Hana Murakami et al. (2005) International Worm Meeting "Aging of associative learning paradigms in C. elegans."

Shin Murakami, Karalee Murray, Jason Hellmann, Hana Murakami

[International Worm Meeting, 2005]

Associative learning in C. elegans includes chemosensory learning and thermotaxis learning. In the associative learning paradigms, worms learn to associate paired stimuli and alter the behavioral output. Conditioned worms with or without food (unconditional stimulus, US) show either preference or avoidance to the conditional stimulus (CS). Recently, slowing responses to food, called basal and enhanced slowing responses, are suggested to be a type of associative learning (Hobert, 2003). We have investigated aging effects on the three learning paradigms. The learning behaviors showed different kinetics of learning declines. The long-lived mutants, age-1 and daf-2, dramatically improves this type of learning behavior. In the mutants, there was a three-fold extension of the young period that allows a high level of thermotaxis learning. In contrast, the mutants had little or modest effects on aging of basal and enhanced slowing responses. Aging of slowing responses is likely caused by age-related changes in serotonin signal (see the other abstract; Murakami et al). Aging of chemosensory learning appears to show different kinetics during aging. In the poster we will summarize aging of the three learning paradigms.

Hana Murakami et al. (2005) International Worm Meeting "Behavioral aging modulated by serotonin signal in C. elegans."

Hana Murakami, Jason Hellmann, Shin Murakami, Karalee Murray, Gregory Luerman

[International Worm Meeting, 2005]

Dopaminionergic and serotonergic neurons are susceptible to the age-related neurodegerative diseases such as Parkinson disease and Alzheimers disease. In C. elegans, dopamine and serotonin signal mediate experience-dependent behaviors, called basal and enhanced slowing response, respectively. Here we investigated as to whether dopamine and serotonin signal show age-related changes, leading to altered slowing responses. With increasing age, basal slowing response became enhanced, resulting in a diminished difference between the two slowing responses. This diminished behavioral plasticity was not correlated with increased oxidative stress during aging, suggesting that oxidative stress is unlikely a cause of the diminished behavioral plasticity. Interestingly, we found that levels of serotonin were increased during aging. Since serotonin is known to reduce locomotion and cause enhanced slowing response, the age-dependent increase in the serotonin level explains the change in basal slowing response. Consistently, a serotonin signal inhibitor restored basal slowing response in old animals, seemingly in a neuron specific manner. We propose that age-related changes in serotonin signal lead to not only declines in locomotion rate and altered slowing responses, but also cause the age-related changes in a variety of behaviors. Importantly, the results point out that sacropenia, or age-dependent dysfunction in muscle tissues, is not the only cause of declines in locomotion behaviors.

Dernburg AF et al. (1997) International C. elegans Meeting "Cytological investigation of homologous pairing during meiotic prophase"

Dernburg AF, Villeneuve AM

[International C. elegans Meeting, 1997]

Pairing and recombination between homologous chromosomes are essential for their faithful segregation during the first meiotic division. The mechanisms leading to homologous chromosome pairing and synapsis are still only poorly understood in any experimental organism. C. elegans provides a highly advantageous model system in which genetic and cytological approaches can be combined to study this process. We are developing tools and methods to enable us to examine chromosome pairing in the light microscope in order to address several specific questions: Do special sites on each chromosome (particularly the genetically-defined !pairing centers!) become associated earlier than others? Do chromosome rearrangements that act as crossover suppressors disrupt homologous pairing as dramatically as genetic evidence seems to suggest? Are gene products that are required for meiotic recombination associated with particular regions of the chromosomes? To answer these and other questions, we are using several approaches, including fluorescence in situ hybridization (FISH) methods, and labeling of chromosomes in living worms using the GFP-Lac repressor binding method developed by Aaron Straight, Andrew Murray, and Andy Belmont for other experimental systems. By applying high-resolution 3-dimensional microscopy, we hope to obtain a better understanding of rules governing the organization and interaction of chromosomes in the meiotic prophase nucleus.

Stegeman, Gregory et al. (2011) International Worm Meeting "Variation in temperature-dependent behaviours among natural isolates of Caenorhabditis briggsae."

Shin, Jiwon, Ryu, William, Cutter, Asher, Bueno De Mesquita, Matthew, Stegeman, Gregory, Lin, Nan

[International Worm Meeting, 2011]

Natural genetic variation allows the discovery of new gene functions and novel alleles for genes already known to act in biologically important processes. We are applying this approach to temperature-dependent behaviours in nematode worms in order to better understand the genetics behind behaviour. We focus on Caenorhabditis briggsae because most wild caught individuals fall into two genetically distinct clades that correspond approximately with northern temperate or with tropical latitiudes. Interestingly, strains from the tropical clade have higher fecundity when reared at higher temperature than do the temperate strains, suggesting local adaptation to climate variables like temperature (Prasad et al. 2011). Movement through its thermal landscape is the main way for nematodes like C. briggsae to regulate body temperature, so we also expect to see heritable differences in temperature-dependent behaviours. Here we quantify for the first time classic thermal-response behaviours among several C. briggsae wild strains from different haplotype groups using assays like accumulation on a linear thermal gradient, isothermal tracking, and a new droplet based thermal gradient assay. We demonstrate that C. briggsae shows thermotaxis and isothermal tracking similar to C. elegans but with some differences. We also identify heritable differences among strains from wild genetic backgrounds within C. briggsae. We will continue to develop higher throughput assays for temperature-dependent behaviour in order to carry out a quantitative trait loci mapping project using recombinant inbred lines derived from tropical and temperate parental strains. Prasad, A., M. Croydon-Sugarman, R.L. Murray & A.D. Cutter. 2011. Temperature-dependent fecundity associates with latitude in Caenorhabditis briggsae. Evolution. 65: 52-63.

Murray AN et al. (2013) Protein Sci "Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes."

Kelly JW, Palhano FL, Murray AN, Bieschke J

[Protein Sci, 2013]

The accumulation of cross--sheet amyloid fibrils is the hallmark of amyloid diseases. Recently, we reported the discovery of amyloid disaggregase activities in extracts from mammalian cells and Caenorhabditis elegans. However, we have discovered a problem with the interpretation of our previous results as A disaggregation in vitro. Here, we show that A fibrils adsorb to the plastic surface of multiwell plates and Eppendorf tubes. This adsorption is markedly increased in the presence of complex biological mixtures subjected to a denaturing air-water interface. The time-dependent loss of thioflavin T fluorescence that we interpreted previously as disaggregation is due to increased adsorption of A amyloid to the surfaces of multiwell plates and Eppendorf tubes in the presence of biological extracts. As the proteins in biological extracts denature over time at the air-water interface due to agitation/shaking, their adsorption increases, in turn promoting adsorption of amyloid fibrils. We delineate important control experiments that quantify the extent of amyloid adsorption to the surface of plastic and quartz containers. Based on the results described in this article, we conclude that our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity. Thus, we correct the two prior publications reporting that worm or mammalian cell extracts disaggregate A amyloid fibrils in vitro at 37C (see Corrigenda in this issue of Protein Science). We apologize for misinterpreting our previous data and for any confounding experimental efforts this may have caused.

Sarov, Mihail et al. (2009) International Worm Meeting "The C. elegans TransgeneOme: a fosmid transgene resource for genome scale protein function analysis."

Zhong, Mei, Snyder, Michael, Sarov, Mihail, Reinke, Valerie, Kim, Stuart, Waterston, Robert, Hyman, Anthony, Murray, John

[International Worm Meeting, 2009]

Our ultimate goal is to understand the molecular principles of development through systematic description of the physical interactions and localization of all proteins encoded in the C. elegans genome. To that end we have developed a transgene-based platform for protein tagging with fluorescent/affinity epitopes [1]. We use in vivo homologous recombination based DNA engineering (recombineering) in E. coli to seamlessly insert a tag coding sequence into genomic fosmid clones containing the gene of interest in its native genomic environment. Stable integration of these large constructs into the worm genome result in reliable, near endogenous levels and patterns of gene expression. Through evaluation of multiple tags we found a cassette that is well tolerated by most proteins and works well for both affinity purification and localization. We have now scaled up the construction of fosmid transgenes using an efficient 96 well format liquid culture recombineering, which delivers a extremely high throughput without compromise in quality. As part of the NIH funded modENCODE project [2] we have applied this approach to the transcription factor proteins, coupled with high resolution protein localization [3] and chromatin immunoprecipitation to map binding sites in the genome. We are currently focusing on extending this approach to further functional sets (Chromatin, cell division and G protein coupled receptors) and eventually to the rest of the genome. The TransgeneOme resource will be another powerful tool for the C. elegans research community. The clones will be made available on the condition of sharing the stable worm lines. We believe that this would be an efficient way of solving the bottleneck step of strain generation through distributed community effort. [1] Sarov et al. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nat Methods (2006) [2] http://www.modencode.org/ [3] Murray et al. Automated analysis of embryonic gene expression with cellular resolution in C. elegans. Nature Methods (2008).

Henriksson, Johan et al. (2013) International Worm Meeting "Endrov, a general imaging framework, to visualize C. elegans gene expression, 4D models, and lineage."

Hench, Jurgen, Luppert, Martin, Burglin, Thomas R., Abou-Zied, Akram, Tang, Lois, Henriksson, Johan, Baillie, David, Tong, Yong-Guang

[International Worm Meeting, 2013]

Events that specify cell fates during embryogenesis are highly dynamic. We have developed an image analysis and microscopy framework, Endrov (www.endrov.net), and used it to create multi-channel 4D recordings of live GFP expression throughout embryogenesis. The software can increase the dynamic range of recordings by changing the camera exposure time over time. We have applied this to homeodomain transcription factors and have so far recorded over 60 homeobox gene expression patterns using DIC and promoter::GFP. We have selected one of these, ceh-5 for in-depth analysis (see poster by Gangishetti et al.). Our data complements the EPIC dataset (Murray et al., Genome Res 2012), with 15 genes in common. We also observed differences, e.g., ceh-14 is detected earlier in our dataset. We have also shown that our recordings correlate with microarray data (Yanai and Hunter, Genome Res 2009). Genes expressed at similar location or time point can be found from clusterings. A website with all the data is currently being set up. Endrov is a general open source image analysis framework, but has many features for the worm community. It allows gene expressions to be extracted and shown on single-cell level, in 3D and on the lineage. In addition to the embryo model (Hench et al., Dev Biol 2009) the 3D EM model of the adult larvae (C. Grove, pers. com.) can be displayed and linked to the lineage. Endrov is also capable of importing the gene expression patterns from the EPIC dataset. Several genes can be overlapped and visualized. Coordinates made using SIMI Biocell can be imported. Lineaging can be done manually and with automatic lineaging algorithms. Endrov can be used with almost any computerized DIC/fluorescent microscope, making it a versatile tool for many labs. Gene expression and morphology can be studied in wild-type or mutant embryos. Endrov allows quantitative comparison of many expression patterns simultaneously, in 3D and on the lineage.

Hutchison L et al. (2019) Bioinformatics "Meta-analysis of C. elegans single-cell developmental data reveals multi-frequency oscillation in gene activation."

Hutchison L, Berger B, Kohane I

[Bioinformatics, 2019]

MOTIVATION: The advent of in vivo automated techniques for single-cell lineaging, sequencing, and analysis of gene expression has begun to dramatically increase our understanding of organismal development. We applied novel meta-analysis and visualization techniques to the EPIC single-cell-resolution developmental gene expression dataset for C. elegans from Bao, Murray, Waterston et al. to gain insights into regulatory mechanisms governing the timing of development. RESULTS: Our meta-analysis of the EPIC dataset revealed that a simple linear combination of the expression levels of the developmental genes is strongly correlated with the developmental age of the organism, irrespective of the cell division rate of different cell lineages. We uncovered a pattern of collective sinusoidal oscillation in gene activation, in multiple dominant frequencies and in multiple orthogonal axes of gene expression, pointing to the existence of a coordinated, multi-frequency global timing mechanism. We developed a novel method based on Fisher's Discriminant Analysis (FDA) to identify gene expression weightings that maximally separate traits of interest, and found that remarkably, simple linear gene expression weightings are capable of producing sinusoidal oscillations of any frequency and phase, adding to the growing body of evidence that oscillatory mechanisms likely play an important role in the timing of development. We cross-linked EPIC with gene ontology and anatomy ontology terms, employing FDA methods to identify previously unknown positive and negative genetic contributions to developmental processes and cell phenotypes. This meta-analysis demonstrates new evidence for direct linear and/or sinusoidal mechanisms regulating the timing of development. We uncovered a number of previously unknown positive and negative correlations between developmental genes and developmental processes or cell phenotypes. Our results highlight both the continued relevance of the EPIC technique, and the value of meta-analysis of previously published results. The presented analysis and visualization techniques are broadly applicable across developmental and systems biology. AVAILABILITY: Analysis software available upon request. SUPPLEMENTARY INFORMATION: Supplementary data are available at the publisher's website.

Riddle DL et al. (1980) Worm Breeder's Gazette "announcements"

Swanson MM, Riddle DL

[Worm Breeder's Gazette, 1980]

The Caehorhabditis Genetics Center (CGC), sponsored by the National Institute on Aging, was funded on September 30, 1979. Dr. Don Murray is the NIA Project Officer. As you know, the CGC is to be responsible for strain acquisition, banking and distribution. Strains will be available without cost to all qualified investigators pursuing genetics-related studies with C. elegans. Other CGC functions will include maintenance of the genetic map, coordination of genetic nomenclature and maintenance of a current data bank on all strains, including bibliographic information. During the first year, we will be developing a computerized system for storage and retrieval of this information. The program will be designed to allow other labs with CRT terminals remote access to the computer data files. We have recently mailed a questionnaire regarding your anticipated use of the Center's services. Since your responses may help us to modify the design of the services to be offered, we would appreciate your returning those questionnaires as soon as possible if you have not already done so. If you did not receive our mailing and anticipate using the CGC, please contact us. We plan to maintain a complete library of articles on Caenorhabditis, so we would appreciate it if you also would send us one copy of each of your Caenorhabditis publications. We have recently done a retrospective search of the literature and will soon be distributing a complete C. elegans bibliography as a supplement to this Newsletter. The new bibliography contains about 350 references, and we plan to update it with each future issue of the Newsletter. We would like to thank Bob Herman for the excellent job he has done as a volunteer stock distribution center. The immensely valuable work that Bob Herman and Bob Horvitz have done on the genetic map surely is apparent to everyone. Bob Herman will be supplying us with many strains he has received from other laboratories and we hope to have a relatively up-to-date C. elegans collection in a few months. The CGC is scheduled to be fully operational by the Fall of 1980, but some services are available now.

Warner, Adam D. et al. (2013) International Worm Meeting "Determining the time and tissue specific expression of genes during embryogenesis."

Huynh, Chau, Waterston, Robert H., Warner, Adam D.

[International Worm Meeting, 2013]

Embryonic development is a tightly controlled and regulated process in many species, with a complex network of transcription factors regulating gene activity. In C. elegans, the process is so tightly controlled that the lineage of cell divisions is invariant and has been fully mapped (Sulston et al, 1983). The set of genetic messages at an embryo's earliest stage is completely maternal, transitioning over time to being generated completely from its own DNA. Determining which genes become active at specific time points provides insight into when key regulatory events occur, and determining the total network of gene expression in each cell type over time will help map out developmental processes unique to each tissue. Previous studies have utilized a combination of FACS, SAGE, and microarrays (Meissner et al, 2009; Spencer et al, 2011) to gain insight into what genes are expressed in late and mixed stage embryonic tissues. More recently, RNA-Seq has been used for expression studies, producing gene expression data with advantages over SAGE and microarrays such as a broader range of expression levels, differentiation between gene isoforms, and information on every transcript expressed (reviewed in Wang et al, 2009). We have previously shown the ability to isolate time synchronized whole embryos at specific time points throughout embryonic development (see abstract from Max Boeck), and we aim to do the same with isolated tissue types by utilizing a collection of GFP and mCherry labeled C. elegans strains (Murray et al, 2012; Sarov et al, 2012) that label individual cells and tissues. Using FACS to isolate specific cell subsets at discrete time points and RNA-Seq for transcript identification and quantification, we will be able to create a map of embryonic gene expression not only specific to individual cells and tissues, but specific to those cell and tissue types over time. This will build upon and complement previous data by adding temporal information and added sensitivity. Clustering of genes with similar expression patterns to genes with known roles in development, combined with emerging ChIP-Seq data, will help identify novel genes involved in specific developmental processes.