC gene clh-3 . The biophysical characteristics of CLH-3 are virtually indistinguishable from heterologously expressed mammalian ClC-2. CLH-3 and ClC-2 share 40% amino acid identity. We have suggested that CLH-3 and ClC-2 are orthologs. The amount of swelling required to activate CLH-3 varies by >50-60 fold between different oocytes. CLH-3 in small, early stage oocytes requires substantially more volume increase to activate compared to larger, later stage oocytes. Upon completion of growth, oocytes undergo meiotic maturation and are ovulated and fertilized. In full-grown, maturing oocytes, CLH-3 is constitutively activated. Mean whole-cell Cl - currents at -70 mV were -3.2 +/- 0.3 pA/pF and -14.6 +/- 2.9 pA/pF in non-maturing and full-grown, maturing oocytes, respectively. These findings suggest that either completion of growth or induction of maturation activate the channel. To determine the role of cell growth versus meiotic maturation in CLH-3 activation, we carried out patch clamp studies on oocytes isolated from fog-2 mutant worms. Mutations in certain C. elegans sex determination genes such as fog-2 block sperm production in hermaphrodites ( Schedl and Kimble, Genetics 119:43-61, 1988 ). The presence of sperm is required for normal progression of meiotic maturation. Late-stage fog-2 oocytes reach full-grown size, but arrest in diakinesis for many hours to days ( McCarter et al., Dev Biol 205:111-128, 1999 ). Since ovulation is triggered by maturation, full-grown oocytes accumulate in the gonad of fog-2 worms. We reasoned that if completion of growth activates CLH-3, then the channel should be active in late-stage fog-2 oocytes. However, fog-2 oocytes exhibited no channel activity. Meiotic maturation was induced in fog-2 oocytes by injecting recombinant Major Sperm Protein (MSP)38 into the uterus. CLH-3 was constitutively activated in late stage, maturing oocytes from MSP38-injected fog-2 worms (Cl - current = -39.7 +/- 1.5 pA/pF). These results indicate that CLH-3 is activated by induction of meiotic maturation. To further examine the role of maturation in channel activation, we carried out a series of experiments on wild type oocytes undergoing maturation in vitro . Shortly before maturation begins, the nucleus increases in size and migrates to the cell periphery. Meiotic maturation is marked by Nuclear Envelope Breakdown (NEBD). Oocytes with centrally located nuclei were never observed to undergo maturation after isolation from the gonad. However, when oocytes with off-center nuclei were isolated and placed in a bath chamber, all of them underwent maturation within 2-10 min (mean time to beginning of NEBD = 6.2 +/- 1 min; n=9). Cell volume did not change significantly prior to and during the maturation process. In separate experiments, we patch clamped oocytes with off-center nuclei and oocytes that had undergone NEBD in vitro . CLH-3 activity was not detected in oocytes with intact nuclear envelopes ( Cl - current = -2.1 +/- 0.3 pA/pF ). However, in oocytes undergoing maturation in vitro , CLH-3 activity was detected immediately upon obtaining whole-cell access. After membrane rupture, CLH-3 continued to activate until the current reached a stable plateau level ( Cl - current = -44.4 +/- 6.2 pA/pF ). Taken together, our results demonstrate clearly that CLH-3 is activated during meiotic maturation rather than after completion of oocyte growth. We are currently characterizing the signaling pathways responsible for channel activation.

Yukimasa Shibata et al. (2007) International Worm Meeting "A putative histone-binding protein, BET-1, maintains distinct gene expression patterns between closely related cells in C. elegans."

Hisako Takeshita, Noriko SASAKAWA, Hitoshi Sawa, Yukimasa Shibata

[International Worm Meeting, 2007]

Progenitor cells divide to produce a variety of differentiated cells that express specific sets of genes. Such characters of differentiated cells must be kept stable, possibly by maintaining chromatin structures to keep transcriptional state of each gene. We isolated bet-1 mutants by abnormal T cell lineage phenotype. bet-1 encodes an evolutionally conserved protein containing the bromodomain that is known to binds to acetylated histones. bet-1 mutants showed defects in maintenance of gene expression pattern. Each descendants of the wild-type neural progenitor cell, V5.pa, start to express a cell type specific marker shortly after its birth (for example, dat-1::gfp and osm-6::gfp for PDE), then those expressions are maintained at later stages. In bet-1 mutants, the descendants that normally do not express those markers frequently start expressing them ectopically at much later stages. In addition, osm-6::gfp-positive neurons frequently lose its expression. These results indicate that BET-1 functions to keep gene expression pattern by chromatin factors including histones. In addition to the V5.pa lineage, ectopic expression of a DTC marker, lag-2::gfp, or AVM/PVM marker, mec-4::gfp, was also observed in the Z1/Z4 or Q lineages, respectively. Ectopic lag-2::gfp or mec-4::gfp expression also started at much later stages than start of the normal expression. These results suggest that BET-1 is involved in maintenance of gene expression pattern in multiple cell lineages. In bet-1, all the cells that ectopically express the marker genes are produced from the same progenitor cells with the normally expressing cells. In addition, in the V5.pa lineage, ectopic osm-6::gfp expression was observed more frequently in the sister or its progeny of PDE than in its cousins. These results suggest that distinct gene expression patterns were fixed progressively through a series of asymmetric cell division, so that differences between sister cells are more highly dependent on bet-1 than those between cousins. Among distantly related cells, differences of gene expression are maintained in bet-1-indpenedent manner.

Nicholas J D Gower et al. (2002) European Worm Meeting "Regulation of inositol 1,4,5-trisphosphate receptor gene (itr-1) expression by pha-4"

Nicholas J D Gower, Howard A Baylis

[European Worm Meeting, 2002]

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a central role in the organisation of calcium signals by regulating intracellular calcium flux. IP3Rs in C. elegans are encoded by a single gene, itr-1 (also called lfe-11 and dec-42). We have shown previously that the itr-1 gene has three putative transcriptional start sites3. Sequence comparisons to the C. briggsae itr-1 gene supported the hypothesis that itr-1 has three promoters (pA, pB and pC), each of which gives rise to an alternative mRNA and hence unique protein. By using transgenic GFP reporter constructs we showed that these promoters direct cell-specific expression of itr-1 variants and further that this results in adjacent cells in the same tissue containing different IP3R isoforms4.