Honer, Madison (2021) International Worm Meeting "Identifying the mechanisms of NLP-14/Orcokinin signaling during sleep."

Honer, Madison

[International Worm Meeting, 2021]

Despite sleep being essential and conserved, an understanding of its mechanisms are lacking. We recently described a role for NLP-14/orcokinin neuropeptides during the regulation of sleep, most notably during stress-induced sleep (SIS). Orcokinins are found in ecdysozoan animals and have been shown to regulate circadian rhythms and molting in insects. Despite their conservation, their downstream mechanisms are not well understood. Taking advantage of the observation that over-expression of nlp-14 induces a strong sleep phenotype, we conducted a forward genetic screen for suppression of the sleep phenotype. Our experimental approach uses 3D-printed chambers which allow for the sorting of animals using gravity. Mutagenized animals are induced to fall asleep by nlp-14 over-expression. Those who fell asleep were trapped in the lower chamber, while the rare suppressors swam against gravity to the upper collection platform. We have isolated multiple mutants, which we call orcokinin suppressors (orcs), and have sequenced their genomes. Currently, we are identifying the causative alleles. Two of these unmapped mutants orcs(327) and orcs(330) display drastically reduced levels of SIS. These new mutants may represent novel sleep genes essential for SIS regulation.

Cassada RC et al. (1995) International C. elegans Meeting "TS EMB-MUTANT PHENOTYPES: PATERNAL & OTHERS WITH EARLY DEFECTS (G-SET)"

Wolke U, Fiebich B, Cassada RC

[International C. elegans Meeting, 1995]

A: The parental effects of mutants emb-27 and emb-30 can be resolved into maternal and paternal effects (Madison Abst. '93) with temperature sensitive periods in meiosis. The phenotypes are variable and complex, so we are glad to have lots of help from D. Shakes' group on emb-27 and S. Siddiqui's on emb-30. Both mutants appear to have problems in centrosome segregation, giving post-fertilization anomalies. In the 1-cell maternal arrest, P-granules collapse around the central nuclei, but segregate normally in paternal effect embryos. B: Altered early division patterns in certain mutants of the Goettingen set (Denich et al.) suggest blastomere identity changes which we are starting to examine more directly: emb-13: E like C; emb-34: all lineages identical except AB (& random orient.); emb-5: all identical (extra P mit.); emb-21: double posterior (AB-less bicaudal?); emb-22: slow clock C: Finally, antibodies to mammalian protein kinase c (PKC) also stain the CDR's described by Waterston's lab.

Kevin Eliceiri et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "A Swept-Field Confocal for Cell Biology Studies"

Kevin Eliceiri, Mike Szulczewski

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

We describe a novel instrument, The Swept-Field Confocal Microscope (SFC) that combines high resolution pinhole imaging with slit imaging. Unlike spinning disk confocal systems that have their pinhole apertures embedded in a spinning disc, the SFC's 32 pinhole array remains stationary. Galvonometric and piezo controlled mirrors sweep the image of the pinholes across the sample. The emission photons are de-scanned and focused through a complementary set of pinholes onto a CCD camera. This results in a high resolution image that can be collected at up to 100 frames/second in the pinhole mode and greater than 1000 frames per second in the slit mode. The ability for the scientist to match the optical recording with the temporal biological fluorescence response has great promise for cell and developmental biology studies where live dynamic events must be captured quickly in high resolution. In this poster we describe the optical path of the microscope and present some early data in collaboration with John White's group at the UW-Madison of using the SFC to image GFP constructs in C .elegans to investigate cell division in the early embryo.

Dasgupta, Krishnakali et al. (2009) International Worm Meeting "Understanding the role of PKC-1 signaling pathway in regulating Dense Core Vesicle secretion."

Sieburth, Derek, Dasgupta, Krishnakali

[International Worm Meeting, 2009]

Synaptic vesicles (SV) and dense core vesicles (DCV) both exhibit calcium dependent neurotransmitter release. However, differences in their biogenesis, release properties, and recycling suggest that each type of organelle may use some unique proteins to carry out their functions. PKC-1, a protein kinase C ortholog in worms, is one such candidate for a unique DCV regulating molecule, since it has been shown to regulate neuropeptide secretion, while possibly not affecting SV release.(1) PKC-1 is most similar to the human eta and epsilon isoforms which are activated by the second messenger DAG via their C1 domains. In order to identify additional components that act in the PKC-1 pathway to regulate DCV secretion, we have used a constitutively active PKC-1 mutant which lacks the autoinhibitory domain and is predicted to be active in the absence of DAG. Expression of this transgene specifically in motor neurons causes hypersensitivity to an acetylcholinesterase inhibitor, aldicarb (Hic) and loopy locomotion. We screened for suppressors of either the loopy or Hic phenotype of animals expressing the constitutive PCK-1, and identified ~30 suppressors that restore movement or aldicarb response. We are in the process of mapping them and characterizing their effects on neuropeptide secretion. Presently, we have identified one aldciarb resistant candidate allele, vj3, which shows decreased uptake of NLP-21::YFP by coelomocytes and increased accumulation of DCVs in axons. However, vj3 mutants do not cause an accumulation of the SV marker at synapses, suggesting that vj3 mutants specifically reduce DCV secretion. vj3 maps to a small interval on LG I that does not contain any known neurotransmitter secretion genes. We are currently in the process of fine mapping, and tesing candidate genes for aldicarb resistant phenotypes by RNAi. 1.PKC-1 regulates secretion of neuropeptides; Derek Sieburth1, Jon M Madison & Joshua M Kaplan; Nature Neuroscience 10, 49 - 57 (2007).

Hideaki Hiraki et al. (2003) International Worm Meeting "Semi-automatic system for creation of cell shape model in C. elegans embryogenesis"

Hideaki Hiraki, Yuji KOHARA

[International Worm Meeting, 2003]

Cells change their shapes and arrangements in embryogenesis. 4D microscopy of C. elegans is useful to observe the process. Time-lapse recording of multiple optical sections with Nomarski DIC microscope and tools to analyse the results have been developed and used successfully, especially for cell lineage analysis because cell nuclei of C. elegans are evident in DIC images. Though cell shape models of early embryo can be reconstructed from DIC images, delineating cell boundaries become harder as the cell number increases. On the other hand, cell shapes can be directly visualized by confocal fluorescent microscopy with vital stain of plasma membranes. We have been developing a computer system to create cell shape models from a time series of confocal images of plasma membranes. In the system, cell shapes are automatically calculated by a seeded region growing algorithm from a 3D image and a set of seed point coordinates. The algorithm segment the image volume to enclosed regions one per a seed. Here a cell boundary is detected as ridges of high fluorescent signal between two seed points. In many cases, the seed points can be automatically placed using results of another time point. But manual editing of the seed sets is necessary where signal to noise ratio is low. The system constructs a pseudo cell lineage from the overlay relationships among the segmented regions over the time course. The inconsistency in the pseudo lineage indicates where editing is required and the editing is assisted by the graphical user interface. We have tested the system on the "dub" data set in WORM 4D CDs, 6/17/97, Bill Mohler, UW-Madison, that is of N2 embryo in 24-200 cell stages. Changes in physical parameters of cell shapes and arrangements, such as cell volumes and cell-to-cell contacting areas, could be estimated from the resultant shape model of a whole embryo under gastrulation. We are planning to apply this system to compare mutant embryos and the embryos from other species closely related to C. elegans.

Hardin JD et al. (1995) International C. elegans Meeting "THE CELLULAR BASIS OF HYPODERMAL ENCLOSURE"

Malik AN, Hardin JD, Williams-Masson EM

[International C. elegans Meeting, 1995]

Our laboratory is using digital "4-d" and confocal microscopy to characterize the morphogenesis of the embryonic hypodermis of C. elegans. The cells of the dorsal hypodermis intercalate to form a single row across the dorssal midline. Concurrent contralateral nuclear migration also takes place in these cells. Based on analysis of unc-83 (e1408) embryos and treatment of embryos with nocodazole, nuclear migration and intercalation are separable. Laser ablation of individual dorsal cells indicates that cells can intercalate when their immediate neighbors fail to migrate. Based on the alignment of cytoskeletal elements that occurs during intercalation, we propose that intercalation may be required for the correct cirumferential alignment of the actomyosin and microtubule cytoskeletal networks re quired for subsequent elongation. Ventral hypodermal cells undergo epiboly to enclose the embryo as they meet at the ventral midline. The bilateral pairs of cells derived from ABpraapp and ABplaapp seem to be particularly important for the advance of the ventral margin; when all four of these cells are ablated early in enclosure, enclosure is prevented. Pairwise ablation of these cells does not prevent enclosure from occurring largely normally, but such embryos develop ventral ruptures during the elongation process. Ablation of margin cells posterior to this quartet causes an immediate retraction (within 5 sec) of the hypodermis back towards the ventral side of the embryo, suggesting that there is substantial tension generated at the free margin of the hypodermis. We propose a two-step model for ventral enclosure in which the four "leading cells" initially pull the ventral margin ventrally, followed by a "purse-string" closure of the posterior. In collaboration with the laboratory of J. Rothman (UW-Madison) we have recovered several phenotypic classes of point mutants and definciency strains that mimic our laser ablation results. These include the deficiency sDf23, in which the "leading" quartet of cells fail to migrate, and the zygotic embryonic lethal mutant ze79, in which head enclosure occurs normally but enclosure of the "torso" is aberrant. Further characterization of these and other mutants is underway.

Paupard M-C et al. (2000) East Coast Worm Meeting "Improved Tissue Preservation Using Metal Mirror Fixation or High Pressure Freezing for TEM"

Macaluso F, Stephney G, Paupard M-C, Hall DH

[East Coast Worm Meeting, 2000]

Recent reports (see below) showed that high pressure freezing (HPF) followed by freeze substitution is superior to chemical immersion fixation for C. elegans. HPF captures a more "life-like" view of the worm's ultrastructure. We compared HPF and a related technique, rapid freezing onto a metal mirror (MMF). For MMF, live animals on a small piece of filter paper are plunged against a metal mirror in liquid nitrogen. Freezing damage is often a problem, but some animals seem to be well frozen throughout. For HPF, we have tried two methods to concentrate live animals into a small metal planchette (see Lavin and McDonald ref's below). Further processing is the same for both methods. While holding at very low temperatures, the samples are freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. By TEM fast-frozen worms reveal excellent views of membrane events and organelles. For instance, we see active endocytosis events that are not captured by chemical fixation. The microtubule network is better preserved and the basal laminae look strikingly different. Sample images are shown at www.aecom.yu.edu/wormem/new.html. HPF and MMF also hold promise for high resolution immunoEM. By reducing the osmium content and adding a dilute aldehyde fixative to the freeze substitution medium, we can better preserve structure than by our microwave technique (Paupard et al., submitted). We have successfully localized epitopes in thin sections from HPF samples. We are conducting HPF trials with Stan Erlandson and Ya Chen at the U. of Minnesota. MMF equipment is available here at Einstein and elsewhere. HPF machines are available to outside users in Madison, Berkeley, Minneapolis, and Albany. As our skills improve, we will offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/coating.html Martin Muller's website at www.em.biol.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Humana Press) 1999.

Stephney G et al. (2000) Midwest Worm Meeting "Improved Tissue Preservation Using Metal Mirror Fixation or High Pressure Freezing for TEM"

Hall DH, Macaluso F, Paupard M-C, Stephney G

[Midwest Worm Meeting, 2000]

Recent reports (see below) showed that high pressure freezing (HPF) followed by freeze substitution is superior to chemical immersion fixation for C. elegans. HPF captures a more "life-like" view of the worm's ultrastructure. We compared HPF and a related technique, rapid freezing onto a metal mirror (MMF). For MMF, live animals on a small piece of filter paper are plunged against a metal mirror in liquid nitrogen. Freezing damage is often a problem, but some animals seem to be well frozen throughout. For HPF, we have tried two methods to concentrate live animals into a small metal planchette (see Lavin and McDonald ref's below). Further processing is the same for both methods. While holding at very low temperatures, the samples are freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. By TEM fast-frozen worms reveal excellent views of membrane events and organelles. For instance, we see active endocytosis events that are not captured by chemical fixation. The microtubule network is better preserved and the basal laminae look strikingly different. Sample images are shown at www.aecom.yu.edu/wormem/new.html. HPF and MMF also hold promise for high resolution immunoEM. By reducing the osmium content and adding a dilute aldehyde fixative to the freeze substitution medium, we can better preserve structure than by our microwave technique (Paupard et al., submitted). We have successfully localized epitopes in thin sections from HPF samples. We are conducting HPF trials with Stan Erlandson and Ya Chen at the U. of Minnesota. MMF equipment is available here at Einstein and elsewhere. HPF machines are available to outside users in Madison, Berkeley, Minneapolis, and Albany. As our skills improve, we will offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/coating.html Martin Muller's website at www.em.biol.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Humana Press) 1999.

Masahiro Ito et al. (1999) International C. elegans Meeting "Toward four-dimensional database of gene expression in C. elegans"

Tomoko Motohashi, Masahiro Ito, Yohei Minakuchi, Yuji KOHARA

[International C. elegans Meeting, 1999]

One of the most challenging targets in the post genomics era is computer simulation of development. We feel C.elegans is the most suitable system for this purpose, since the complete parts list of cells has been identified and the expression patterns and functions of all genes are being analyzed systematically. Aiming at the final goal and at integrating such information, we are constructing a computer graphics (CG)-based four-dimensional (3D + time course) database of gene expression in C.elegans . Thus far, we have made a CG for early embryogenesis up to 100 cell stage based on 4D Nomarski image data including the data kindly supplied by Kevin O'Connel at the University of Wisconsin at Madison. Since it was very hard for conventional image processing software to extract the contour of cells from the Nomarski images, we adopted a 'low-tech' procedure; firstly we traced the contour of individual cells and nuclei by hand-writing on thousands of the original images of the 4D microscopic data and then reconstructed 3D structures by piling up and making relationship among the contour data from different focal planes. The resulting 3D structures of various stage embryos were arranged along the time course of development and connected each other by an interpolating method to finally generate the CG of early embryogenesis. The graphics is faithful to the real embryogenesis, thus, it provides various information such as the volume of individual cells, the extent of cell-cell contact and so on. To incorporate the information of gene expression patterns into the 4D graphics database, we have developed a procedure to superimpose a fluorescent-confocal data of the distribution of mRNA and proteins onto the CG. In the procedure, we use triple color staining, namely, DAPI and Cy-3 for nuclei and POS-1 (P-lineage specific) as internal markers for superimposing, respectively, and Cy-5 for a query gene product. By this fashion, we are incorporating the immunostaining patterns of maternal gene products. We are planning to make the 4D database accessible over the internet.

Shiraishi, Hirohisa et al. (2013) International Worm Meeting "Genetic requirements of the pentose phosphate pathway for the intestinal granule formation in C. elegans."

Shiraishi, Hirohisa, Asanuma, Yumi, Tanabe, Mari, Oikawa, Takato, Aoyama, Reiko, Nishikori, Kenji, Ohashi-Kobayashi, Ayako, Ishibashi, Maya, Tanji, Takahiro

[International Worm Meeting, 2013]

We have focused on intestinal granular organelles observed from late larval to early adult stage in C. elegans since a major subset of them dynamically emerge or evanesce in an age- and nutrient-dependent manner (Nishikori K., et al., C. elegans Topic Meeting 2012, Madison, WI). Such organelles are non-acidic granules to which HAF-4 and HAF-9, ABC transporters homologous to a mammalian lysosomal peptide transporter ABCB9, co-localize. The deletion mutants for haf-4 and haf-9 exhibited the loss of the HAF-4/HAF-9-positive granules and some physiological defects such as slow growth and reduced brood size, suggesting that the granules play important roles in the storage and supply of some nutritional components during reproductive period. To uncover the physiological roles and the regulatory mechanisms of the intestinal granules, we have performed comprehensive RNAi screening using C. elegans RNAi Feeding Library (Open Biosystems). To achieve the quick and reliable evaluation of RNAi effects, we adopted a high-powered digital microscope for observation. This system magnifies live worms on the rearing agar plate up to 5,000 times, which facilitates screening for the presence of intestinal granules. So far several tens of candidates with reduced granular number have been identified among approximately 2,000 genes tested. In this meeting, we report tkt-1, an orthologous gene for mammalian transketolase (TKT), as one candidate. TKT acts in the pentose phosphate pathway (PPP), which contributes to fatty acid synthesis and oxidative stress prevention via NADPH production as an alternative to glycolysis pathway. We found that RNAi of some PPP-related genes and the glutathione-disulfide reductase gene results in the reduction of the HAF-4::GFP-positive granules and the DHS-3::GFP-positive lipid droplets, but not of the GLO-1::GFP-positive lysosome-related organelles. The NADPH-producing oxidative pathway of PPP might be necessary for the normal formation of specific subsets of the intestinal granules in C. elegans.