10 Papers found (0.01 seconds)

Carmona, Javier et al. (2017) International Worm Meeting "2D worm tracking line-confocal microscope with virtual reality."

Wang, Charles, Carmona, Javier, Baldo, Anthony, Madruga, Blake, Mendoza, Steve, Jin, Suying, Arisaka, Katsushi, Liu, Larry, Thatcher, Joseph

[

International Worm Meeting,

2017]

Calcium dynamic imaging and free motion tracking coupled with external stimulations allow for in depth analysis of C. elegans behavior. We have developed an integrated platform for monitoring and controlling C. elegans under a variety of external stimulations, including thermal, electrical, and photo stimuli. This innovative platform combines rapid volumetric (20 volume/s) diffraction limited dual line-confocal microscopy (0.5 um x 1 um x 5 um voxel) to determine the neural pathways different external stimuli induce, while tracking worm's two dimensional motion. Never before has dynamic signal propagation, from neuron to neuron, been observed for C. elegans in free motion at such high volume scanning rate. External stimuli are computer controlled with < 10 ms resolution for precise spatio-temporal synchronization with free motion behavior and whole-brain calcium dynamics. Physical linear and circular thermal gradients were implemented using customized temperature plates with thermal fluctuations of less than 0.05 deg C. In addition, thermal stimulation was applied via a 1490 nm infrared laser to create virtual temperature conditions, synchronized with head motion. Infrared laser stimulation allows C. elegans' thermoreceptor (AFD neuron) to perceive temperature fluctuations exclusively in the time domain, thereby allowing for the complete virtual manipulation of the nematode's thermal environment. Electrical responses were induced using a technique that involves applying a linear or spatially alternating electrical field through a gelatin sample with fields ranging from 4 to 14 V/cm. Photon stimulation was implemented using a 405 nm laser with intensities ranging from 0 to 10 mW/mm^2. Volumetric Calcium imaging of QW1217 has also allowed for the complete mapping of the neurons responsible for each of the aforementioned stimuli. The microscope and software accommodate multiple simultaneous stimuli applications, such as electrical and photon simulations. Also tested were the neural pathway differences between infrared and photo avoidance behavior due to their very similar behavioral responses.

Apfeld J et al. (1996) West Coast Worm Meeting "A GENETIC SCREEN FOR LONG-LIVED WORMS."

Hsin H, Dorman JB, Kenyon CJ, Apfeld J, Albinder B

[

West Coast Worm Meeting,

1996]

We are interested in finding genes that control lifespan in C. elegans. We are currently doing a clonal screen for mutations that lead to an increase in lifespan. We mutagenize a temperature-sensitive sterile strain, clone out F2 progeny, allow these worms to self fertilize, clone some of their progeny into a new plate at the restrictive temperature, and screen these for increased lifespan. So far we have screened 360 F2 clones and are in the process of retesting 3 of them. Javier Apfeld is also studying the gene daf-2. The e1370 allele of this gene has multiple phenotypes: at 25°C daf-2(e1370) worms develop into dauer larvae (a developmentally arrested, stress resistant, long lived alternative larval form); at 20°C daf-2(e1370) worms do not form dauer larvae, but live 2.5 times longer than wild-type (N2) worms. Javier wants to know where daf-2 functions and how it acts, and is currently doing mosaic analysis to address these questions.

Madruga, Blake et al. (2017) International Worm Meeting "Development of a SLM-based sheet illumination microscope for rapid, large-scale, video-rate 3-D neurodynamic observation in C. elegans."

Mendoza, Steve, Madruga, Blake, Carmona, Javier, Thatcher, Joe, Arisaka, Katsushi

[

International Worm Meeting,

2017]

Sheet Illumination microscopy has made a large impact on the microscopy community due to its many inherent advantages. Increased photonic efficiency allows for lower power light sources, which in turn reduce phototoxic damage to the sample while providing an increased signal to noise ratio. To take advantage of such technique, a type of phase modulator, known as a Spatial Light Modulator (SLM) is used to generate a deep-penetrating, extremely long and narrow Bessel beam interference pattern. Through the use of an SLM, one can easily modulate the phase characteristics of an illumination beam in real time. This property enables greater flexibility and aberration compensation at the sample. The Bessel beams are generated through a bitmap image of the beam's modulation transfer function, which is then displayed on the SLM with a prism phase rotation. This provides a much longer region of micron-order uniformity along the beam axis compared to conventional Gaussian geometries, while shifting the modulated beam away from the higher diffraction orders. The beams are then mapped onto the readout of two scientific CMOS cameras for rapid multi-channel imaging. A piezoelectric objective collar is used to enable rapid z-scanning, thereby creating 4D image volumes with adequate time resolution to characterize and observe active neural dynamics in C. elegans. A long working distance, high numerical aperture (NA), refractive-corrected objective lens is used to study neurobiology and participate in ratiometric calcium imaging. Tools of such flexibility will enable the study of whole-brain-scale neuronal activity and structure under various controlled conditions in C. elegans, over a variety of temporal and spatial scales.

Thatcher, Joseph et al. (2017) International Worm Meeting "Development of a three dimensional fluorescence tracking microscope for the motional analysis and behavioral stimulation of C. elegans."

Lam, Brian, Madruga, Blake, Carmona, Javier, Shrestha, Ahis, Jin, Suying, Mendoza, Steve, Thatcher, Joseph, Arisaka, Katsushi, Niaki, Shayan

[

International Worm Meeting,

2017]

Extensive advances have been made in understanding the behavior of C. elegans in two dimensional environments. However, they impose substantial constraints on the worm's motion and ultimately restrict the set of possible natural behavioral states it can demonstrate. Addressing limitations encountered by previous efforts in three dimensional imaging, we designed and built a microscope capable of tracking the motion of C. elegans via a set of motorized stages while navigating freely within a sample volume. The variation of gelatin concentration (1% - 4%) and the utilization of temporally controlled ultraviolet photo-stimulation (405 nm) were also incorporated into the system. The addition of a refractive index mismatch correction chamber and fluorescence detection enable novel opportunities for observation and categorization of motion. Preliminary data of photoavoidance response in three dimensions was acquired and demonstrates the added complexity present in an unconstrained response. A novel use of fluorescence enables the identification of C. elegans' absolute orientation with respect to the ventral nerve cord. A model of motion based on sinusoidal wave propagation was applied to C. elegans' forward locomotion, thereby categorizing a set of three dimensional body states inhabited. From this analysis, we have identified three distinct motional states: one of which is sinusoidal in the worm's ventrodorsal plane, another which is sinusoidal in their lateral plane, and a final state that is helical in shape. Fitting this parametric model allows the extraction of a variety of wave-based parameters including wavelength, frequency, wave speed and phase difference which may then be correlated with other dynamic quantities and gelatin concentrations. Namely, the phase difference acts as a direct indicator of the degree to which the worm's posture is planar or helical, allowing the ability to parameterize its general motional form with a single number. Furthermore, from pre-existing, established data of the C. elegans' connectome, we hypothesize a neuronal mechanism for rhythmic signal generation based on the SMD motor neurons which predicts the motional states observed.

Rabichow, Laura et al. (2017) International Worm Meeting "Photo avoidance reflex influenced by C. elegans' own motional state."

Punjabi, Nihal, Yardley, Reule, Bhagawat, Chaiti, Lee, Diane, Nguyen, Tiffany, Carmona, Javier, Kardoust, Arash, Mohnot, Kushal, Santana, Joslyn, Mendoza, Steve, Rabichow, Laura, McAdam, Meera, Fu, Daniel, Ross, Evan, Mahavni, Anika, Arisaka, Katsushi

[

International Worm Meeting,

2017]

Self-awareness is a critical aspect related to the effective navigation of a complex environment. For a given input stimulus to a sensory structure, behavioral output seems closely related to an organism's own motion. An example may be drawn from a relativistic pressure applied to a C. elegans' mechanosensory structure in time, based on two conditions. When considering a mechanical pressure driven by the worm's own motion (as if a worm struck a stationary barrier) the response is dramatically different to one of similar magnitude unrelated to the worm's motion (if a stationary worm was lightly touched with an eyebrow hair). Interestingly, these conditions apply the same force to a given sensory structure in time, but result in a much higher probability of avoidance response if the worm is stationary at the time of the input. To more closely evaluate this complex phenomenon, the well characterized, highly deterministic photo-avoidance behavior was implemented in a number of experimental wave speed conditions. We postulate that a lower avoidance response probability will be found in quickly moving C. elegans as compared to those with slow wave speeds. Gelatin concentration was altered from 1 - 4 % in order to shift the wave speed accordingly, while phototaxis stimulation acted as our input force; provided from a well-calibrated 405nm laser. A custom-built line confocal microscope supplied a triggering signal to the laser stimulus while recording worm position and orientation over experimental trials. Offline, a MatLab program was used to analyze the data and sinusoidally fit an analytic function to the worm's body in time. Wave speed and center of mass velocity were extracted from this data, and avoidance probability was investigated as an independent variable. Preliminarily, observations suggest that lower gelatin concentrations (i.e. faster wave speed) correspond with lower avoidance probability; perhaps due to a partial inhibition of the AVA interneuron through sensorimotor integration.

Tan, Trudy et al. (2017) International Worm Meeting "Thermotaxis navigational modeling and virtual reality investigation."

Yen, Jessica, Tan, Trudy, Jin, Suying, Zadoorian, Arbi, Arisaka, Katsushi, Huang, Rebecca, Narain, Shreya, D'Orazio, Etta, Yamada, Mandi, Mai, Phat, Park, Jane, Yang, Karen, Carmona, Javier, Kim, Ted, Liu, Junliang, Mendoza, Steve, Watson, Sonya

[

International Worm Meeting,

2017]

Caenorhabditis elegans exhibit a well-characterized host of thermosensory behaviors necessary for efficient navigation, foraging, and survival in the natural environment. Many of the neural structures responsible for temperature sensing have evolved to exceptional sensitivity over time, giving rise to cognitively complex, deterministic behaviors such as bias orientation and 90 degree turning. In order to better comprehend the underlying neural circuitry responsible for such behaviors, multiple PID-controlled hardware systems have been constructed to generate and control various thermal gradient conditions within 0.05 degrees C, over durations of ~ 30 minutes. Additionally, an IR laser-based thermosensory system has been fabricated, to provide spatiotemporally controlled thermal stimulus at a highly localized region of the worm's body, enabling the establishment of a virtual thermal environment on which the worm can behave. Using these tools, we have investigated several intricacies of C. elegans' response to temperature, including the mechanism governing 90 degree turns and biased orientation during negative thermotaxis. Making use of line confocal calcium imaging microscopy methods, the dynamics of the AFD sensory neuron and the AIY interneuron were observed, yielding multiple noteworthy datasets. Custom-written MatLab image processing tools based on Goodman and NEMO were utilized to conduct a systematic motional analysis in a variety of experimental thermal conditions. This poster aims to outline the some of these recent advancements in thermotaxis investigation in the Elegant Mind Club, along with preliminary supporting datasets, on a case-by-case basis.

Dasa Longman et al. (2006) European Worm Meeting "A Systematic Analysis of Nonsense Mediated mRNA Decay in C.elegans"

Iain L. Johnstone, Dasa Longman, Javier F. Caceres

[

European Worm Meeting,

2006]

Dasa Longman1, Iain L. Johnstone2 and Javier F. Caceres1. Nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs). Seven genes (smg1-7) essential for NMD were originally identified in the nematode Caenorhabditis elegans, and orthologs of these genes have been found in many other species. A crucial aspect of NMD is the mechanism of PTC definition. In human cells, it has been established that pre-mRNA splicing plays a critical role in defining PTCs, and this is mediated by the components of the exon junction complex (EJC) assembled upstream of exon-exon boundaries. In Drosophila cells, definition of a PTC occurs independently of exon boundaries, and accordingly, the components of the EJC are dispensable for NMD (Gatfield et al., 2003, EMBO J., 22:3960). Although the requirement for smg genes for NMD in C.elegans is well documented, little is known about the mechanism of PTC definition in this organism. Here, we have conducted a systematic analysis of the cis-acting sequences and trans-acting factors that are required for PTC recognition in the nematode C.elegans. We have used transgenic C. elegans strains expressing GFP/LacZ reporter, either in a wild-type version, or harboring premature termination codon (PTC). We show that introduction of a PTC into the reporter induces a robust NMD response, as determined by the lack of GFP expression and decreased level of the corresponding mRNA. Use of cDNA reporters established that introns are not required to define a PTC, and we show that accordingly EJC components are not essential for NMD in this organism. We will discuss the use of these reporters in a genome-wide RNAi-based screen to identify novel genes that contribute to the process of nonsense-mediated decay.

Huang, Rebecca et al. (2017) International Worm Meeting "Isothermal behavior in Caenorhabditis elegans - the neural origin and development of sensorimotor integration."

[

International Worm Meeting,

2017]

Caenorhabditis elegans have an ability to exhibit isothermal behavior up to a remarkable precision of a 0.05 degree difference. However, the neural circuitry underlying isothermal behavior remains elusive as it is very difficult to track neuronal activity in freely moving specimen. Furthermore, the computational power of their nervous system is often overlooked due to the small number of neurons - hundreds compared to a mammal's billions. Isothermal tracking appears to be a deterministic behavior that may be a product of a more complex processing system - a behavior facilitated by a network. There is strong evidence sensorimotor integration is occurring; the C. elegans are tracking temperature differences and coordinating their movements to the local temperature in a highly deliberate and precise manner. In this experiment, we created an assay plate over a thermal gradient and physically tracked the worm's movements using a high-powered line confocal microscope with a motorized stage and fluorescently labeled neurons. We were able to track the neural activity of the whole nervous system in real time as the worm moved over the thermal gradient. This eliminates the need for laser ablation or immobilization and allows us to observe how a Caenorhabditis elegans would move in its natural form. We conducted this experiment with worms in the L1 stage as well as worms in the L4 stage to observe possible synaptogenesis or synaptic plasticity.

Ginger R Miley et al. (2004) Mid-west Worm Meeting "Analysis of LIN-1, an ETS protein, involved in vulval development."

Ginger R Miley, Kerry Kornfeld

[

Mid-west Worm Meeting,

2004]

A highly conserved RTK/Ras/MAP kinase pathway promotes the primary vulval cell fate by regulating the ETS transcription factor LIN-1. Although LIN-1 plays a critical role in vulval precursor cell (VPC) fate determination, the regulatory mechanisms that control LIN-1 activity and the mechanism of action of LIN-1 have not been extensively characterized. Fifty alleles of lin-1 have been isolated in a variety of screens for vulval defects conducted in several laboratories. These alleles include loss-of-function and gain-of-function mutations of different severities. We used DNA sequencing to identify the molecular lesion in 27 loss-of-function alleles. The loss-of-function alleles include eleven missense mutations that affect nine different conserved residues in the ETS domain. Most of these mutations cause a severe loss-of-function, although the ga56 mutation causes a partial loss-of-function. We examined the DNA-binding properties of the wild-type and mutant ETS domains using purified extracts in an electrophoretic mobility shift assay (EMSA). The wild-type LIN-1 ETS domain displays sequence specific binding to a consensus ETS binding site. These mutant proteins were severely defective in DNA binding. These finding indicate that the DNA binding activity of LIN-1 is essential for function in vivo . Target genes that are directly regulated by LIN-1 not been characterized. We utilized our EMSA to test for LIN- 1 binding sites in candidate genes involved in vulval development. One likely candidate is lin-39 , which encodes a homeobox gene transcription factor. Javier Wagmaister and David Eisenmann have characterized the lin-39 promoter and identified a 1.3Kb region that displays Ras responsive expression in transgenic animals. This region contains multiple GGA motifs that are potential ETS binding sites. EMSA experiments demonstrated that LIN-1 can bind some but not all fragments of the 1.3 Kb region. We are currently performing mutational analysis to identify the minimal regions that are necessary and sufficient for LIN-1 binding.

Robert Ferguson et al. (2007) International Worm Meeting "Identification of transcription factors that control the temporal and spatial expression of the C.elegans Hox gene lin-39 using a Yeast-1-Hybrid system."

Robert Ferguson, David M. Eisenmann

[

International Worm Meeting,

2007]

Hox genes encode a large group of evolutionarily conserved transcription factors that function in providing regional identity along the metazoan anterior-posterior body axis. lin-39 is one of six C. elegans Hox genes and it is involved in the patterning of the mid-body region of the nematode, where the vulva is formed. In larvae, lin-39 is expressed in the six VPCs, Pn.a derived ventral cord neurons, sex muscles and the QR neuroblast descendents. Our lab and others have identified several transcription factors and chromatin regulatory proteins that regulate lin-39 expression. In addition, previous work by Javier Wagmaister divided the lin-39 regulatory region into 10 fragments that were used to generate GFP reporter constructs. The expression patterns of these constructs identified several cis-acting regions involved in the regulation of lin-39 expression. The goal of my project is to identify the trans-acting factors necessary for proper spatial and temporal regulation of lin-39 expression in both embryos and larva, and to locate the cis-acting sites that they act through. I intend to do this using the Yeast-1-Hybrid system. I am cloning the 10 fragments defined by Wagmaister et al into lacZ and HIS3 reporter constructs that will be integrated into yeast. This yeast will be transformed with a transcription factor library (gift of Walhout lab) containing approximately 800 C. elegans transcription factors to identify transcription factors that bind to that particular lin-39 regulatory region. GFP reporter constructs of the identified transcription factors will be generated and injected into C. elegans to see if their expression is localized to the same cells that express lin-39. In addition, mutants of the identified transcription factors or RNA interference will be used to determine whether loss of transcription factor function influences lin-39::GFP. From these experiments I hope to identify transcription factors that are important in activating or controlling the expression of lin-39.