Ting Zeng et al. (2006) European Worm Meeting "Regulation of C. elegans Behaviour by the Metabotropic Glutamate Receptors"

Ting Zeng, James Dillon, Neil A. Hopper, Vincent O'Connor

[European Worm Meeting, 2006]

Glutamate is a major neurotransmitter in the mammalian brain, signalling via ionotropic glutamate receptors, which act as ion channels, and metabotropic glutamate receptors (mGluRs), which couple to G proteins. Glutamate was first implicated as a neurotransmitter in C. elegans through the discovery of glutamate gated chloride channels. More recently, glr-1 and nmr-1, which encode AMPA and NMDA class ionotropic glutamate channels respectively in C. elegans, have been shown to regulate C. elegans behaviours such as the forward versus backing locomotory behaviour. Three mGluR-like genes [mgl-1, mgl-2 and Y4C6A.2a (referred as mgl-3)] have been identified in C. elegans. Our aim is to understand the role of mgl receptors in regulating C. elegans behaviour and we sought to test whether they are involved in behaviours known to be dependent upon glutamatergic signalling. We have obtained deletion mutants of each of the three mgl genes from the knock-out consortium. All three backcrossed mutant strains appear superficially wildtype (N2) and display apparently normal locomotion on agar and thrashing behaviour in liquid. However, mgl-1(tm1811) shows a defect in backing and moves forward for longer than the wild type when placed on unseeded NGM plates. Mutants deficient in ionotropic glutamate receptors, which back more, are known to be deficient in their ability to move towards a point food source. We hypothesized that the decreased frequency of backing in mgl-1 mutants might impact on this behaviour. However, we were surprised to note that both mgl-1(tm1811) and mgl-3(tm1766) behaved normally in this foraging assay. In contrast the mgl-2 mutants, which move normally on unseeded plates, were significantly retarded in their ability to locate and/or move towards a point food source. Further experiments in which the backward and forward movement was assayed in individual N2 and mutant strains placed on plates with or without a point food source helped delineate sub-behavior underlying this deficiency. As expected we observed that N2 and mgl-3(tm1766) worms indeed backed less as they directed themselves towards the food. This change in backing behaviour was apparent even when the worms were some distance (>5cm) away consistent with worms readily detecting food. mgl-1(tm1811) back infrequently even in the absence of food and the presence of food had little effect on this rate. Finally we found that mgl-2(tm355) worms, which backed at the same frequency as N2 worms in the absence of food, failed to alter this behavior in the presence of food. This result implies that mgl-2(tm355) worms are deficient in their ability to sense food or in their ability to integrate the detection of food to modify locomotory behaviour. We are currently using modality specific assays to understand at what level the mgl-2(tm355) phenotype might occur, as the preliminary expression patterns of mgl-2 are consistent with both hypotheses. Taken together our analysis highlights distinct neuromodulatory functions of mgl-1 and mgl-2 and provides a platform to investigate the molecular basis of the circuits and behaviours that utilize glutamatergic transmission in the worm.

Lee, S. et al. (2017) International Worm Meeting "The Novel Utility of the Central Pattern Generator within C. elegans."

Arisaka, K., Lee, S., Hydari, S., Zeng, C.

[International Worm Meeting, 2017]

Central pattern generators, or CPGs, are intrinsic neural rhythms generated in the absence of sensory information. The CPG underlies several diverse and essential behaviors, functionally streamlined and localized for their non-divorceable utility. Traditionally, the CPG has been understood unjustly in the confines of rhythmicity and motor processes. Beginning and framing the C. elegans as a foundational model for the incredible versatility of the basic CPG, we delve into the CPGs unconventional essentiality in sensorimotor integration, substantiation of the physical principles of causality and locality, and its novelty in the context of understanding more complex behaviors, such as human saccadic eye movement. We then discuss the global trends and emergent properties of such rhythms across several organisms that characterize the manifestation of a meaningful patterned neural activity, premised again, in the lens of the highly elucidative neural system of the C. elegans. Penultimately, we demarcate the morphological evolution of the neural information code and neuromodulatory vehicles that biological pressures have enforced on the increasingly complex design of organisms, starring the C. elegans, once again, as the basis and transitional prism for the evolution of this information code from an amplitude based phenomena (graded potential code) to a frequency based phenomena (action potential code). Ultimately, the thematic motif of this article demonstrates the immense utility in understanding the overlooked 302-neuron nervous system of the C. elegans towards unveiling powerful truths in the evolutionary ladder and trending homologies in more complex organisms- of particular interest here, pattern generated behaviors. Our analyses incepts and extends from a compilation of personal lab discoveries and extrinsic literary interpretations of the idiosyncrasies of the C. elegans nervous system, and has been protracted homologies across various Animalia and processes, including the aforementioned saccades in human oculomotor codes.

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.

Pereirinha, Joana et al. (2021) International Worm Meeting "Investigation of the role of the protein complex PETISCO in C. elegans embryonic viability"

Cordeiro Rodrigues, Ricardo, Ketting, Rene, Pereirinha, Joana

[International Worm Meeting, 2021]

The PIWI/piRNA pathway is a highly conserved small RNA pathway. Central to the pathway is an RNA silencing complex made of an Argonaute protein of the PIWI clade and PIWI-interacting RNAs (piRNAs), which target transposons in animal germlines. In C. elegans, piRNAs also target endogenous protein-coding genes, making the PIWI/piRNA pathway an important regulator of gene expression. C. elegans piRNAs are referred to as 21U RNAs since they are 21 nucleotides long and have a 5' bias for uridine monophosphate. We recently identified a protein complex driving 21U RNA biogenesis, which we named PETISCO (Cordeiro Rodrigues et al., 2019). However, we found that PETISCO can have different functions depending on whether it interacts with the proteins PID-1 (PiRNA Induced silencing Defective) or TOST-1 (Twenty-One U antagoniST). PETISCO:PID-1 complex participates in the 21U RNA biogenesis by interaction and stabilization of the 21U RNA precursors. While being important for gene regulation, the loss of PID-1 is not embryonic lethal. In contrast, PETISCO:TOST-1 is not involved in 21U RNA biogenesis but is nevertheless essential for embryonic development of the subsequent generation (Cordeiro Rodrigues et al., 2019; Zeng et al., 2019). The molecular function of this complex as well as the observed lethality effect is not yet understood. Based on preliminary data, we hypothesize that the PETISCO:TOST-1 complex regulates replication-dependent histones mRNA expression. In this study we want to characterize the PETISCO::TOST-1 complex and dissect its role in embryogenesis. This study would highlight the role of 21U RNA pathway factors in gene expression and C. elegans embryonic development.

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.

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.

Siddiqui, Shahid S. et al. (2011) International Worm Meeting "Fat Burning in Sleeping Worms: Regulation of Multiple Signaling Pathways by Kruppel Like Factors."

Gozal, David, Yang, Chen, Zhang, Jun, Siddiqui, Shahid S., Dhansingh, Immanuel, Hashmi, Sarwar

[International Worm Meeting, 2011]

Sleep is an important behavior across diverse species, regulated by conserved signaling pathways. OSAS (obstructive sleep apnea syndrome) is characterized by episodic hypoxia, affecting about 5% of adult American population and may lead to hypertension, if left untreated. In C. elegans post-embryonic development is accompanied by four larval stages [Sulston et al., 1983] that are interrupted by periods of inactivity called lethargus, and has been suggested to represent 'sleep' [Raizen et al., 2008]. The cAMP-CREB pathway has been shown to affect sleep-wake cycle in Drosophila, C. elegans and mammalian systems [Zimmerman et al., 2008], and cGMP dependent protein kinase expression is regulated by Rho and KLF4 [Zeng et al., 2006]. Genetic analysis has suggested the role of LIN-3 and LET-23 (the EGF like-ligand and EGF receptor, respectively) in sleep like behavior in C. elegans [Van Buskirk and Sternberg, 2007]. Overexpression of LIN-3 results in quiescent behavior during periods of normal activity, through the activity of ALA neuron that is regulated by paired (a family of Kruppel like transcription factors) and LIM class of transcription factors CEH17, CEH-14/Chx10, and CEH17/Phox2 through a feedback regulatory pathway [Van Buskirk and Sternberg, 2010]. We have shown the role of KLF-3 in beta oxidation of fatty acids in C. elegans that results in accumulation of fat in intestinal cells in klf-3(ok1975) homozygous loss of function mutants, arrested development and poor fecundity [Hashmi et al., 2008; Zhang et al. 2011 submitted], and may also affect sleep cycle by affecting the lethargus timings during larval molts. Interestingly, KLF4 in mammals not only mediates adipogenesis but also regulates kallistatin expression [Shen et al., 2009] that regulates kidney protein expression during episodic hypoxia (EH), by inhibiting vasodilation mediated by kallikrein-Kallistatin signaling pathway [Thonhboonkered et al., 2002]. The sleep regulating egl-4 gain of function mutation in cyclic GMP also results in the accumulation of fat in the intestine [Raizen et al., 2006]. Thus accumulating results suggest that klf-3 signaling pathway analysis in C. elegans may elucidate fat utilization (see the abstract by Hashmi et al., this meeting), sleep, and reproductive behavior.

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.