Jaklitsch, Erik et al. (2021) International Worm Meeting "Immobilization of C. elegans by thermoelectric cooling for high-throughput microscopy"

Jaklitsch, Erik, Grooms, Noa, Chung, Samuel, Wang, Yao

[International Worm Meeting, 2021]

Microscopy, surgical techniques, and imaging in vivo are broadly utilized for model organisms. However, despite widespread usage, these strategies for multicellular organisms remain low-throughput and require significant manual involvement. Here, we report the implementation of a novel cooling stage to immobilize Caenorhabditis elegans on typical agar cultivation plates for these purposes. This device can effectively cool C. elegans to between 1-2 degrees Celsius for immobilization and maintain the temperature with minimal fluctuations. We demonstrate the ability to perform imaging and surgical techniques without classic anesthetic agents like sodium azide. This technique decreases animal processing time while maintaining organism viability and fecundity, using an intuitive device built with attainable materials. Our thermoelectric cooling stage, which is highly effective and built to combine with standard microscopy setups, can enable high-throughput microscopy and surgical techniques with decreased manual and chemical interventions.

Wang, Yao et al. (2019) International Worm Meeting "High-throughput Microscopy Platform toward a Roadmap for Accelerating Vertebrate Neuroregeneration Research."

Jaklitsch, Erik, Chung, Samuel, Wang, Yao

[International Worm Meeting, 2019]

Statement of Purpose Lesion conditioning to a neuron activates cellular mechanisms to enhance neuro-regeneration. Our prior study established the roundworm C. elegans as a powerful model for the rapid discovery of genes underlying lesion conditioning, which is impractical in vertebrates (Chung, S. H., et al. (2016).) Even so, a complete search for regeneration genes requires surgery and reimaging of a fluorescent neuron in >30,000 C. elegans, which represents significant efforts for the gene screening. A high-throughput surgery and reimaging platform, or specifically, a new integrated microscope platform for rapid immobilization, imaging, and optical surgery of C. elegans, would enable this gene search. Method Used Achievement of this goal requires the acceleration of two time-consuming steps: animal immobilization and precise neuronal microscopy. This can be accomplished through two specific goals: 1. Develop a microscope stage to readily immobilize many animals for in vivo imaging. 2. Enable robust automation of microscopy by improving neuron-background contrast. For Goal 1, C. elegans will be immobilized by thermoelectrically cooling them directly on their cultivation plates, instead of mounting them to slides. For Goal 2, patterned illumination approaches will enhance contrast by reducing the excitation power that illuminates extremely bright cell bodies. This patterned illumination will reduce the bright haze around the cell body from obscuring the axon and dendrites, and exposes the axon and dendrites near the cell body. Summary of Result We finished the thermal simulation of the cooling stage and tested thermoelectric performance under experimental conditions, which confirmed the feasibility of immobilizing worms on their cultivation plates. We invented a new coarse segmentation algorithm and patterned the illumination by employing a spatial light modulator, which validates our contrast improvement approaches. The accomplishment of our goals will pave the path to the high-throughput microscope platform for C. elegans research, which can be used in numerous neuroscience labs worldwide. This platform also enables our regeneration screen. Affiliations: Funding provided by Northeastern University

Kim J et al. (2017) MicroPubl Biol "A quantitative trait locus for nictation behavior on chromosome V"

Lee D, Lee J, Kim J

[MicroPubl Biol, 2017]

Lee et al., 2017 showed that CB4856 has a lower nictation ratio (the number of nictating dauers over the number of moving dauers on a micro-dirt chip) than N2, and that a genetic locus (nict-1) on chromosome IV mediates this phenotype difference. Although this paper had no evidence of any other genetic loci for nictation behavior, we have studied other quantitative trait loci (QTL) using different linkage-based mapping strategies. Here, we report another genetic locus that was identified using introgression lines on chromosome V. N2 x CB4856 introgression lines were tested using a population nictation assay (Lee et al., 2015). We measured the nictation ratios of the ewIR65, ewIR67, ewIR68, and ewIR70 lines, which contain N2-type genome in all chromosomes except for CB4856-type chromosome V segments (Figure 1A). Among them, only ewIR70 line showed significant lower nictation ratio. Therefore, we narrowed down the chromosome V right region. We also measured nictation ratios of ysIR50, ewIR74, and ysIR51 lines (Figure 1B). ysIR50 and ysIR51 were derived from N2 x ewIR70. ysIR50, ewIR74, and ysIR51 lines showed significantly lower nictation ratios than that of N2. Because the putatively responsible QTL region is covered by another near-isogenic region, eanIR158, which was made independently by Erik Andersens laboratory, we measured the nictation ratios of eanIR158 and jyIR3 lines and found that they did not show any significant difference from N2 (Figure 1C). eanIR158 and jyIR3 lines were built with QX1430 x N2 (personal communication) and QX217 x N2 (Balla et al., 2015), respectively, and QX1430 and QX217 were made by Leonid Kruglyaks laboratory (Andersen et al., 2015; Rockmen and Kruglyak, 2009). However, ewIR70 and ewIR74 were built by Jan Kammengas laboratory (Doroszuk et al., 2009). Thus, we concluded that the two laboratories may have used different N2 or CB4856 strain derivatives, which may have altered genetic variations in the introgression lines. A take-home lesson is that it is important to examine NILs derived from common original strains in order to follow QTL without being affected by differences in background genotypes. Jan Kammenga kindly provided their N2 x CB4856 introgression lines for chromosome V, which include ewIR65, ewIR67, ewIR68, ewIR70, and ewIR74, and Erik Andersen also gave us many introgression lines, which include eanIR158 and jyIR3 covering 15-19 Mb of chromosome V. We appreciate their help.

Crowell T et al. (1995) International C. elegans Meeting "SYNAPTOTAGMIN-DEFICIENT (SNT-1) MUTANTS OF C. ELEGANS: MOLECULAR AND PHENOTYPIC ANALYSIS"

Crowell T, Rand JB, McManus JR

[International C. elegans Meeting, 1995]

Synaptotagmin is a major synaptic vesicle protein; it contains 2 apparent calcium-binding (PKC-CII) domains and seems to be involved in both exocytosis and endocytosis. The cloning of snt-1 and the isolation of mutants were reported by Nonet et al. (1993). We have now analyzed 17 independent spontaneous snt-1 mutations isolated in our laboratory, as well as alleles isolated by Leon Avery and Erik Jorgensen. Molecular analysis indicates that 12 of the alleles are associated with DNA rearrangements (3 Tc1 insertions, 1 non- Tc1 insertion and 8 deletions), with the remaining alleles due to point mutations. Some of the deletions provide guaranteed molecular null alleles; null mutants are quite uncoordinated, but viable. Phenotypic analysis of snt-1 mutants included quantitative behavioral studies (pharyngeal pumping and head thrashing) and immunoblots. Several of the mutants clearly retain some gene function, and it was possible to establish an alleleic series of phenotypic severity. Partial gene function is retained not only by some of the point mutants, but also by a 66-bp (inframe) deletion which eliminates most of the first CII domain, and by a 9-bp deletion which disrupts the second CII domain. Supported by a grant from MDA

Duerr JS et al. (1995) International C. elegans Meeting "ANATOMY OF CHOLINERGIC NEURONS IN NORMAL AND MUTANT C.ELEGANS AND IN ASCARIS SUUM"

Spaulding D, Johnson CD, Rand JB, Duerr JS

[International C. elegans Meeting, 1995]

Special thanks to Tony Stretton and Judy Donmoyer for assistance with Ascaris We are studying the anatomy of the cholinergic nervous system using two proteins as markers, choline acetyltransferase (ChAT), the acetylcholine synthetic enzyme, and UNC-17, a synaptic vesicle acetylcholine transporter. In C. elegans, antibodies to UNC-17 appear to label synaptic regions, while antibodies to ChAT are enriched in synaptic regions, but also faintly label somas. ChAT and UNC-17 are found in many putative excitatory motor neurons in C. elegans, including the ventral cord motor neurons DA, DB, AS, VA, and VB, as well as VC. Interestingly, the ventral and dorsal sublateral cords show regular punctate staining with anti-UNC-17, suggesting that they have cholinergic synaptic regions. Motor neurons and interneurons in the pharynx are also labeled. Few, if any, purely sensory neurons contain significant levels of these cholinergic proteins. In A.suum, anti-UNC-17 or ChAT also show punctate labeling in a subset of neurons. Labeled neurons include motor neurons that are known from biochemical and physiological studies to be cholinergic. We have begun to examine the level and distribution of these proteins in mutants of C. elegans and find differences in ric-7 (courtesy of Erik Jorgensen), unc-18, unc-33, unc-44, unc-58, unc-75, and unc-104. Supported by grants from OCAST and NIH.

Victor L Jensen et al. (2008) European Worm Meeting "The maternal-effect Daf-c gene daf-25 is expressed in ciliated neurons and functions in neurosecretion"

Victor L Jensen, Sharon Bishop-Hurley, Marco Gallo, Donald L Riddle

[European Worm Meeting, 2008]

We isolated four alleles of daf-25 in screens for new temperature-sensitive. Daf-c (dauer-constitutive) mutants. At restrictive temperature, daf-25. mutants form dauer larvae even when food is abundant. The Daf-c phenotype. can be rescued by maternally supplied daf-25(+). Compared to N2, daf-25. worms failed to avoid sucrose and high concentrations of NaCl, indicating. that they are defective in sensing osmotic gradients. There is also a. reduction of brood size when worms are shifted to the restrictive. temperature (25degreesC) at L4, despite normal levels of progeny at the. permissive temperature (15degreesC). Epistasis results put DAF-25 function. downstream of DAF-6, but upstream of DAF-10. This implies that DAF-25. function requires cilia because DAF-10 is necessary for proper cilia. production. DAF-6 is required for proper formation of the amphid channel,. which connects the ciliated neurons to the environment. Indeed, a daf-. 25p::GFP reporter is expressed in the ciliated neurons among other tissues.. Because daf-25 dauer larvae are defective in dauer recovery, similar to. unc-31 dauers, we analyzed dense core vesicle secretion. Using a paex-. 3::ANF::GFP (gift from Erik Jorgensen), we observed a secretion defect in a. deletion mutant that affects both isoforms of daf-25. By contrast, a. deletion mutant that only affects one of the isoforms, and has a weaker Daf-. c phenotype, has no defect in ANF::GFP secretion. A model for the role of. DAF-25 in dauer formation will be presented.

Eastman CL et al. (1997) International C. elegans Meeting "Regulation of unc-47 by unc-30"

Eastman CL, Jin YS

[International C. elegans Meeting, 1997]

We are interested in the development and function of the GABAergic D neurons in the ventral cord. These neurons mediate the coordinated sinusoidal motion of the worm by inhibiting body wall muscle contractions. D neurons in unc-30 mutants do not produce GABA, are defective in axonal pathfinding, and fail to make proper synaptic connections(J.White, personal communication). unc-30 encodes a homeodomain transcription factor expressed in all D neurons (Jin Y, Hoskins R and Horvitz HR Nature 372: 780), suggesting that UNC-30 controls transcription of genes that are involved in D neuron differentiation and function. unc-47 mutant worms express higher levels of GABA than wild type animals and display a similar locomotion phenotype to that of unc-30 (McIntire SL, Jorgensen E and Horvitz HR Nature 364:334). unc-47 has been shown to encode the vesicular pump for GABA and is expressed in all GABAergic neurons (K. Schuske and E. Jorgensen, WCWM 1996). The phenotype of unc-47 worms and the expression pattern of unc-47 suggest that it might be regulated by unc-30. We crossed the unc-47-GFP array (gift of Kim Schuske and Erik Jorgensen) into an unc-30 null mutant. In the unc-30 mutant background, unc-47 expression was abolished in the D neurons whereas its expression in other GABAergic neurons was unaffected, suggesting that unc-30 regulates transcription of unc-47 in the D neurons. Experiments to identify the unc-47 promoter elements regulated by unc-30 are underway. We will present this analysis at the meeting.

Lackner MR et al. (1998) West Coast Worm Meeting "egl-8 ENCODES A PHOSPHOLIPASE C BETA ISOFORM THAT IS INVOLVED IN THE MODULATION OF EGG LAYING, LOCOMOTION, AND DEFECATION"

Kaplan JM, Lackner MR

[West Coast Worm Meeting, 1998]

G proteins modulate many of the behaviors exhibited by C. elegans, including locomotion, foraging, and egg laying. However, little is known concerning the effector pathways regulated by these G proteins. We will present evidence that the egl-8 gene encodes a phospholipase C beta isoform that may be an effector of the Gq alpha subunit encoded by egl-30. egl-8 mutations were initially isolated in a general screen for egg laying defective mutations, though many additional alleles have been isolated in screens for mutants defective in various aspects of defecation. We noticed that egl-8 mutants exhibit many phenotypes that resemble those caused by egl-30 mutations, including sluggish locomotion, feeble head foraging movements, and defective egg laying that is resistant to 5 mM serotonin. We also showed that, like egl-30 hypomorphic mutations, an egl-8 mutation suppresses the arecoline hypersensitivity seen in eat-11(ad541) mutants. The genome sequencing consortium recently identified a homolog of vertebrate phospholipase C beta (PLC beta) on the left arm of chromosome V, near the genetic location of egl-8. Because egl-8 and egl-30 share many phenotypes, and because mammalian Gq alpha is known to signal through PLC beta, we hypothesized that egl-8 might encode this PLC beta. Two lines of evidence suggest this is the case. First, injection of a cosmid containing the PLC-beta gene rescues all of the phenotypes seen in egl-8 mutants. Second, sequence analysis of the PLC beta from egl-8 mutants1 revealed that egl-8(n2659) contains a missense mutation in a conserved residue, while egl-8(sa47) mutant DNA contains an amber stop mutation in exon 2. 1We would like to thank Erik Jorgensen and Jim Thomas for providing egl-8 alleles.

Elizabeth Gleason et al. (2008) Development & Evolution Meeting "Developmental Genetics of Secretory Vesicle Acidification during C. elegans Spermatogenesis"

Paul Hartley, Guang-dan Zhu, Steven L'Hernault, Elizabeth Gleason, Suzanne Cordovado, Tim Kroft, Katherine Hill-Harfe

[Development & Evolution Meeting, 2008]

Like the acrosome found in flagellated spermatozoa, amoeboid C. elegans spermatozoa contain a specialized secretory vesicle (MOs) that fuses with the cell surface and makes it competent for fertilization. We have studied MO biogenesis and function in order to better understand its role during spermatogenesis and fertilization in C. elegans. pH sensitive vital dye staining reveals that the MO is acidified in a manner that is tightly correlated with spermatid formation. MO, like acrosome, acidification is blocked by bafilomycin, which is a specific inhibitor of the vacuolar (V) ATPase. The V-ATPase is a multisubunit machine that hydrolyzes ATP to pump protons across a membrane, and it is critical for establishing the low internal pH that characterizes many types of vesicles. Since the V-ATPase is essential, it is a challenge to study its role in a specific tissue of most animal species. Genome wide microarray experiments uncovered one V-ATPase B subunit paralog that is up-regulated during spermatogenesis. We discovered that the spermatogenesis defective spe-5 gene encodes this V-ATPase B subunit. Prior work from our lab showed that spe-5 mutant spermatocytes show a severe disruption of their MOs. A monoclonal antibody to the SPE-5 protein reveals that the V-ATPase localizes to the MO during spermatid formation, consistent with the spe-5 mutant phenotype. The vha-12 gene is located on the X chromosome and encodes another B subunit that has 85% amino acid identity to SPE-5. We found that a transgene (courtesy of Erik Jorgensen's lab) containing vha-12, driven by its own promoter, partially rescues the spe-5 self-sterile phenotype. This suggests that the vha-12 transgene escapes X chromosome transcriptional silencing because the natural (X chromosome located) vha-12 gene does not rescue the spe-5 mutant phenotype. These data show that proper MO morphogenesis requires V-ATPase mediated acidification of this vesicular compartment.

Valerie Robert et al. (2006) European Worm Meeting "MosTIC: A Novel Tool to Engineer the C. elegans Genome by Homologous Recombination"

Erik M. Jorgensen, Valerie ROBERT, Jean-Louis Bessereau.

[European Worm Meeting, 2006]

Valrie Robert, Erik M. Jorgensen1 and Jean-Louis Bessereau. Techniques based on homologous recombination provide a means to engineer custom mutations at specific loci and are widely used to study gene functions in most eukaryotic model organisms. Unfortunately, in Caenorhabditis elegans, homologous recombination between the genome and exogenous DNA fragments is very inefficient and only few examples of genome engineering by homologous recombination have been documented so far. We have developed a novel technique to introduce targeted gene modifications in the C.elegans genome using transgene-instructed gene conversion following Mos1 transposon excision.. Mos1 is a drosophila transposon that can be mobilized by expressing the Mos transposase in the C.elegans germline. Transgene-instructed DSB repair requires two steps: (1) generation of a DSB at the locus of interest by excision of a pre-existing Mos1 insertion and (2) repair of this DSB by gene conversion using an engineered transgene as a template. To establish this technique, we used Mos1 insertions in the unc63 and unc-5 genes. Following expression of the Mos transposase in the germline, we were able to demonstrate gene conversion events between the chromosome and a transgene containing only 3 kb of homologous sequences. This technique, that we called MosTIC (Mos1 induced transgene instructed gene conversion), allowed (i) the introduction of point mutations, (ii) the engineering of deletions, and (iii) the knock-in of gfp in the unc5 gene. All these events were recovered at a frequency ranging from from 5.10-5 to 5.10-4 per generation. Analysis of the conversion tracts indicated that 500 bp on each side of the Mos1 insertion site were copied from the transgene into the chromosome in 50 % of the MosTIC events.. MosTIC requires a Mos1 insertion in the vicinity of the genomic region to manipulate. Such insertions can be recovered in genetic screens using Mos1-mediated mutagenesis. In addition, progress of the NEMAGENETAG consortium to produce a comprehensive library of localized Mos1 insertions will provide a means to manipulate most C.elegans genes with the MosTIC technique.