Burr, A.H.Jay (2017) International Worm Meeting "What is different about the cells comprising the C. elegans stoma and pharynx?"

Burr, A.H.Jay

[International Worm Meeting, 2017]

In a recent article it was shown that the architecture of the tube of epithelial cells that comprise the stoma and pharynx of C. elegans is basically the same as for other nematodes: the sequential order and characteristics of the cell classes are conserved (Burr and Baldwin 2016). The row of radial cell classes pm1-pm5 are paired within each interradial sector and in this way are distinct from the single, unpaired radial cells e1, e3, pm6 and pm7. Immunofluorescence labeling (MH27) showed that marginal cells e2 and mc1 are not separated by the encircling pm1 syncytium, but share a common circumferential apical junction. Here I will add TEM evidence that the encircling pm1 cell syncytium passes peripheral to the adjoining e2 and mc1. Marginal cells e2, mc1, mc2 and mc3 form a continuous row and border with the same classes of radial cells in most nematodes. In this paper I will compare certain features of the pharyngeal cells in C. elegans and closely related taxa that are different from other nematodes. 1) The unpaired radial cells e1 and e3 are not muscles in C. elegans and other taxa within Rhabditina, but do express muscle cytoskeleton in most other nematode taxa. To provide a terminology consistent across Nematoda, the pharyngeal 'epithelial' and 'muscle' cells of the C. elegans pharynx would better be distinguished by topological terms such as 'unpaired radial' cells (e1, e3, pm6-7) or 'paired radial' cells (pm1-5), and 'marginal' cells (e2, mc1-3), rather than by the confusing functional terms. 2) In C. elegans the paired radial cells of the pharynx become fused shortly after hatching or molting - a segment of the apposed plasma membranes that normally separate the pairs is dissolved (Shemer et al. 2004). However the muscle cytoskeleton remains paired. Fusion of these cells occurs only in two clades within the suborder Rhabditina: in Eurhabditis, which includes C. elegans, and in a clade within Diplogasteromorpha. 3) In C. elegans the six pm1 cells are fused within and between sectors to form a syncytium that encircles the stoma (Albertson and Thomson, 1976). Circumferentially fused radial cells have rarely been reported in other taxa. An interesting question: What are the functional advantages of these special features? Albertson and Thomson 1976. Philos Trans R Soc Lond B 275:299-325. Burr and Baldwin 2016. J Morphology 277:1168-86. Shemer, Suissa, Kolotuev and Hall 2004. Curr Biol 14:1587-91.

Leung K et al. (1996) West Coast Worm Meeting "REVERSAL RESPONSES TO LIGHT IN C. ELEGANS: RESULTS WITH MUTANTS"

Leung K, Cheng B, Burr AHJ

[West Coast Worm Meeting, 1996]

In an previous study (Burr, 1985), it was reported that for monochromatic stimuli ranging from 420-680 nm, reversal bouts were elicited at five wavelengths from 520-600 nm (sample sizes were 200-400). At saturating intensities, the 10 s stimulus increased the probability of a reversal to 0.21 from a 0.12 background level due to spontaneous reversals. This increment corresponds to one response elicited for every 11 light presentations, on average. With such a low probability of response on top of a spontaneous background, a large sample size and paired statistics is required to achieve significance. To facilitate further investigation, we have developed a computer-video automated tracking system which scores reversals of up to 23 worms simultaneously and we here report the first results obtained with this system. Time resolution was one second, and sample sizes were on the order of 1000. Habituation was negligible over 15 cycles with 50 s of darkness alternating with a 10-second broad-band (360-610 nm) stimulus at saturating intensity. For statistical analysis, the 10 s stimulus period was paired with the preceding 10 s of darkness. Most responses occurred 1-7 s after light onset with a peak probability at 3 s. The incremental probability of a reversal was of the same order as observed previously. In both this and the earlier study, filtering the stimulus beam with 5 cm water and blocking filters is aimed at eliminating any far- and near-infrared light that could heat the worm or arena. That this was accomplished was supported by tests and analysis in the previous study. To confirm that the response was to light, not a temperature change, we repeated the above experiment under identical conditions except for an additional filter which blocked all wavelengths shorter than 630 nm light while transmitting infrared. The incremental response during the shutter-open period was eliminated, confirming that light elicits reversals. We have initiated experiments with mutants aimed at narrowing the possible candidates for the light-sensitive receptor neuron. So far, we have found that light sensitivity is retained in ttx-1 and lost in daf-19. Considering the known morphological effects of these mutations (Perkins et al., 1986), it would appear that the structural integrity of the fingers in AFD is not essential for light sensitivity but the presence of cilia in one or more ciliated sensory neurons probably is. Burr (1985) Photochem. Photobiol. 41: 577-582. Perkins, Hedgecock, Thomson & Culotti (1986) Dev. Biol. 117:456-487.

Boom J et al. (1991) International C. elegans Meeting "OPSIN-FAMILY GENES OF CAENORHABDITIS ELEGANS."

Burr AHJ, Boom J, Hayes M

[International C. elegans Meeting, 1991]

Hoping to find a gene for the visual pigment responsible for the light sensitivity of C. elegans (Burr, 1985), we have cloned 5 unique genomic fragments which hybridize with cDNA probes derived from the Drosophila ninaE opsin gene CThis is one of four opsin genes expressed in Drosophila eyes). A 1.6 kb cDNA probe provided by Charles Zuker was used to screen a C. elegans genomic library in Lambda Charon 4 phage ( made by Terry Snutch) at moderate stringency (62 C; 1 x SSPE). Thirteen positive phage were grouped into 5 unique classes (designated s401-s405) based on the restriction patterns produced by combined digestion with EcoRI and HindIII. The digests were labelled by either of the two PstI fragments of the coding region of the probe. The 5 classes mapped to widely separated cosmids of the C. elegans genome ( courtesy of Alan Coulson and John Sulston). Since hybridization occurred at moderate stringency levels, there appears to be a greater sequence homology between the ninaE and C. elegans opsin genes than between ninaE and many visual pigment opsin genes for which lower stringency levels are necessary for cross-hybridization. It is also possible that the genes we have isolated could be genes of other members of the family of opsin-like receptor proteins. These include hormone, neurotransmitter and pheromone sensors. However, in investigations of vertebrates these others do not cross-hybridize with opsin genes. We plan to identify the function of the five genes by obtaining their sequence and studying their expression and hope to have some sequence to report by meeting time.

A H Jay Burr et al. (2001) International C. elegans Meeting "Light-increased reversal frequency and spontaneous reversal frequency are affected by starvation."

Ann Chan, Wendell Challenger, A H Jay Burr

[International C. elegans Meeting, 2001]

C elegans was shown previously to reverse direction in response to light (Burr 1985). In that study, 40-80% increases in reversal frequency over the background level varied with light intensity and wavelength in a normal way and were shown not to be due to radiant heating. Because large sample sizes are needed in order to detect small increases on top of the fluctuating frequency of spontaneous reversals, we have developed a computer automated tracking system that makes experimentation possible in a reasonable length of time. Under continuous near-infrared illumination, video images are digitized once each second and processed to find the new coordinates of up to 23 worms. Another program identifies reversals from the coordinates along the track. These are counted during a 10 s light period (1.6 x10 4 uW cm -2 blue-white broadband light) and a preceding 10 s dark period in each 40 s measurement cycle. There are 5 cycles per 200 s run. The number of reversals per 10 s measurement period, averaged over all worms and cycles during a run, counts as one independent measurement. Data are analyzed as a generalized randomized complete block design using the Proc Mixed procedure of SAS version 8. For detecting the effect of light, the difference in reversal frequency between the paired light and dark periods is analyzed in the same way. A new sample of worms was used for each run. About 30 synchronous six day-old, well-fed worms were washed off an OP50 culture plate and sedimented twice through behavior buffer (5.5 mM Tris + 34 mM NaCl). The worms were transferred in a drop of buffer to the edge of a 50-mm behavior plate and the plate was rotated to distribute the worms around the edge. The behavior plates were prepared by making 1.8% agar in 90% behavior buffer then drying to 90% of poured weight. Just before using, about 100 uL distilled water was distributed around the edge and allowed to soak in. The resulting lower osmolarity at the agar perimeter discouraged the worms from crawling near the edge. Worms were allowed to crawl into the 22x28 mm field of view and were tracked within 30-50 min of removal from the food. All procedures were carried out at 19 C. Sensitivity of the system under actual conditions was estimated by simulation, starting with data from an experiment with fed worms during which the light beam was blocked. Reversals were added to the data during the 'light' periods at frequencies of 0.01 to 0.10 per 10 s period (perP), and the data were reanalyzed. The increase in reversal frequency became significant at 0.050 perP (P = 0.037, n = 21 runs), a 45% increase over the background (dark period) frequency. A greater sensitivity would be expected at higher sample sizes. In all our experiments with well-fed worms, light had no significant effect on reversal frequency. To investigate the influence of starvation, we pooled worms from several food plates and put half on a food-free plate and half on a fresh food plate. After a starvation period, the fed and starved worms were tested in alternate runs. Light increased reversal frequency significantly in worms starved 20 h (+0.040 perP, +62%, P=0.049, n=51), but not significantly in worms starved 6 h (+0.013 perP, +12%, P = 0.4, n = 39). In both experiments, light had no significant effect on the fed controls (-0.023 perP, -15%, P = 0.2, n = 58 and -0.015 perP, -9%, P = 0.4, n = 42). In addition to being more responsive to light, starved worms had a significantly lower background (dark period) frequency. For the 0 h, 6 h and 20 h starved worms, average dark frequency (+/- SE) was 0.15 (0.013) perP, 0.11 (0.013) perP and 0.064 (0.012) perP, respectively. Thus, both background reversal frequency and responsiveness to light appear to be affected by starvation. In the previous study in which light responses were observed (Burr 1985), the worms may inadvertently have been starved.