Kao, Michelle et al. (2015) International Worm Meeting "The Elegant Mind Club: Undergraduate Research Club to Explore the Minds of C. elegans."

Atamdede, Sean, Haller, Leonard, Kao, Michelle, Mendoza, Steve, Madruga, Blake, Agarwal, Neha, Nowak, Nathaniel, Sunyoto, Amanda, Sherry, Tim, Jiang, Karen, Cheng, Shirley, Vanmali, Bobby, Trusz, Guillaume, Arisaka, Katsushi, Pellionisz, Peter, Woolfork, De'Marcu, Sagadevan, Addelyn, Kim, Taejoon, Ling, Linsay, Lam, Brian

[International Worm Meeting, 2015]

For students, the study of model organisms presents an opportunity to learn various general science and research disciplines; however, each model organism has it's own set of advantages. Chiefly, Caenorhabditis elegans are excellent model organisms to study neuroscience and biophysics due to its availability, tractability, relatively simple nervous system, and it's patently observable behavior. Here in the Elegant Mind Club at UCLA, we provide undergraduate students a unique hands-on experience working with C. elegans and a chance to present their own scientific methods of interest, allowing them to explore the nature of scientific research and understand the conclusions drawn from their data. In our laboratory, undergraduate students are entirely responsible for maintaining and culturing the worms as well as building and refining their experimental systems. Direct involvement with the biological samples teaches students the discipline of working with chemicals and maintaining sterility. Published papers and online resources such as WormBook, WormAtlas, and Caenorhabditis Genetics Center provide students with a source of well-established methods and techniques to serve as a basis for their own studies. Manual practice in hardware development permits students to personally hone their experiment to be the most controlled and reproducible systems. As of now, systems for thermotaxis, electrotaxis, chemotaxis, phototaxis, durotaxis, as well as behavior within a magnetic field and the absence of stimuli have been reproduced and improved by our members. Our laboratory begun with a few core members and have expanded to accommodate more than 80 students from different universities over the world and we hope to encourage more students to approach scientific research with enthusiasm.

Mendoza, Steve et al. (2015) International Worm Meeting "A Novel Worm Tracking Calcium Imaging System Utilizing a Mobile Microscope."

Sherry, Tim, Lam, Brian, Kao, Michelle, Nowak, Nate, Mendoza, Steve, Kim, Taejoon, Arisaka, Katsushi, Madruga, Blake, Jiang, Karen

[International Worm Meeting, 2015]

Worm tracking of freely moving worms is essential to study the connection between behavior and neural activity. However, these microscopes are often limited in their ability to track worms under various behavioral simulations. We present a fluorescence worm tracking microscope that has an open geometry and thus can track worms even if the behavioral platform is difficult or impossible to move via a motorized stage. As opposed to other automated worm tracking systems, our microscope is fully mobile-where all the optical components are mounted on top of a motorized xy stage-while the sample stage where C. elegans rests is stationary. This platform allows for ratiometric calcium imaging while also tracking a dark field worm image for behavioral analysis, running at 15 frames per second. The current configuration has three cameras, two for each of the YFP and CFP channels, and a dark field image showing the worm body, under a 10x magnification; the microscope can also be adjusted to image at 20x. Being able to track freely moving worms without moving the sample stage, allows our microscope to perform worm tracking in experimental conditions that similar systems have not been able to achieve. In addition, since the sample is stationary, we also avoid introducing confounding effects on the worms due to stage acceleration. To test this novel hardware, we run a thermotaxis experiment tracking a worm without moving the temperature platform. Our worm was labeled with AIY::CAM and we find a correlation between the AIY activity and the temperature of the head location during isothermal behavior. Our methodology could also apply to other behavioral experiments where an external stimulus would be hard to move via a motorized stage, such as an electrotaxis experiment.

Michelle S. Teng et al. (2006) European Worm Meeting "Expression of Mammalian GPCRs in C. elegans Generates Novel Behavioural Responses to Human Ligands"

John McCafferty, Martijn P.J. Dekkers, Andrew G. Fraser, Michelle S. Teng, Bee Ling Ng, Gert Jansen, Suzanne Rademakers

[European Worm Meeting, 2006]

Michelle S. Teng1, Martijn P.J. Dekkers2, Bee Ling Ng1, Suzanne Rademakers2, Gert Jansen2, Andrew G. Fraser1 & John McCafferty1. G protein coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However purification of GPCRs for biochemical study is difficult and most methods of screening receptor-ligand interactions require cultured cells and endotoxin free compounds. In contrast, Caenorhabditis elegans is a soil dwelling nematode that feeds on bacteria and uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds. Here we report that expression of the mammalian somatostatin receptor (Sstr2) and chemokine receptor 5 (CCR5) in gustatory neurons allow C. elegans to specifically detect and respond to human somatostatin and MIP-1? respectively in a simple avoidance assay. The endogenous signalling components involved in this remarkable promiscuity of interaction, spanning 800 million years of evolution, are investigated. This system has practical utility in ligand screening. Using structure:function studies, we identified key amino acid residues involved in the interaction of somatostatin with its receptor. This in vivo system, which imparts novel avoidance behaviour on C. elegans, can therefore be used in screening impure GPCR ligands, including the identification of bacterial clones expressing agonists within recombinant libraries.

Woldemariam, Sarah et al. (2015) International Worm Meeting "Elucidating the role of cGMP dynamics in behavioral plasticity."

Schneider, Martin, Krzyzanowski, Michelle, Bethke, Mary, Nagpal, Jatin, Gottschalk, Alexander, Woldemariam, Sarah, Ferkey, Denise, L'Etoile, Noelle

[International Worm Meeting, 2015]

cGMP is a ubiquitous second messenger implicated in many important biological processes. In neurons, cGMP dynamics can regulate the function of ion channels and kinases, resulting in physiological changes. In the context of learning and memory, these changes result in short-term and long-term behavioral changes based on the organism's experience. We attempt to understand the molecular basis for long-term plasticity by studying the behavioral responses of the nematode C. elegans. Along with our collaborator Michelle Krzyzanowski from the Denise Ferkey lab in SUNY Buffalo, we are interested in how food might modulate behaviors. One food-modulated behavior is repulsion from quinine. This repulsion is mediated by the ASH neuron and it is down regulated by food withdrawal and the cGMP-dependent protein kinase EGL-4. This poses a conundrum since no guanylyl cyclases, which produce cGMP, are expressed in ASH. Genetic evidence suggests that guanylyl cyclases in other neurons are required for the food-modulated repulsion from quinine in ASH and that gap junctions are required for the transmission of cGMP from these neurons to ASH. In order to understand how cGMP dynamics in these neurons are modulated, we need a tool to visualize cGMP. To this end, we are using a cGMP sensor that will allow us to image cGMP dynamics in ASH and other neurons in the living behaving animal in the presence and absence of food.

Link CD et al. (1997) International C. elegans Meeting "PHYSIOLOGICAL EFFECTS OF BETA AMYLOID PEPTIDE EXPRESSION IN C. ELEGANS"

Fay DS, Link CD, Johnson CJ

[International C. elegans Meeting, 1997]

Transgenic worms containing an unc-54/signal peptide/ beta peptide 1-42 minigene (derived from the human Amyloid Precursor Protein, APP) produce intracellular immunoreactive beta peptide deposits that also react with the amyloid specific dye thioflavin S. Animals expressing beta peptide show a distinct vacuolar phenotype and a progressive paralysis as adults. We are investigating both the specificity and the underlying biology of these phenotypes using a combination of genetic, transgenetic, and microscopy techniques. To directly visualize the physiological effects of beta peptide expression, we have used stress-responsive reporter transgenes, such as hsp16-2/GFP, in combination with amyloid transgenes. Double transgenic animals containing independently integrated unc-54/signal peptide/ beta 1-42 and hsp-16/GFP transgenes show muscle-specific induction of GFP independent of heat shock. Interestingly, ultrastructural studies (see Kao et al abstract) indicate that the vacuoles observed in unc-54/signal peptide/ beta 1-42 transgenic animals are not associated with muscles, but instead appear to be associated with hypodermal and neuronal cells. Moreover, these vacuoles have ultrastructural similarities with degenerin-induced vacuoles, suggesting that they may result from neurotoxicity of muscle-secreted beta peptide. The persistance of beta peptide-induced vacuoles is enhanced by ced-5(e1812) (as are degenerin vacuoles); however, these vacuoles are not suppressed by a mec-6 mutation. We are currently examining transgenic animals expressing variant peptides in order to determine the specificity of these physiological effects of beta peptide expression.

Christian Rocheleau et al. (2001) International C. elegans Meeting "GENETIC SCREENS FOR POSITIVE REGULATORS OF RAS SIGNALING"

Mandy Volk, Meera Sundaram, Alissa Goldman, Christian Rocheleau

[International C. elegans Meeting, 2001]

RTK/Ras/Raf-MEK-ERK signaling pathway is a highly conserved signal transduction pathway required for multiple aspects of C. elegans development. The genes ksr-1 and sur-8 positively regulate Ras signaling at a step between let-60 ras and lin-45 raf . To identify positive regulators of the Ras pathway that may function with either ksr-1 or sur-8 , or at another step in the pathway, we have been performing an Enhancer Of Raf (Eor) screen. Screening for enhancers of weak hypomorphic alleles of lin-45 raf has already successfully identified mutations in ksr-1 , sur-6 , and previously unidentified regulators eor-1 and eor-2 (see abstracts by Howard et al. and Kao et al.). In addition, this screen has identified interesting alleles of sem-5 and let-341 , as well as hypomorphic alleles of sur-2 and lin-25 . Although this screen has identified multiple alleles of eor-1 , eor-2 , sur-2 and lin-25 , it is far from saturated. As an alternative approach, we have initiated an Enhancer Of Sur-8 (Eos) screen. Screening in the sur-8 mutant background for mutations that synergize with sur-8 is expected to identify mutations in positive regulators that may function with ksr-1 and sur-6 . We believe this screen will also identify mutations in eor-1 and eor-2 , as well as new loci, while reducing some background mutations identified in the Eor screen. The results of these screens as well as reverse genetic analysis of candidate genes that may function to positively regulate Ras signaling will be presented.

Chen Y et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Insulin Peptides in Nutritional Control of Development in C. elegans"

Chen Y, Kurhanewicz N, Baugh R

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Animals have evolved great ability to adapt to fluctuating nutrient availability by balancing growth and survival. In C. elegans, newly hatched L1 stage larvae remain developmentally arrested until feeding. This phenomenon, called L1 arrest, provides an opportunity to study regulatory mechanisms mediating nutritional control of growth and development. Insulin-like signaling is a key regulator of L1 arrest (Baugh and Sternberg, 2006; Kao et al, 2007). In spite of great interest in the insulin-like pathway given its function in dauer formation, aging and L1 arrest, the function of specific insulin-like peptide ligands is generally not understood. The C. elegans genome encodes 40 insulin-like peptides, and they are thought to function as either agonists or antagonists of the insulin-like receptor DAF-2 and growth (Pierce et al., 2001). We are using a combination of expression and phenotypic analysis to characterize their regulation and function during L1 arrest. Although several insulin-like genes may be broadly redundant, we propose that there is specificity to their function in timing and site of action, and that they comprise regulatory network that responds to nutrient availability and controls growth and development. From our expression data we identify several putative agonists, including ins-4, ins-5, ins-6, and ins-7, which are up-regulated by feeding, and several putative antagonists, including ins-17, ins-18 and ins-24, that are up-regulated by starvation. Transcriptional YFP reporters of them show that the agonists and antagonists are expressed in different subsets of amphid sensory neurons in L1 worms, and emphasize the intestine as a site of nutrient-dependent transcriptional regulation. Moreover, phenotypic analysis of growth rate, starvation survival and early postembryonic cell division on single and multiple deletion mutants will help determine their role in promoting growth and development.

Richardson, Claire E. et al. (2009) International Worm Meeting "The IRE-1 Branch of the Unfolded Protein Response Functions in C. elegans Pathogen Resistance During Larval Development."

Kim, Dennis H., Kooistra, Tristan, Richardson, Claire E.

[International Worm Meeting, 2009]

The Unfolded Protein Response (UPR), a mechanism to sense and respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), has an important role in development and stress resistance from C. elegans to mammals. We asked if the UPR functions in C. elegans immune defense against the bacterial pathogen Pseudomonas aeruginosa. We found that the conserved IRE-1-XBP-1 branch of the UPR is required specifically for immunity during larval development. IRE-1 is an endoribonuclease that activates the transcription factor XBP-1 by splicing a short exon from its mRNA. Using quantitative PCR, we show that the relative amount of IRE-1-spliced xbp-1 transcript increases after pathogen exposure. Furthermore, a fluorescent reporter for IRE-1 activation is induced after exposure to Pseudomonas specifically at the site of infection - the intestine. We analyzed the relationship between the IRE-1 branch of the UPR and the PMK-1 pathway, which is known to promote C. elegans immunity, and found that IRE-1 activation in response to Pseudomonas is downstream of the PMK-1 pathway. The IRE-1-XBP-1 branch of the UPR has been recently shown to be required for cellular defense against pore-forming toxins, also functioning downstream of PMK-1 (Bischoff et al, 2008). Our data point to a more general role for the UPR in innate immunity against infection. Since PMK-1 pathway activates transcription of putative pathogen resistance proteins, we suggest that the IRE-1 branch of the UPR promotes pathogen resistance by accommodating a PMK-1-driven influx of pathogen resistance proteins to the secretory pathway. Bischof, L.J., Kao, C.-Y., Los, F.C.O., Gonzalez, M.R., Shen, Z., Briggs, S.P., van der Goot, F.G., Aroian, R.V. (2008) Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo. PLOS pathogens, 4, 1-11.

Ryan Baugh et al. (2007) International Worm Meeting "Nutritional control of larval development: DAF-16 is required for transcription of cki-1 and repression of lin-4 during L1 arrest."

Ryan Baugh, Paul Sternberg

[International Worm Meeting, 2007]

C. elegans larvae initiate post-embryonic development only in the presence of food, although they can survive starvation for weeks in a developmentally arrested state (L1 arrest). In contrast to the well-studied dauer arrest, L1 arrest occurs without morphological modification, although larvae in L1 arrest are more resistant to environmental stress than developing larvae. Consistent with its role in dauer formation and aging, we and others have shown that insulin/insulin-like growth factor (IGF) signaling regulates L1 arrest (Baugh and Sternberg, Cur. Biol. 2006; Fukuyama et al, Cur. Biol. 2006; Kao et al, Cell 2007). Our results indicate that daf-2 insulin/IGF receptor mutants have a constitutive L1 arrest phenotype when fed and extended survival of L1 arrest when starved. Conversely, daf-16/FOXO mutants have a defective arrest phenotype, failing to arrest development and dying rapidly when starved. We have shown that DAF-16 is required for transcription of the cyclin-dependent kinase inhibitor cki-1 in lateral epidermal seam cells in response to starvation, accounting for the failure of daf-16 mutants to arrest somatic cell division during L1 arrest. Other developmental events such as cell migration, cell fusion, and expression of the microRNA lin-4, a temporal regulator of post-embryonic development, are also observed in starved daf-16 mutants. These and other results implicate insulin/IGF signaling in the nutritional control of development during the first larval stage in C. elegans, although additional DAF-16-independent pathways must also participate. In addition to performing a genome-wide characterization of mRNA expression during L1 arrest, we are currently investigating the insulin ligands and their sites of action as well as additional candidate signaling pathways that regulate L1 growth and arrest in response to changing environmental conditions.

Judy, M. et al. (2013) International Worm Meeting "Mutations in progranulin and cell death genes confer organismal stress resistance."

Nakamura, A., Judy, M., Grant, H., Huang, A., Kenyon, C., McCurdy, H., Kao, A.

[International Worm Meeting, 2013]

As animals move about their world, they encounter changing and sometimes harsh environmental conditions. To cope with environmental stress, animals have evolved protective mechanisms. At the local or cellular level, these responses include apoptosis to remove damaged cells. On a systemic or organismal level, animals can alter metabolism or gene expression to promote stress resistance. Because neurodegeneration involves cell death and dysfunction modulated by both genetic and environmental factors, we are interested in the link between stress response and programmed cell death (PCD). Mutations in the progranulin gene link to familial forms of the neurodegenerative disease frontotemporal lobar degeneration. Earlier, we showed that C. elegans lacking progranulin (pgrn-1) exhibit accelerated clearance of apoptotic cells (Kao, PNAS 2011). More recently, we found that pgrn-1 mutants were specifically resistant to heat, osmotic and ER stress, but not to oxidative, UV or pathogen stress. Because pgrn-1 normally regulates PCD, we wondered whether other cell death mutations affected the stress response. Indeed, a similar pattern of stress resistance was induced by mutations that either block cell death (ced-3, ced-4) or slow apoptotic cell engulfment (ced-1, -2, -5, -6, -7). Double mutants of pgrn-1 and ced-3 or engulfment mutants were not additive in ER stress resistance, suggesting a common mechanism of enhanced stress response. These results demonstrate that mutations perturbing PCD in different ways have similar effects on environmental stress resistance. We speculate that these cell death regulators converge on a common stress regulatory mechanism. We are currently using transcriptional analysis to examine the genetic pathways altered in cell death mutants and investigating whether cleavage of progranulin into granulins is changed in cell death mutants. These findings may lead to new therapeutic targets in the treatment and prevention of neurodegenerative disease.