Kerr, Rex A. (2013) International Worm Meeting "Chemotaxis follows the nose."

Kerr, Rex A.

[International Worm Meeting, 2013]

Worms can reliably chemotax: that is, follow a gradient of an attractive chemical (soluble or volatile) to its peak. In addition to the well-known pirouette and weathervane mechanisms, high throughput behavioral analysis identified at least nine other apparently distinct behavioral modifications which, at least in theory, aid chemotaxis behavior. This suggests that the control of behavior by chemotaxis is particularly broad-ranging. Recent imaging results from a number of labs have implicated individual sensory and inter-neurons involved in chemotaxis in driving head swings. I therefore asked: is the profusion of different strategies actually just a reflection of simple regulation of head swings that is not apparent when performing traditional center-of-mass analysis of chemotaxis? To a large extent, at least, the answer is yes; there seem to be only four fundamental behaviors which are regulated in simple ways by concentration changes at the nose, namely head swing size, reversal probability, reversal size (a novel finding), and movement speed. Other behaviors are in large part and perhaps completely a consequence of these. That reversal size and probability are at least partially independently regulated is confirmed by multi-sensory integration experiments. In response to tap, worms execute a reversal with characteristic size and probability. When tap is delivered during a chemotaxis assay, the probability of reversal is essentially unaffected by whether the animal is traveling in the right or wrong direction in the gradient. However, the reversal distance is strongly modulated, and both results hold during habituation to tap. Thus, chemotaxis can be viewed as a small set of coordinately regulated simple behaviors that nonetheless can sensibly integrate with other sensory cues.

Kerr, Rex (2015) International Worm Meeting "Testable hypothesis generation via directed analysis of plausible information flow in the connectome."

Kerr, Rex

[International Worm Meeting, 2015]

C. elegans is the only animal with an approximately complete connectome. Although initial hopes that connectivity alone would allow a deep understanding of nervous system function have gone unfulfilled, the connectome has proven to be an essential tool for the experimentalist in generating hypotheses and planning experiments. Nonetheless, the majority of computational analysis of the connectome has focused either on attempts at whole-animal simulation, or on topology-based graph-theoretic measures that apply to graphs of all sorts. Neither approach seeks to emulate the directed inquiry of the experimentalist.Graph-theoretic measures take no advantage of our knowledge of the function of a nervous system as a time-varying computational system that propagates information, and thus discards much valuable information about the system. But the success of a full simulation depends on detailed knowledge of the biophysical properties of neurons and synapses that exceeds what we have, and it is unclear whether optimization techniques can suffice to fill in the missing pieces. Here, a middle path is taken: the connectome is used to define routes of plausible informationflow, which neither discards information we have nor requires information that we do not.Information flow is then used to compute a variety of metrics of particular interest to experimentalists. Flow-similarity groups neurons that seem to have a similar pattern of impact on the nervous system, suggesting that they function in similar behaviors. For example, ASE and AWC, which sense salt and volatile attractants respectively, are scored with very high similarity, as we would hope given that they both drive chemotaxis behavior. Flow-compensability measures the extent to which information flow to muscles can be preserved after ablating one or more neurons by up- or down-regulating the responsiveness of other neuron(s); as expected, right and left neurons are typically very good at compensating for reach other. Flow-persistence detects neurons in recurrent loops where ongoing state may be maintained; this metric gives high scores to command neurons. Thus, these and other similar computation-based metrics provide a rich view of possible neuronal function, and are a promising source of novel hypotheses for experimentalists.(Note: some of this work was performed at, and all of it was supported by, the Howard Hughes Medical Institute's Janelia Farm Research Campus.).

Kerr, Rex et al. (2019) International Worm Meeting "Bridging the gap from raw data to result in large-scale aging experiments."

Kenyon, Cynthia, Kerr, Rex, Kimmel, Jacob, Xue, Eddie

[International Worm Meeting, 2019]

Animals exhibit declining behavioral capacity as they age. These declines typically include a reduction of speed and strength of movement, slowed reactions, and loss of coordination. However, the genes involved in preserving youthful behavior or setting the pace and nature of the decline have not been extensively studied, in large part due to the difficulty of conducting and analyzing experiments on a sufficient scale. We have created an automated system, the C. elegans Observatory, to simultaneously characterize hundreds of strains of worms as they age by computing a variety of behavioral metrics. However, analyzing the resulting data becomes an intractable burden for a typical experimentalist. We therefore have constructed a series of automated analyses, ranging from low-level through to near-publication-quality graphs for key behaviors, which run on the fly as data is generated. This enables the same kind of rapid understanding and iterative experimentation in a big-data style experiment as is typical for a benchtop experiment. Examples of the analyses, using data from ongoing experiments on wild-type and long-lived mutants, will be used to illustrate the process.

Rex Kerr et al. (2004) West Coast Worm Meeting "Tracking Individual Behaviors in a Population"

Catharine H Rankin, Rex Kerr, William R Schafer

[West Coast Worm Meeting, 2004]

Many behaviors exhibited by C. elegans , including chemotaxis, thermotaxis, and tap habituation, are most easily detected at the population level. However, plate-based assays typically collect very little information about behavior at the individual level, and assays that look only at single worms can be very labor-intensive since the assay must be repeated until a sufficiently large population is sampled. In particular, tap habituation is best scored by examining the change in reversal frequency with repeated taps [Rankin et al., Behav. Brain Res. 37:89, 1990], with reversals scored by eye on a single worm at a time. The time-consuming nature of this assay makes it essentially impossible to screen for tap habituation defective mutants. To overcome this limitation, we have developed a multiworm tracker (MWT) that uses a relatively inexpensive high-resolution camera to simultaneously image an entire population of worms on a single plate. Using a variety of rapid image processing techniques, the MWT tracks the position and bearing of up to 64 worms simultaneously and can automatically detect reversals. Thus, the habituation phenotype of a strain can be assayed in the time it takes to run the protocol once (five minutes in the case of habituation to thirty taps with 10s ISI). We will discuss the design of the MWT and compare MWT results with those from traditional single-worm scoring. In the future, we hope to use the MWT to screen for tap habituation mutants and examine its utility for assaying individual behavior in population-based assays for chemotaxis or thermotaxis.

Rex Kerr et al. (2002) East Coast Worm Meeting "In vivo imaging of neuronal responses to touch and taste."

Massimo Hilliard, HIROSHI SUZUKI, Rex Kerr, Dan Sloan, Jian Xue, Monica Driscoll, William Schafer, Christian Frokjar-Jensen

[East Coast Worm Meeting, 2002]

Genetic analysis of behavior has provided imporant insights into the mechanisms underlying behaviors such as touch avoidance and chemotaxis. However, to fully under-stand these processes at the molecular and cellular level, it is necessary to determine how specific gene products affect the activity of identified neurons, and to correlate the activity of these neurons with behavior. Genetically-encoded optical sensors, such as the FRET-based, ratiometric calcium-sensitive protein cameleon, have many potential advantages for cell-specific non-invasive neural imaging in C. elegans; however, because of their relatively slow kinetics and small signal size, it has been difficult to use indicators like cameleon in excitable cells. We have recently overcome these hurdles and developed imaging methods that have allowed us to detect and measure in vivo calcium transients in mechanosensory touch receptor neurons in response to sensory stimulation. Using this technique, we have found that application of a mechanical stimulus near the sensory dendrite of ALM or PLM leads to a rapid increase in intracellular calcium in the process and cell body of that neuron. Recordings of calcium transients from mutants defective in the putative mechanotransduc-tion channel MEC-4 failed to show calcium influx in response to a "light touch" stimulus (a rapid poking with a flexible probe) but often showed calcium transients in response to a "harsh touch" stimulus (a slower poking with a solid probe). These results suggest that MEC-4 plays a specific role in mechanotransduction, and that the touch neurons may con-tain a mec-4-independent sensory modality involved in harsh touch detection. We have also used cameleon-based calcium imaging to detect responses to soluble repellant stimuli by the polymodal ASH sensory neurons. Large calcium transients were detected in response to several aversive soluble tastants (quinine, SDS, and copper ions) and to high osmotic strength (glycerol); as in the touch neurons, the calcium increase was rapid and the return to baseline more gradual. Altering the duration of the repellant stimu-lus strongly affected the duration but not the amplitude of the ASH calcium transient. The ASH neurons showed significant sensory adaptation in their calcium responses to both copper and glycerol; the extent of this adaptation was highly dependent on both the duration of the stimulus and the interstimulus interval. We have begun to investigate the effects of molecules thought to participate in ASH sensory transduction (OSM-9) and adaptation (TAX-6) on chemosensory calcium transients; results of these experiments will be pre-sented.

Rex Kerr et al. (2001) International C. elegans Meeting "In Vivo Optical Imaging of Neuronal and Muscle Cell Activity"

Roger Y Tsien, Rex Kerr, Christian Froekjaer-Jensen, William R Schafer, HIROSHI SUZUKI

[International C. elegans Meeting, 2001]

Genetically-encoded optical sensors, such as the FRET-based, ratiometric calcium-sensitive protein cameleon, have many potential advantages for cell-specific non-invasive neural imaging. The use of optical indicators is particularly attractive in C. elegans due to the animal's transparency, the ease with which transgenic animals can be generated, and the difficulty of electrophysiological methods. However, because of their relatively slow kinetics and small signal size, it has been difficult to use genetically-encoded sensors like cameleon in excitable cells. We have recently overcome these hurdles and developed imaging methods that have allowed us to detect and measure in vivo calcium transients in neurons and muscle cells. While developing the technique, we initially focused on detecting and measuring the calcium influx accompanying contraction of the pharyngeal muscle. We expressed various cameleons under control of the pharyngeal-specific promoter myo-2 , and imaged the fluorescence ratio emitted by the pharyngeal muscle cells. We observed prominent peaks in these ratiometric traces characteristic of calcium transients, which precisely accompanied muscular contractions and which were accompanied by an increase in FRET. Using this approach, we have made the surprising discovery that mutations eliminating the activity of UNC-36, the conserved alpha-2 subunit of the calcium channel, cause a significant increase in the magnitude of the pharyngeal calcium transient. This suggests either that the alpha-2 subunit functions in muscle channels to negatively regulate calcium influx or that it acts indirectly on the muscle by altering synaptic transmission in pharyngeal neurons. Experiments are in progress to distinguish between these hypotheses. We have also used cameleon to detect calcium transients in neurons. Direct electrical stimulation using an extracellular electrode produced reliable responses in cameleon-expressing neurons. Preliminary experiments indicate that the neurons of unc-36 animals may be less easily excited than in wild-type, suggesting that the alpha-2 subunit may promote calcium influx in neurons. We have recorded from the mechanosensory PLM neurons using cameleon driven by the mec-7 promoter and have observed responses to a train of regularly spaced mechanical stimuli. In collaboration with the Driscoll lab, we are currently analyzing the effects of mutations in the putative mechanotransduction channel MEC4/MEC10 to determine their effects on mechanically-activated neural activity. We also plan to use these imaging methods to investigate the activity patterns of interneurons receiving synaptic input from PLM. By simultaneously imaging the activity of sensory neurons and their post-synaptic partner, we hope gain insight into the mechanisms underlying the integration and processing of sensory information in these simple sensory circuits.

Rex Kerr et al. (2002) East Coast Worm Meeting "Cameleon-reported Insights into Touch Receptor Channel Function in Vivo"

Monica Driscoll, Dan Slone, William Schafer, Rex Kerr, HIROSHI SUZUKI, Jian Xue

[East Coast Worm Meeting, 2002]

Mechanical signaling, such as underlies the sense of touch, is the least understood type of signal transduction. Elegant genetic analyses of gentle body touch sensation conducted by Chalfie and colleagues have provided data supporting a molecular model of touch transduction. In this model, a candidate mechanically-gated touch-transducing channel (made up of MEC-4 and MEC-10 subunits of the DEG/ENaC channel superfamily) associates with additional MEC proteins inside and outside the cell. These contacts tether the channel to extracellular and intracellular proteins in associations that exert gating tension. A real challenge in testing the working model of touch transduction is in finding an appropriate system for electrophysiological testing of mechanosensitive gating. Expression in heterologous systems such as oocytes is problematic because several proteins, both inside and outside the cell, must contact the channel to deliver gating precise forces. Direct patching of touch neurons is also technically difficult given that tiny neurons are tightly tied to the cuticle within a specialized extracellular matrix. For this reason, we have been interested in in vivo assays of channel function, such as made possible by the work with gene-based sensors of neuronal activity such as calcium sensing cameleons. In previous work, Kerr et al. (Neuron 26:583-594) reported effective use of a cameleon to report transient calcium changes in muscle and neurons. The cameleon houses a YFP and a CFP connected by a calcium binding domain. When calcium is bound to the cameleon, a conformational change allows for fluorescence resonance energy transfer so that ratiometric measurements of YFP and CFP signals can be used to indicate changes in calcium concentrations.

Rex A Kerr et al. (2003) International Worm Meeting "Mechanosensory neuron activity in behavior and habituation."

William R Schafer, Rex A Kerr, HIROSHI SUZUKI, Katie S Kindt

[International Worm Meeting, 2003]

The gentle touch neurons in C. elegans are the primary sensory neurons that detect mechanical stimuli such as localized gentle body touch and nonlocalized tap (vibration). Habituation, a reduction in the behavioral response after repeated stimulations, is observed with each of these stimulus protocols. However, until recently, it has not been possible to assess the effect of gentle touch stimuli on the activity of the primary sensory neurons. Thus it has been difficult to determine which aspects of behavior and habituation are a result of the properties of the sensory neurons themselves, and which aspects depend on downstream interneurons. We are using the fluorescent calcium indicator protein cameleon to provide an indirect readout of the activity of the gentle touch neuron ALM. This technique has allowed us to characterize the response properties of ALM. ALM appears to function primarily as a motion sensor: a constantly moving stimulus can evoke a strong response for at least 5.0s, but application of a constant deflection of the cuticle only causes a strong response for approximately 0.5s. This observation is consistent with the effectiveness of tap in inducing a mechanosensory response; one would expect tap-induced vibrations to be effective at exciting a motion-sensor. Interestingly, ALM shows desensitization in response to repeated applications of longer (>1s) stimuli, but not to brief (0.2s) stimuli. This suggests that habituation of the behavioral response to tap may in part be due to desensitization of the sensory neurons. We will present the results of ongoing experiments comparing the timecourse and magnitude of habituation of behavior to that of desensitization of ALM. In particular, ALM appears also to desensitize in response to a single very long stimulation, a protocol that one would not expect to cause classical behavioral habituation.

Judith Jans et al. (2005) International Worm Meeting "Recruitment Elements on X (rex) Target Hermaphrodite X Chromosomes for Repression by the Dosage Compensation Complex."

Patrick McDonel, Judith Jans, Barbara J Meyer

[International Worm Meeting, 2005]

The essential process of dosage compensation ensures that males (XO or XY) and females (XX) express equal levels of X-linked gene products, despite their difference in X chromosome dose. In C. elegans, a specialized dosage compensation complex (DCC) assembles along the entirety of both hermaphrodite X chromosomes to reduce transcript levels by half. Though many components of the DCC have been defined, the molecular mechanism by which the complex recognizes X and regulates gene expression is not known. We therefore investigated the properties of X that target it for repression by the DCC. Our approach was to define the cis-acting DCC recruitment sites and the trans-acting factors that bind them directly. Using X chromosome duplications 2-3 Mb in size, we previously showed the DCC is recruited to X chromosomes by multiple cis-acting recruitment sites, which nucleate spreading of the DCC along X and thereby establish a repressive chromatin state (Science 303:1182-1185). Our recent in vivo studies using transgenes of X sequences identified ten such sites, which we named Recruitment Elements on X (rex). We mapped all of these rex sites to single cosmids, and have further refined four (rex-1, rex-2, rex-3, and rex-4) to sequences 33-411 bp in length. Sequence comparisons among rex-1 through rex-4 revealed two short (6-7 bp) conserved sequence motifs, one of which occurs in all ten rex sites. Mutation of either motif abolishes DCC recruitment to rex-1. The role of these motifs in other rex sites is under analysis. Remarkably, additional X chromosome sequences containing both motifs within close proximity also recruit the DCC in vivo. This predictive success combined with our functional analysis of mutant rex motifs demonstrates we have uncovered critical features of the cis-acting information that targets X chromosomes for repression by the DCC. We are currently investigating what additional shared properties of rex sites might collaborate with primary DNA sequence to specify X chromosome identity, and we are using the smallest rex sites to purify the trans-acting factors that bind directly to these sequences.

Ragipani, B. et al. (2019) International Worm Meeting "Specificity of long-range interactions between DCC recruitment sites in C. elegans."

Albritton, S. E., Ercan, S., Kim, J., Jimenez, D. S., Ragipani, B.

[International Worm Meeting, 2019]

Condensins are conserved ring-shaped protein complexes that are essential for development and are involved in organization of chromosomes in animals. In C. elegans, there are three types of condensins: condensin I, II, and DC. Condensin DC interacts with other proteins to make up the dosage compensation complex (DCC). DCC binds to the two X chromosomes and represses both in XX hermaphrodites by half to match the expression of the single X in XO males. The DCC binds to short genomic recruitment elements on the X (rex) from which they spread to promoters and enhancers. DCC regulates X chromosome organization in part by mediating long-range 3D interactions between rex sites. To understand the mechanisms that confer specificity to the rex interactions, we applied 4C-seq analyses in C. elegans. Next, I am using CRISPR/Cas9 to ectopically insert a rex element in two orientations. I will conduct ChIP qPCR to determine if rex sequence orientation is important for DCC recruitment and then perform comparative 4C-seq analysis to determine the dependence of 3D interactions on sequence, position, and orientation of the rex element. This information will be important for understanding the mechanisms behind condensin-mediated organization of chromosome structure.