Papp, Andy et al. (2013) International Worm Meeting "Investigation of Simplified Dual-fluorophore Dissecting Stereomicroscopes."

Aldrich, Chris, Papp, Andy

[International Worm Meeting, 2013]

In previous studies (Papp et al., 2009, Papp et al. 2011) we demonstrated that high-power Light Emitting Diodes (LEDs) could be combined with good quality filters to visualize the expression of fluorescent transgenes and produce a low-cost fluorescent dissecting stereomicroscope system. Two of the cost saving methods were: (1) the use of oblique rather than epi- illumination and (2) putting emission filters into the eyepieces and/or camera port rather than using filter cubes with dichroic mirrors. While this system was shown to work well for multiple fluorophores (eGFP, dsRed, and mCherry), it took about 1 minute to change lamps and eyepieces, making it impractical to screen individual moving worms for two fluorophores expressed in the same location (we were able to view eGFP and dsRed simultaneously using a single excitation and emission filter set, but one is likely to obscure the other if they are co-localized). In the present study, we investigate the use of multiple LEDs and filters in the same lamp assembly and filter sliders to reduce the switchover time between screening for two fluorophores. Preliminary data suggest that the switching can be done in under 3 seconds (a similar time scale to switching filter cubes in a conventional epifluorescence system) without a significant increase in cost or complexity as compared with the original low-cost system. Papp, Andy et al. (2009) International Worm Meeting "Investigation of Low-cost GFP Microscopy." Papp, Andy et al. (2011) International Worm Meeting "Investigation of Low-cost Fluorescence Microscopy.".

Papp, Andy et al. (2011) International Worm Meeting "Investigation of Low-cost Fluorescence Microscopy."

Aldrich, Chris, Perry, David, Papp, Andy

[International Worm Meeting, 2011]

Small genes encoding fluorescent proteins (e.g. EGFP, mCherry and dsRed), are used extensively as visible markers to study the location and timing of gene expression. Fluorescent fusion proteins are especially well-suited to studying in vivo gene expression patterns in the transparent C. elegans (Tursun et al., 2009). Perhaps one of the most significant limitations to their use is the high cost of the equipment needed to observe fluorescent proteins microscopically - the fluorescence dissecting stereomicroscope for screening and picking mutant worms and epi-fluorescent compound microscopes for more detailed studies. Commercially available fluorescence dissecting stereomicroscopes typically cost between US$12,000 and US$50,000. They are offered by only the "high-end" microscope companies, based upon their most expensive dissecting scopes, and incorporate premium-priced mercury arc-lamp illuminators, power supplies and epi-fluorescence modules. In a previous study, we demonstrated that significant cost savings could be achieved in EGFP detection by substituting high-flux blue spectrum LEDs for the mercury arc-lamps, and incident illumination for epi-illumination, if appropriate high-quality optical filters were employed (Papp et al., 2009). The resultant US$7,000 prototype system was compared with a US$50,000 top-of-the-line Leica fluorescence dissecting stereomicroscope system using the same plate of EGFP positive worms and the systems gave similar results. In the current study, we investigate the possibility of extending our findings to fluorophores with different spectral properties, such as dsRed and mCherry. We examined various LEDs combined with different excitation and emission filters. While the commercially available high-flux green spectrum LEDs that we studied do not have emission peaks that line up well with the excitation peaks of the red fluorophores, we were still able to demonstrate a highly usable level of detection by using appropriate optical filters. Since the human eye and fluorescence cameras can detect very low light levels, the absolute fluorescence level is not as important as the fluorescence signal-to-noise ratio. We will investigate sources of noise and present data (possibly with a demonstration) regarding techniques to improve the signal:noise ratio by reducing background fluorescence.

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

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

[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.

S Golik et al. (1999) International C. elegans Meeting "FMRFamide-related peptides and egg-laying"

S Golik, W Schafer, L Hardaker

[International C. elegans Meeting, 1999]

The egg-laying motorneurons release multiple neurotransmitters, each of which appears to control a specific aspect of egg-laying behavior. Under favorable conditions, wild-type worms fluctuate between alternative behavioral states for egg-laying: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs are retained. Switching between states, as well as egg-laying within the active state, are stochastic, Poisson-like processes. We showed previously that serotonin, released primarily from the HSNs, facilitates the switch from the inactive to the active state, while acetylcholine, released from both the HSNs and VCs, promotes egg-laying within the active state. Li and Chalfie have shown that both the HSNs and VCs contain one or more FMRFamide-related peptides, and provided pharmacological evidence that these neuropeptides may control of egg-laying behavior. To investigate the roles of these peptides in more detail, we have analyzed the egg-laying behavior of deletion mutants (kindly provided by Chris Li) that are defective in production of specific FaRP precursors. We have found that one of these mutants, defective in the gene flp-1, have dramatically lengthened inactive phases, but completely normal egg-laying within the active phase. Moreover, although these mutants are stimulated to lay eggs by serotonin with approximately normal dose responses, their rate and pattern or egg-laying on serotonin are abnormal. Thus, we hypothesize that the FLP-1 peptides and serotonin act in parallel to affect the same step in egg-laying behavior--inducing the switch between the inactive and active phases. Experiments to investigate the cellular and molecular mechanisms for the FLP-1 peptides' affects on egg-laying will be described. Thanks to Chris Li for discussions and for providing strains.

Crisp A et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Age-related Cholinergic Neurodegeneration by Overexpression of apl-1 in C.elegans"

Crisp A, Pierce-Shimomura J

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Alzheimer's disease (AD) is the most common cause of dementia. It is characterized by selective degeneration of cholinergic neurons involved in memory. Dying neurons are surrounded by dense plaques primarily composed of beta-amyloid peptide (Aβ). Aβ is just one cleavage product from the protein APP. Mutations in APP that affect the processing of Aβ result in early-onset AD; however, a single additional wild-type copy of APP can also lead to AD, as seen in all individuals with Down syndrome. Aβ plaques were originally thought to be the primary cause of neurodegeneration, but new research suggests the role of the APP in AD is more complex than originally appreciated. The labs of Dr. Chris Li and Dr. Chris Link have pioneered the use of C. elegans to study APP function and dysfunction. For instance, overexpression of human Aβ in C. elegans muscle leads to the formation of Aβ aggregates(1). Overexpression of multiple copies of the APP-related gene, apl-1, in C. elegans leads to gross phenotypes, including partial lethality, arrested development, and vacuolization(2). We are testing whether expression of only one additional wild-type copy of apl-1 results in AD-related phenotypes. DIC and fluorescence microscopy were used to evaluate worms for signs of neurodegeneration. We have discovered that overexpression of apl-1 in a single copy results in the age-related degeneration of a specific subset of cholinergic neurons in C. elegans. To quantify the impact of degeneration, we track the deterioration of two natural behaviors (swimming and egg-laying) that rely on these neurons. These worms displayed no obvious defects in early adulthood but began to display defects in egg-laying and swimming by the third day of adulthood ('middle age'). In addition, ablation of these specific neurons in wild-type individuals recapitulates the behavioral defects observed in egg-laying and swimming. These results suggest that the behavioral defects observed in the overexpression strain are primarily due to the selective degeneration of these neurons. The quantifiable link between these behaviors and neurodegeneration allows us to test strategies to recover the function mediated by the degenerated cholinergic neurons. We are now studying the mechanisms by which apl-1 overexpression causes death of cholinergic neurons in the worm and in the process, generate novel hypotheses about the mechanism of Alzheimer's disease. 1. Link CD. (1995) PNAS 92, 9368-9372. 2. Hornsten A. et al. (2007) PNAS 104, 1971-1976.

Havrylenko, Svitlana et al. (2013) International Worm Meeting "Actin-based cell motility in developing C. elegans: dissecting actin assembly factors."

Havrylenko, Svitlana, Noguera, Philippe, Plastino, Julie

[International Worm Meeting, 2013]

Protrusive cell shape changes powered by actin assembly are one of the bases of cell motility observed in both normal and pathological processes. Actin assembly factors Enabled/Vasodilator-Stimulated Phosphoprotein (Ena/VASP) and the Wiskott-Aldrich Syndrome Protein (WASP) families of proteins are among numerous biochemical factors that affect actin cytoskeleton network architecture and dynamics. Mechanism of action of these multidomain proteins and interplay between them remain poorly understood. Developing C. elegans, and in particular ventral enclosure, a collective cell migration event during embryogenesis, offer a system to study the role of Ena/VASP and WASP proteins in mediating protrusive activity of the cells in the complex in vivo environment. We are constructing transgenic strains expressing different mutant forms of Ena/VASP and WASP proteins in the corresponding genetic knock-out background, while visualizing actin filaments with LifeAct:GFP expressed under the control of a tissue-specific promoter. In parallel we are testing the same modified forms of the Ena/VASP and WASP proteins in a stripped-down in vitro bead system in order to understand their mechanism of action. Our preliminary results indicate that the F-actin binding capacity of Ena/VASP, its tetramerization domain and its recruitment of the actin monomer binding protein, profilin, are necessary for enhanced bead motility in vitro and for efficient ventral enclosure in vivo. All together these results suggest that Ena/VASP acts during ventral enclosure to increase protrusion via multiple interactions with growing actin filaments.

Jiang, Hongbing et al. (2017) International Worm Meeting "Identification of an evolutionarily conserved pathway essential for virus infection using Orsay virus infection of C. elegans."

Jiang, Hongbing, Sandoval, Luis, Wang, David, Chen, Kevin, Leung, Christian

[International Worm Meeting, 2017]

Many fundamental biological discoveries have been made in <em abp="1258">C. elegans</em>. The discovery of Orsay virus has enabled studies of host-virus interactions in this model organism. To identify host factors critical for virus infection, we designed a forward genetic screen that utilizes a virally induced GFP reporter. Following chemical mutagenesis, two <em abp="1259">viro</em> (virus induced reporter off) mutants that failed to express GFP were mapped to <em abp="1260">sid-3</em>, a non-receptor tyrosine kinase, and <em abp="1261">B0280.13</em>, an orthologue of human WASP (Wiskott-Aldrich Syndrome Protein). Both mutants yielded 5-6 logs lower Orsay RNA levels than the parent strain, which approximates residual input Orsay levels, demonstrating the essential roles of these genes. However, Orsay virus replication initiated from an endogenous transgene bypasses the requirements for both SID-3 and B0280.13 suggesting that these two genes function at an early step in the virus lifecycle. Strikingly, the human ortholog of SID-3, activated CDC42 associated kinase (ACK1/TNK2), is capable of phosphorylating human WASP, suggesting that B0280.13 may be a substrate for SID-3 in <em abp="1262">C. elegans</em>. In support of this model, fluorescently tagged SID-3 and B0280.13 co-localized to the apical membrane of C. elegans intestinal cells. Further genetic ablation of TNK2 through CRISPR-Cas9 in two human cell lines, A549 (an adenocarcinomic human alveolar basal epithelial cell line) and Hap1 (a near haploid human cell line derived from myeloid leukemia patients), showed reduced infection by polio virus. Thus, an unbiased genetic screen in <em abp="1263">C. elegans</em> identified critical roles in virus infection for evolutionarily conserved genes in a known human pathway.

David Harris et al. (2006) Development & Evolution Meeting "Progress Towards Cloning mab-10, a Heterochronic Gene Required for the Proper Timing of the L4/Adult Transition in Males"

David Harris, Bob Horvitz

[Development & Evolution Meeting, 2006]

The study of heterochronic mutants has revealed a complex genetic pathway that regulates the timing of many developmental events in C. elegans. Mutations that disrupt this pathway fall into two classes: precocious mutants, which cause the premature expression of developemntal fates, and retarded mutants, which cause the reiteration of developmental fates. One abnormality often associated with the retarded heterochronic mutant phenotype is the execution of supernumerary molts. For example, supernumerary molts are observed in strains with loss-of-function mutations in let-7 or lin-29. In an attempt to further our understanding of the regulation of developmental timing, we are extending the characterization of mab-10(e1248), a mutation that confers a male-specific supernumerary molt. mab-10(e1248) was identified by Jonathan Hodgkin in a screen for mating-defective males1. mab-10 mutant males have a swollen bursa, are mating-defective and undergo a highly penetrant supernumerary molt approximately 18 hours after the L4-to-adult transition. While mab-10 hermaphrodites do not undergo an additional molt, they are slightly Pvl at 20 degrees C and often burst from the vulva as young adults when raised at 25 degrees C. Initial mapping performed by Chris Link placed mab-10 to the right of the gene cluster on linkage group II, between unc-4 and rol-12. By following the Pvl phenotype, we have mapped mab-10 to a 70 kb region using a combination of deficiency and polymorphism mapping. Currently, we are attempting rescue experiments using cosmids that span this interval. Additionally, we are performing further phenotypic characterization of mab-10 males and mab-10 hermaphrodites.

Tiewen Liu et al. (2005) International Worm Meeting "Neuropeptide modulation of C. elegans male mating behavior"

Tiewen Liu, Maureen M Barr

[International Worm Meeting, 2005]

C. elegans male mating behavior comprises the steps of response, backing, turning, location of vulva, spicule insertion and sperm transfer. Cell ablation experiments have identified sensory neurons responsible for each step (1), making this behavior an attractive model for studying synaptic transmission in a defined neural circuit. Neuropeptides are a large and diverse set of non-classical neural transmitters. Neuropeptides modulate synaptic transmission by regulating presynaptic neurotransmitter release and/or postsynaptic neurotransmitter receptor responsiveness. The C. elegans genome encodes 23 FMRFamide-like neuropeptide (flp) genes (2) and we are interested in studying their possible roles in the male nervous system. First we characterized flp expression patterns in adult C. elegans males using GFP reporters. Several flp genes are expressed in male-specific neurons, suggesting a role in male mating. Then we characterized male mating behavior of a number of flp mutants. Four of them, flp-8, flp-10, flp-12 and flp-20, exhibit an abnormal turning phenotype during mating. We also found that C. elegans proprotein convertase gene egl-3 (3), when mutated, causes a similar but much more severe turning defect. These results support the ideas that proprotein convertase EGL-3 is the master procesing enzyme for FMRFamide-like neuropeptides in C. elegans and several FLPs may act redundantly to modulate turning behavior. Work aiming to determine the cellular basis of this male turning defect is in progress.We are grateful to Dr. Chris Li for the flp::GFP strains and flp mutant strains. 1) Liu and Sternberg 1995, Neuron 14, 79-89. 2) Li et al 1999, Ann. NY Acad. Sci. 897, 239-252. 3) Thacker and Rose 2000, Bioessays 22, 545-553.

Guenther C et al. (1997) International C. elegans Meeting "HAM-1 and asymmetric cell division"

Garriga G, Hawkins NC, Guenther C

[International C. elegans Meeting, 1997]

We have characterized the gene ham-1 with the goal of understanding the molecular mechanisms responsible for establishing asymmetries during cell division. ham-1 mutations disrupt asymmetric cell divisions in five embryonic lineages, including the HSN/PHB neuroblast division. In wildtype, the HSN/PHB neuroblast divides asymmetrically to produce a smaller anterior daughter that dies and a larger posterior daughter, the HSN/PHB precursor. Direct observation of the dividing HSN/PHB neuroblasts in ham-1 embryos revealed that the anterior daughter is transformed into a second HSN/PHB precursor which can survive and divide to produce extra HSN and PHB neurons. HAM-1 encodes a novel protein that is asymmetrically localized in many dividing cells during embryogenesis. In the HSN/PHB neuroblast, HAM-1 is restricted to one side of the cell periphery and is predominately inherited by the posterior daughter cell upon division, the HSN/PHB precursor. This result, coupled with the observation that loss of ham-1 function affects the fate of the daughter cell that does not normally inherited HAM-1, has led us to propose a model in which ham-1 acts prior to cell division to segregate factors that confer specific fates to daughter cells. To identify determinants segregated by HAM-1, and proteins necessary for the asymmetric localization of HAM-1, we have conducted a yeast two hybrid screen. Two intereacting proteins of interest were identifed; a dishevelled (dsh) homolog on chromosome II and a protein with similarity to the Wiskott-Aldrich (WAS) protein on chromosome IV. In Drosophila, dsh has been shown to function in the wingless (wnt) signalling pathway, and in C.elegans, wnt signalling has been shown to orient asymmetric cell divisions. Thus, ham-1 might represent a novel member of this pathway. The human WAS gene encodes a multifunctional protein that can interact with both the plasma membrane and actin cytoskeleton. The role of these two interacting proteins is currently under further investigation.