Lasse, Samuel et al. (2015) International Worm Meeting "Genetic Polymorphisms and the Relationship Between Ambient Temperature and Behavioral Performance in Caenorhabditis elegans."

Goodman, Miriam B., Lasse, Samuel, Andersen, Erik C., Hoelscher, Victoria, McPherson, Kevin P.

[International Worm Meeting, 2015]

Temperature and muscle function are highly correlated. For instance, marathon runners' winning times decrease significantly in a range of 10 to 15degC [1]. To learn more about how genes might influence the temperature dependence of behavioral performance, we turned to C. elegans egg laying as a model. This behavior has several features that make it ideal for the studies currently in progress. First, egg laying involves a simple motor circuit and well-defined set of muscles. Second, the output of this motor program is straightforward to measure. Third, we find that Caenorhabditis elegans, egg-laying rates increase gradually with heating until a maximum is achieved and then decline sharply, similar to observations reported for other behaviors in other ectotherms. Additionally, egg-laying rates vary between C. elegans strains isolated from different environments. For example, the maximal egg-laying rate of the Bristol (N2) hermaphrodites is about three-fold higher and occurs at a lower temperature than that of Hawaiian (CB4856) hermaphrodites (see Lasse, Koulechova & Goodman, IWM 2015).We are investigating the possibility that genetic polymorphisms account for differences in egg-laying rates by scoring a panel of recombinant inbred lines between N2 and CB4856. In brief, we measured egg-laying rates by placing three young adult (two-day old) hermaphrodites per well in a 96-well plate held at defined temperatures and recording rate as eggs/worm/hour during a two-hour assay time. This preliminary QTL analysis points to loci on chromosome IV that may account for variations in egg-laying rates based on measurements at a fixed temperature (25degC). In parallel, we investigated the possibility that ANOH-1 and ANOH-2, C. elegans orthologs of calcium-activated and temperature-sensitive chloride channels anoctamin-1 and anoctamin-2 found in humans [2], might contribute to the temperature sensitivity of egg-laying in N2 hermaphrodites. For these experiments, the egg-laying rate in anoh-1 and anoh-2 mutants was compared with that of wild-type (N2) hermaphrodites at temperatures between 5 C and 35degC. The resulting rate-temperature curves were indistinguishable, suggesting that neither anoh-1 nor anoh-2 plays a significant role in regulating the temperature-dependence of egg laying. References: [1] N Maughan, Scand J Med Sci Sports. 20 (2010), p. 95-102; [2] Y Wang et al., Am J Physiol Integr Comp Physiol. 11 (2013), p. R1376-R1389. .

David Alan Wharton (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "Panagrolaimus davidi, an Antarctic nematode model for the survival of extreme environmental stress"

David Alan Wharton

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Nematodes are found in almost all environments, including those where they are often exposed to extreme environmental stress. Panagrolaimus davidi is an Antarctic nematode living associated with moss and algae in terrestrial habitats on the Victoria Land coast that are free of snow and ice for part of the year. It has to survive very variable thermal and hydric environments where liquid water and temperatures suitable for growth are only periodically available. P. davidi can survive complete water loss (anhydrobiosis) and is the only organism that has been shown to survive intracellular ice formation throughout its tissues. It has several cold tolerance strategies, including; freeze avoidance, cryoprotective dehydration, freezing tolerance and anhydrobiosis. The mechanisms involved may include the production of trehalose, ice active proteins and the control of ice nucleation. Do the different survival strategies of P. davidi represent the expression of different gene sets or does the production of stress-related compounds provide protection against a variety of environmental challenges? Other nematodes, including Caenorhabditis elegans, are not so resistant to desiccation and freezing. Comparing the genomes of P. davidi and C. elegans may thus highlight the adaptations that are necessary for the survival of extreme environmental stress.

Zhu X et al. (1995) International C. elegans Meeting "INTEGRIN EXPRESSION PATTERN IN C. elegans"

Zhu X, Hedgecock EM

[International C. elegans Meeting, 1995]

Integrins are a family of extracellular matrix receptors involved in cell adhesion, signal transduction and cytoskeletal organization. Three genes for integrin subunits have been identified in C. elegans to date. Two a-subunit genes were discovered by the genome sequencing project. One b-subunit gene was cloned by degenerate PCR based on the known insect and vertebrate sequences, later identified as the product of pat-3 (Gettner et al., 1992). Genetic and immunohistochemical studies indicate that pat-3 is essential for muscle development, canal outgrowth and some cell migrations. These and other studies in higher organisms have led us to speculate that pat-3 might play diverse roles in development, both embryonically and post- embryonically. To further study bPAT-3 function, we sought to establish the spatial and temporal expression pattern in more detail using A. victoria GFP as a tag. We have inserted GFP into the bPAT-3 reading frame immediately upstream of the normal terminator. When this construct was injected into pat-3 null mutants, the animals were fully rescued by a heritable extrachromosomal array. Rescued animals had bright green fluorescence in specific patterns including most sites previously detected by immunofluorescence with anti-integrin antibodies. Hence, the fusion protein bPAT-3::GFP is functional and behaves much like the wildtype protein. bPAT-3::GFP is expressed continuously in muscle cells in larvae and adults. In body wall muscles, it is localized at Z-disc (dense body), M-line and dense plaque attachments. Vulval, uterine , anal depressor and sphincter muscles, but not pharynx, also express strongly. Muscle arms are also seen projecting to the nerve cords and ring. In addition, sheet-like structures lining the inner surface of the nerve ring are visible. Tentatively, these are the processes of mesoglial cells GLR. bPAT-3::GFP is also expressed transiently in many tissues. Larval neuroblasts such as ALM and Q descendants express during migration. Weaker signals are seen in the gonad, including distal tip cells and sheath cells. Finally, various cells express transiently in the late embryo. In conclusion, our data shows that both transient and continuous expression of pat-3 play roles in many different cell types.

Papp, Andy et al. (2009) International Worm Meeting "Investigation of Low-cost GFP Microscopy."

Papp, Andy, Aldrich, Chris, Bangalore, Srikanth, Perry, David

[International Worm Meeting, 2009]

The Green Fluorescent Protein (GFP) gene from the fluorescent jellyfish Aequorea victoria, and its variants (such as EGFP), are used extensively to study the location and timing of gene expression in transgenic animals and plants (Chalfie et al, 1994). Resultant GFP fusion proteins are especially well-suited to studying in vivo gene expression patterns in the transparent nematode, C. elegans and the transparent larva of the fly D. melanogaster. Perhaps one of the most significant limitations to its use in large-scale genetic screens is the high cost of equipment needed to observe GFP microscopically - namely the Fluorescence Dissecting Stereomicroscope for screening and picking mutants and upright and inverted epi-fluorescent compound microscopes for more detailed studies. Commercially available Fluorescence Dissecting Stereomicroscopes typically sell in the midrange between US$10,000 and US$20,000. They are offered by only the "high-end" microscope companies, based upon their most expensive dissecting scopes, and incorporate their premium-priced mercury arc-lamp illuminators, power supplies and epi-fluorescence modules. This leads us to wonder whether it is possible to cut corners without sacrificing utility, and which corners can be cut. Since the inception of GFP-based expression screens, a variety of researchers have produced custom-made and home-made GFP dissecting scopes. These include, for example, Welcome Bender (Harvard Medical School, pers. comm.) and Ian Chin-Sang (Chin-Sang, 2004). There are several possibilities to consider toward lowering the cost of a Fluorescence Dissecting Stereomicroscope. It may be possible to get sufficient light from relatively inexpensive, long-lived, lower-power-consuming Light Emitting Diodes (LEDs) vs. mercury arc lamps. Depending upon the spectral specificity of the LEDs, filters or dichroic mirrors might be omitted. Getting enough light intensity of the precise wavelengths that excite GFP fluorescence focused onto the sample is important. The exciting beam can be provided directly or focused backward through the microscope using "epi-illumination". We will explore these possibilities and report (and possibly demonstrate) the results. Chin-Sang, Ian. (2004) GFP Stereoscope Using LED light source. Queen''s University, Kingston, ON, Canada. http://130.15.90.245/gfp_stereoscope.htm.