Kislyak Y et al. (2000) East Coast Worm Meeting "Biochemical Characterization of C. elegans RNA Helicase A and Malate Dehydrogenase"

Kislyak Y, Walstrom KM, Do IP

[East Coast Worm Meeting, 2000]

In my laboratory, we are overexpressing and biochemically characterizing two different proteins from C. elegans. RNA helicase A (RHA-1) is essential for proper development in many organisms, including C. elegans. Human RHA is involved in RNA metabolism. For example, it is capable of mediating an association between CREB binding protein (CBP) and RNA polymerase II and is suggested to be required for the activation of transcription by CBP (Nakajima et al. (1997) Cell 90, 1107-1112). Human RHA is also required for RNA export from the nucleus (Tang et al. (1999) MCB 19, 3540-3550). We are attempting to determine the RNA binding targets of RHA-1 in the cell in order to elucidate its function in C. elegans. In another project, malate dehydrogenase (MDH), the last enzyme in the citric acid cycle, has been overexpressed and purified. This enzyme is active, and the enzyme kinetics of C. elegans MDH are being studied. Recent results from both projects will be presented. We are grateful to Yuji Kohara for providing the cDNA clones of these enzymes.

Peter Swoboda et al. (2002) European Worm Meeting "A multifaceted approach to elucidate the regulation of ciliogenesis in C. elegans"

Evgeni Efimenko, Peter Swoboda

[European Worm Meeting, 2002]

Cilia are evolutionarily conserved subcellular organelles functioning in cell motility, movement of extracellular fluids, sensory perception (e.g. smell) and determination of left-right asymmetry. While a great deal is known about the structure, function and motility of cilia, very little is known about the molecular mechanisms that regulate ciliogenesis in a cell-type specific and developmental manner. How do cilia become functional? How can they do what they do?

Rafael P Vazquez-Manrique et al. (2007) International Worm Meeting "PLC-1/Phospholipase C epsilon regulates embryonic morphogenesis through the IP3 receptor in Caenorhabditis elegans."

Sung Ly, Howard A Baylis, Olivia Bales, Rafael P Vazquez-Manrique, James C Legg

[International Worm Meeting, 2007]

Signalling through the second messenger IP₃ is required at several stages of embryonic development in C. elegans. Previous work has demonstrated a role for IP₃ signalling in the rearrangement of epidermal cells during morphogenesis. However little is known about the molecules acting in this pathway. IP₃ is produced by phospholipase C enzymes (PLC). In order to identify signalling pathways upstream of IP₃ production, in epidermal morphogenesis, we sought to identify which of the five active PLCs in C. elegans is important for this process. We tested the function of PLC genes using RNAi and mutant strains, and we found that depletion of PLC-1/PLC-<font face=symbol>e</font> produced substantial embryonic lethality. 96% of these arrested embryos show morphogenetic defects, which include aberrant dorsal intercalation and defective ventral migration. As a consequence plc-1 loss of function results in ruptured embryos with Gex phenotype (gut in the exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. However null or severe loss of function plc-1 mutants do not show the same level of embryonic lethality as some itr-1 mutants. We therefore tested if other PLCs may act redundantly with PLC-1. Using double mutants of PLCs together with plc-1, we show that PLC-3/PLC-<font face=symbol>g</font> and EGL-8/PLC-<font face=symbol>b</font> can compensate for the lack of PLC-1 activity. Finally, we demonstrate that PLC-1 acts through the IP₃ receptor of C. elegans (ITR-1), a molecule known to be involved in the regulation of C. elegans morphogenesis.

Lee M-H et al. (1997) International C. elegans Meeting "TOWARD THE IDENTIFICATION OF IN VIVO RNA TARGETS OF GLD-1."

Lee M-H, Schedl TB

[International C. elegans Meeting, 1997]

gld-1 is a female germ cell specific tumor suppressor gene that is essential for normal oocyte differentiation and meiotic prophase progression (Francis et al., 1995a,b). GLD-1 is a member of a small family of proteins, including the mouse/human Sam68 and Quaking proteins, that are highly related over an ~ 200 amino acid region which contains a 70 to 100 amino acid RNA binding domain called the KH motif (Jones and Schedl, 1995). Extensive mutational analysis of gld-1 has demonstrated the importance of conserved sequences for its in vivo function. GLD-1 is a cytoplasmic protein and therefore is likely to control mRNA translation or RNA stability in the cytoplasm (Jones et al., 1996). Currently, no obvious RNA targets have been identified from genetic analysis. We have employed a biochemical approach to identify in vivo RNA targets of GLD-1. The strategy is based on the ability of anti GLD-1 antibodies to immunoprecipitate (IP) GLD-1 from a worm lysate and the strong likelihood that GLD-1 present in the lysate is functional and bound to RNAs. GLD-1 was IPed with affinity purified polyclonal antibodies from a cytosol extract of adult hermaphrodites or with rabbit IgG as a control. RNAs co-IP with GLD-1, as well as with rabbit IgG, were converted into cDNAs. Non-specifically trapped RNAs from the GLD-1 IP were eliminated by subtracting cDNAs from the GLD-1 IP with cDNAs from the control IP. The difference product after 4 rounds of subtraction (DP4) was used to screen a Lambda ZAP II cDNA library constructed with the cDNAs from the GLD-1 IP. Duplicate filters were also screened with a probe made from control IP cDNA. Clones that are positive only with the DP4 probe are currently being characterized. Francis et al., 1995a Genetics 139, 579-606; Francis et al., 1995b Genetics 139, 607-630; Jones and Schedl, 1995 Genes Dev. 9, 1491-1504; Jones et al., 1996 Dev. Biol. 180, 165-183. This work was supported by NIH Grant HD25614.

P Dal Santo et al. (1999) International C. elegans Meeting "The IP3 Receptor is a Timekeeper for the Defecation Cycle Rhythm"

EM Jorgensen, P Dal Santo, MA Logan, AD Chisholm

[International C. elegans Meeting, 1999]

The C. elegans defecation cycle is characterized by the activation of three distinct muscle contractions every 45-50 seconds. How is cycle time maintained? To address this question we identified and characterized mutants with abnormal cycle times. Specifically, we cloned the dec-4 gene and demonstrated that it encodes the inositol trisphosphate receptor (IP 3 receptor). Mutations in the IP 3 receptor alter the timing of the defecation cycle. Null mutations eliminate the cycle, hypomorphic mutants have a slow cycle, and overexpression of the protein results in a fast cycle time. The IP 3 receptor is expressed in several different tissues including the pharynx, the somatic gonad, and the intestine. Using mosaic analysis we demonstrated that IP 3 receptor expression in the intestine is necessary and sufficient for normal timing of the cycle. This indicates that the periodic muscle contractions can be controlled non-autonomously from the intestine. IP 3 receptors are localized to the endoplasmic reticulum and are known to function as calcium release channels. Activation of the channel by a phospholipid signaling pathway leads to the production of temporally and spatially regulated calcium oscillations. Our current data predicts a model in which calcium oscillations in the intestine trigger the defecation motor program. To test this model we injected calcium-sensitive dyes into the posterior intestinal cells of adult animals. Our results demonstrate that calcium oscillations occur in the intestine. The frequency of calcium oscillations are consistent with the timing of the defecation cycle and peak calcium levels occur just before the onset of the muscle program. We conclude that periodic calcium release in the intestinal cells initiates the muscle contractions of the defecation cycle and that the IP 3 receptor is a central component of the timekeeping mechanism that regulates this behavioral rhythm.

Irene Kalchhauser et al. (2008) European Worm Meeting "The role of GLD-1 in the maintenance of germ cell totipotency"

Rafal Ciosk, Irene Kalchhauser, Mathias Senften, Min-Ho Lee

[European Worm Meeting, 2008]

The conserved C.elegans translational regulator GLD-1 is a germline tumor. suppressor that is enriched in the germline granules. Together with another. conserved translational regulator, MEX-3, GLD-1 maintains germline. totipotency. In mex-3 gld-1 mutant worms, germ cells transdifferentiate. into somatic cell types such as neurons or muscles. Several defects,. including loss of germline granules, have been implicated in. transdifferentiation. To understand how GLD-1 contributes to germline. totipotency and to the maintenance of germline granules, we are looking for. GLD-1 mRNA targets whose depletion affects transdifferentiation. Currently,. we are testing candidates obtained from RNA-Co-IP and microarray analysis. by RNAi and are validating their regulation by GLD-1. We expect new. insights on the interconnection between translational regulation and cell. fate choice.. Do not add objects such as pictures, boxes, headers, footers, footnotes,. etc.

Min-Ho Lee et al. (2005) International Worm Meeting "GLD-1 mRNA Targets and Binding Specificity"

Valerie Reinke, Ting Wang, Tim Schedl, Min-Ho Lee, Gary Stormo

[International Worm Meeting, 2005]

The germline is an excellent system for investigating translational control as it is a major mechanism for regulating gene expression. GLD-1 is a germline specific, maxi-KH motif containing RNA binding protein that controls multiple aspects of C. elegans germ cell development, suggesting that it regulates multiple mRNAs. To understand how GLD-1 regulates mRNAs to control germ cell development, we have previously identified multiple in vivo mRNA targets of GLD-1 that were co-immunoprecipitated (IP) with GLD-1 from cytosol extracts and recovered following subtractive hybridizationa, b. These target mRNAs are preferentially expressed in the germline and several have essential functions in oocyte differentiation and early embryogenesis. GLD-1 is a translational repressor, based on the analysis of mRNA and protein levels of five mRNA targets (rme-2, puf-5, cep-1, oma-1, and oma-2) in wild-type and gld-1 null germlines. However, it has been clear that we do not have the complete list of GLD-1 mRNA targets. Thus, we used microarray analysis to identify additional targets that are co-IPed with GLD-1. We identified 129 genes that are enriched more than two fold (p<0.05) in the GLD-1 IP over the control IP. Essentially all of our previously identified targets are recovered in the new screen. To determine the in vivo binding specificity of GLD-1 or GLD-1 containing complexes, we have used GLD-1 from wild-type cytosol extracts to identify 12 GLD-1 binding regions in six targets thus far. GLD-1 can bind the 5UTR, 3UTR or in the coding region depend on the target. Using the sequences of 13 GLD-1 binding regions (12 in the six targets and the tra-2 3UTR) and the corresponding, orthologous C. briggsae regions, we found 3 potential GLD-1 binding motifs with PhyloCon, a program that identifies regulatory motifs among co-regulated orthologous gene setsc. One of the motifs is essentially identical to the hexanucleotide consensus that was identified in the tra-2 3UTR by Ryder et al, 2004d. Interestingly, the tra-2 3UTR has only this motif four times. Most other binding regions have two different motifs and three regions do not contain the hexanucleotide consensus. Biochemical analysis of the motifs indicates that two motifs, including the hexanucleotide consensus, are important for GLD-1 binding and synergistic interactions between motifs likely occur. Therefore, the data from the IP/microarray analysis and the binding studies suggest that, in addition to the hexanucleotide consensus, GLD-1/GLD-1 containing complexes likely utilize other sequences to achieve specificity. a, b Lee and Schedl, Genes Dev, 2001 & 2004; c, Wang and Stormo Bioinformatics, 2003; d, Ryder et al. Nat Struct Mol Biol, 2004

Chihara, Daisuke et al. (2009) International Worm Meeting "Internalization of primordial germ cells during C. elegans gastrulation."

Chihara, Daisuke, Nance, Jeremy

[International Worm Meeting, 2009]

Gastrulation in C. elegans begins when the endodermal precursor cells constrict their apical surfaces and ingress into the interior of the embryo. Subsequently, mesodermal cells and the two primordial germ cells (PGCs) ingress in a spatial and temporal sequence that is highly orchestrated. Proper ingression of endodermal precursor cells requires zygotic transcription and a PAR protein-mediated polarity that distinguishes the contacted and contact-free surfaces of the ingressing cells. By contrast, embryonic gene expression has not been observed in the PGCs at the time that they ingress, and these cells do not develop the contact/contact-free asymmetry of PAR proteins that polarizes somatic cells. Therefore somatic cells and the PGCs might utilize distinct mechanisms to ingress during gastrulation. We hypothesized that forces exerted by surrounding cells might internalize the PGCs. Using time-lapse videomicroscopy, we first identified ingressing somatic cells cells that make contact with ingressing PGCs. Ingressing descendants of the MS and D lineages contacted the PGCs on the surface of the embryo, and endodermal precursor cells, which ingressed earlier during gastrulation, contacted the PGCs from below. We used laser operation to determine which of these cells is important for PGC ingression. Our results suggest that PGC ingressions can occur when ingression of the flanking MS or D descendants is blocked by laser-killing of these cells, but PGCs do not ingress if the endodermal precursors are killed. In addition, the PGCs do not ingress in end-1 end-3 double mutant embryos, in which endodermal cells are born in their normal position but do not ingress properly and do not form endoderm. We imaged embryos expressing transgenes that label PGCs and endodermal precursor cells and observed that these cells maintain a close association prior to, during, and after PGC ingression. Our experiments suggest that signals or forces from the endodermal precursor cells are critical for ingression of the PGCs.

Daisuke Kanematsu et al. (2002) Japanese Worm Meeting "Is IP3 Receptor or Ryanodine Receptor Involved in a Transient Increase of Cytosolic Ca2+ - Concentration during Fertilization of C. elegans Oocyte?"

Yasuji Sakube, Hideyo Kuroda, Hiroaki Kagawa, Ritsu Kuroda, Daisuke Kanematsu

[Japanese Worm Meeting, 2002]

Transient increases of intracellular calcium ion concentration ([Ca 2+ ] i ) have been observed during fertilization of eggs, from invertebrates to mammals. Caenorhabditis elegans seems to be a hopeful model organism to study the signaling pathway from a sperm-egg fusion to a transient increase in [Ca 2+ ] i . Since we have not succeeded to bring about the fertilization in vitro , we measured the [Ca 2+ ] i changes of oocytes during fertilization in vivo . A Ca 2+ indicator, Calcium-Green 1 dextran (10,000 MW) was injected into the distal portion of the ovary of adult worms. Two hrs after injection, the oocytescontaining the indicator lined up at the proximal portion of the ovary. Even in the worm anesthetized with tricaine and tetramisole, the oocytes moved against the proximal end of the ovary and entered into spermatheca in order. We collected the [Ca 2+ ] i images of oocytes in the ovary or spermatheca with a cooled CCD camera. We observed the fluorescent change of hermaphrodite oocytes during self-fertilization. Several seconds after passing through the valve between the ovary and spermatheca, an oocyte showed a transient increase in [Ca 2+ ] i . These results suggest that sperm of C. elegans fertilizes oocytes in the spermatheca and bring about a transient change in [Ca 2+ ] i of oocytes. In mammal eggs, the inositol 1,4,5-trisphosphate receptor/channels (IP 3 R) of endoplasmic reticulum membrane are responsible for these Ca 2+ -changes. The Ca 2+ -transients of sea urchin eggs seems to depend on the synergistical release via both of IP 3 R and ryanodine receptor/channel (RyR). To examine the contribution of IP 3 R or RyR to the Ca 2+ -transient of C. elegans , we examined the changes in [Ca 2+ ] i during self-fertilization of mutant strains of two genes, unc-68 encoding RyR and itr-1 encoding IP 3 R. Both in oocytes of unc-68 and itr-1 , similar changes to the Ca 2+ -transient in N2 oocytes were recorded. These results suggest two possibility, the Ca 2+ -transient during fertilization depend on (1) the synergistical release of Ca 2+ via RyR and IP 3 R from endoplasmic reticulum, or (2) the Ca 2+ -influx through a Ca 2+ channel of the plasmamembrane. To examine these possibilities, we are trying the RNAi of itr-1 and unc-68 .

Haji Adineh, S et al. (2019) International Worm Meeting "The tempo of paternal mitochondrial transmission in C. briggsae hybrids."

Jorgensen, C, Chahal, G, Haji Adineh, S, Ortega, B, Aguilar, A, Ross, J

[International Worm Meeting, 2019]

Although mitochondria are usually maternally inherited, substantial evidence from multiple taxa reveals that hybridization often promotes transmission of sperm-borne mitochondrial into the fertilized oocyte. Given prior reports of such paternal "leakage" of mitochondria in intra-species C. briggsae crosses, we conducted new experiments to refine our understanding of the genetic basis and tempo of paternal mitochondrial transmission (PMT). Analysis of paternal mitochondrial haplotypes in replicate hybrid lines revealed persistent but transient heteroplasmy that never persisted beyond ten generations. Thus, PMT appears to occur regularly following hybridization, but heteroplasmy is usually resolved in favor of the maternal mitotype. Additionally, no paternal mitotypes were detected among a sample of existing C. briggsae advanced-intercross recombinant inbred lines (AI-RIL), which could suggest that using recombination to disrupt a potentially complex genetic mechanism that regularly acts to prevent paternal transmission does not result in sustained homoplasmy of the paternal mitotype. However, crosses involving AI-RIL pseudofemales do occasionally result in PMT, further implicating the role of a hybrid maternal nuclear genome in facilitating PMT. Current efforts in C. elegans are testing the hypothesis that PMT is dependent on somatic sex, such that hermaphrodites do not perform paternal mitochondrial elimination during self-fertilization but do eliminate non-self sperm mitochondria when mated with a male.