David M Raizen et al. (2003) International Worm Meeting "Long-term observations of L1 rest/activity cycles"

Joan C Hendricks, Allan I Pack, David M Raizen, Meera Sundaram

[International Worm Meeting, 2003]

Diurnal environmental cues consisting of light and temperature changes play an important role in control of animal behavior. Behavior of food-deprived L1s, a long-lasting developmental state, may be particularly adaptive to daily changes in conditions, and may therefore be a useful system to study diurnal changes in behavior. Using time-lapse video analysis of L1s in the absence of food, we have been observing movements of L1 for several days. We transfer ~3000 worms in solution onto an agar surface of a 3 cm plate. We videotape the worms at 30-minute intervals for 2 minutes under red light, and then analyze the videos in time-lapse mode. For some experiments, the worms are exposed to white light for 12 hours alternating with darkness for 12 hours. We have observed two distinct behavioral states. In one state, the worms are swimming rapidly across the agar surface with few changes in direction. This state is reminiscent of the roam state defined for adult worms in the presence of food by Fujiwara et al (Neuron 02). In the other state, which is reminiscent of the dwell state defined by Fujiwara et al, the worms make little to no net movement over the two minutes of observation. We define worms that had made at least one net body length movement in the two-minute observation bin as active and other worms as resting. L1s populations remain active continuously for the first day after transferring from solution onto an agar surface. After approximately one day of continuous activity, there is decay in worm activity over the next several days. This decay in activity is unlikely to be explained simply by fatigue or sickness of the worms for the following reasons: (1) the rest state is not permanent. (2) Survival of L1s maintained on an agar surface in the absence of food does not drop significantly until 7-10 days after plating, yet activity drops significantly by the second day after plating. (3) After spending a week on one agar plate, the fraction of active worms drops to approximately 0.3. If these worms are then washed off this plate and transferred to a fresh plate, the fraction active increases immediately back to nearly 1.0. We suggest that this activity decay process reflects habituation to a novel environment. Our second observation of activity relates to the rest/activity patterns within each day. We have found the when the day is divided into 12 hours of light and 12 hours of darkness, worm activity is significantly higher during the light (L) phase than during the dark (D) phase. FFT analysis of rest/activity cycles reveals a peak at a period of 24.5 hours, indicating that there is a diurnal rhythm of rest/activity under L:D conditions.

Conroy, Brian et al. (2013) International Worm Meeting "The neuronal basis of food choice behavior in Caenorhabditis elegans."

Morabe, Maria, Chambers, Melissa, Conroy, Brian, Macfarlane, Rachel, Haynes, Lillian, Glater, Elizabeth

[International Worm Meeting, 2013]

Caenorhabditis elegans uses chemosensation to distinguish among various species of bacteria, their major food source (Ha et al., 2010; Shtonda and Avery, 2006). Although the neurons required for the detection of specific food-odors have been well-defined (Bargmann, 2006), less is known about the sensory circuits underlying the discrimination among the mixtures of odors released by bacteria. We plan to examine the neural machinery underlying bacterial preference among a diverse set of bacterial species. Does bacterial choice use one common neuronal mechanism or a diversity of mechanisms depending on the bacteria? Do some bacterial choices involve a single sensory neuron and others involve multiple sensory neurons? To address these questions, we are testing the food preferences of C. elegans for bacteria found in their natural habitats (kindly provided by Marie-Anne Felix, Institut Jacques Monod, Paris, France). We have found that C. elegans strongly prefers the odors of Providencia sp., Alcaligenes sp., and Flavobacteria sp., to Escherichia coli HB101, a commonly used food source for C. elegans. We have identified that the olfactory neuron AWC is necessary for this preference. We intend to test whether other amphid sensory neurons are also necessary for bacterial preference. In the future we will extend our analysis to other bacterial species to determine the diversity of the underlying neuronal mechanisms. Bargmann, C.I. (2006). http://www.wormbook.org. Ha, H.I., Hendricks, M., Shen, Y., Gabel, C.V., Fang-Yen, C., Qin, Y., Colon-Ramos, D., Shen, K., Samuel, A.D., and Zhang, Y. (2010). Neuron 68, 1173-1186. Shtonda, B.B., and Avery, L. (2006). J Exp Biol 209, 89-102.

Donato, A. et al. (2015) International Worm Meeting "Molecular and cellular mechanisms regulating axonal compartmentalization in C. elegans RIA interneurons."

Ren, J., Donato, A., Zhang, Y., Hilliard, M. A.

[International Worm Meeting, 2015]

Neurons are highly compartmentalized cells consisting of three main structural and functional domains, cell body (soma), axon, and dendrites. In the last decade, increasing experimental evidence has revealed that additional levels of compartmentalization are in place within the processes, providing higher computational potential than previously thought. However, little is known on the molecular and cellular mechanisms that regulate the establishment of these subcellular compartments during development and their maintenance during the lifetime of the animals. A recent study from Yun Zhang's laboratory has shown that the unipolar RIA interneurons display distinct compartmentalized activity in different axonal domains. Remarkably, calcium activity in specific axonal domains is correlated with the direction of the dorsal-ventral head bending and the compartmental activity regulates the amplitude of the head bending (Hendricks et al., 2012). These results reveal the properties and function of RIA axonal compartmentalization. Here, we propose to elucidate the underlying molecular and cellular mechanisms. First, we tested the hypothesis that the functional compartmentalization of RIA results from compartmentalized localization of the muscarinic acetylcholine receptor GAR-3. Previously, it has been shown that the axonal compartmentalized activity of RIA requires the synaptic output of the cholinergic head motor neurons SMDD/V and the function of GAR-3 in RIA (Hendricks et al., 2012). Thus, we analysed the distribution of the GAR-3 receptor in RIA, and found that its localisation is diffused and does not correlate with compartmentalized calcium activity, suggesting different underlying mechanisms. Second, we tested the hypothesis that specific localization of mitochondria in RIA may regulate RIA compartmental activity by buffering calcium waves in certain axonal domains. We found that mitochondria distribution in RIA is not consistent with a potential role in generating calcium compartmentalisation in this cell. Finally, using the GFP reconstruction across synaptic partners (GRASP) (Feinberg et al., 2008), we have specifically labelled the synaptic contacts between SMDs and RIAs, which will allow us to investigate if the calcium compartmentalisation is governed by the precise synaptic organisation in the RIA axon and how changing in the synaptic organisation can affect neuronal activity and therefore the animal behaviour. .

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Nishida, Y. et al. (2013) International Worm Meeting "Design of the microchip device to dissect the neural circuit based on thermotactic behavior in C. elegans."

Kobayashi, K., Jaehoon, J., Nakajima, M., Takeuchi, M., Fukuda, T., Mori, I., Nishida, Y.

[International Worm Meeting, 2013]

Animals cope with environmental stimuli by altering behaviors. Previous studies have characterized the thermotactic neural circuit, in which temperature is sensed and processed through the coordination of thermosensory neurons AFD and AWC, and interneurons AIY, AIZ and RIA in C. elegans (Mori et. al., 2007). This simple circuit is profitable to dissect neural activities of the respective neurons in response to temperature stimuli. However, it is unknown how the activities mediate the behavior to cope with environmental temperature. The recent study shows that RIA interneurons, which receive sensory inputs, encode head movement (Hendricks et. al., 2012). This finding provides a clue to tie the neural activities to not only the thermosensory inputs but also the behavioral outputs.In this study, we aim to study how the thermotactic neural circuit processes the temperature and mediates the behavior by monitoring the neural activities of the respective neurons for thermotactic behavior. We are now designing the microchip device to control both temperature stimuli and head movements. First, we are optimizing the shape of microfluidic channel for stable fixation of the animals' body to ensure free head movements. We are planning to fabricate a device for handling head movements by manipulation of a micro-probe attached to head region of the animals. We are also fabricating temperature control system to present the animals with accurate temperature stimuli in the microchip device. Our study surely supports understanding how temperature is sensed and processed through the neural circuit to mediate thermotactic behavior.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.

Kiontke, Karin C. et al. (2009) International Worm Meeting "New Caenorhabditis species: phylogeny and evolution."

Fitch, David H. A., Felix, Marie-Anne, Kiontke, Karin C.

[International Worm Meeting, 2009]

Recently, nine new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to nineteen, eleven of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10 and 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Although none of them is the sister species of C. elegans, C. sp. 5 and C. sp. 9 are close relatives of C. briggsae. C. sp. 9 can hybridize with C. briggsae in the laboratory. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. Three of these species, C. sp. 7, C. sp. 9 and C. sp. 11 have been chosen for genome sequencing. Four further new species branch off before C. japonica within a monophyletic clade which also comprises C. sp. 3 and C. drosophilae. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and seven from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. Other characters, like the shape of the stoma and the male tail, introns, susceptibility to RNAi and genome size are being evaluated in the context of the phylogeny. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last few years. There is no indication that we are even close to knowing all species in this genus.

Xiaodong Wang et al. (2003) International Worm Meeting "Evolution of a novel gene expression pattern in C. elegans"

Xiaodong Wang, Helen M Chamberlin

[International Worm Meeting, 2003]

Previous studies have shown that C. elegans ovo-related gene lin-48 expresses in a small number of cells including the excretory duct cell. In the related species C. briggsae, the expression is conserved in all cells except the excretory duct. This lin-48 expression difference affects excretory duct morphogenesis. In C. briggsae, as well as in C. elegans lin-48(sa496) mutants, the excretory duct is more anterior than in C. elegans wild type. This indicates that C. elegans lin-48 (Ce-lin-48) is involved in duct morphogenesis and positioning, but this gene function is absent in C. briggsae (1). We have made reporter transgenes composed of the lin-48 regulatory sequences from C. elegans or C. briggsae driving expression of green fluorescent protein (GFP). Tests of these clones in each species showed that only the Ce-lin-48 is expressed in excretory duct cell in C. elegans animal. These results indicate that there are differences in both cis-regulatory sequences and trans-acting proteins between the two species. By creating chimeric reporter transgenes including C. elegans and C. briggsae regulatory sequences, we have found that one difference between the two species is the presence of regulatory sequences in Ce-lin-48 that respond to the bZip protein CES-2 (1). The lin-48 gene expression differences between C. elegans and C. briggsae could result from loss of excretory duct expression in the C.briggsae lineage or acquired expression in the C. elegans lineage. To distinguish between these possibilities, we have analyzed three additional Caenorhabditis species (C. remanei, C. sp. CB5161 and C. sp. PS1010). We found these species have a duct morphology similar to C. briggsae indicating the C. elegans morphology is unique to this species. For comparison to C. elegans and C. briggsae, we have isolated the lin-48 gene from C. remanei and C. sp. CB5161. Alignment of the lin-48 regulatory sequences reveals that the sequences are more conserved among C. briggsae, C. remanei and C. sp. 5161. Several conserved domains are absent from C. elegans, whereas the previously identified CES-2 binding sites are absent from the other species. Currently, we are creating lin-48::gfp reporter transgenes for each species to observe the gene expression patterns. Further experiments with these transgenes will allow us to test whether the differences between C. elegans and the other species result from a loss of repressor elements or gain of activator elements in the C. elegans gene. (1)X. Wang and H. M. Chamberlin (2002) Genes & Development 16: 2345-2349.