Tetsuhiro, Kikuchi et al. (2011) International Worm Meeting "Mutations Affecting Cessation of Gonadal Leader Cell Migration in C. elegans."

Kiyoji, Nishiwaki, Yukimasa, Shibata, Tetsuhiro, Kikuchi

[International Worm Meeting, 2011]

Organs are often formed by elongation and branching of epithelial tubes. Although regulation of initiation and direction of epithelial tube migration is an important aspect in organogenesis, there must be mechanisms that terminate epithelial migration to generate organs of appropriate size. These mechanisms remain mostly unexplored, however. The gonadal development in C. elegans offers a simple model system to study the morphogenesis by epithelial tubes. The migration of the anterior and posterior gonadal leader cells called distal tip cells (DTCs) promote directional elongation of gonad arms during larval development and form a pair of U-shaped gonad arms in rotational symmetry. We placed Venus under the control of the DTC specific mig-24 promoter and fluorescently visualized DTCs. In the wild type adult worms, two DTCs stopped around the dorsal side of the vulva. We isolated two mutants, tk102 and tk107 by EMS mutageneis. About 80% of DTCs in these mutants overshot the vulval region, but fewer than 10% of DTCs did in the wild type. Both of these mutants are recessive and they partially complement each other. These mutants seem not to affect growth rate, animal size and behavior. To understand the difference in migration modes of DTCs between the wild type and the mutants, we analyzed the timing of DTC turns and migration speed of DTCs. We found that DTCs in these mutants make two turns at the same timing as in the wild type, but that the deceleration rates of mutant DTCs after the second turn were decreased compared to the wild type. Thus the DTCs in the mutants fail to stop at the normal timing. Interestingly, we found that the expression of mig-24::Venus, which is normally weakened after stopping of DTCs at the young adult stage, was retained to be strong even in the 2-day-old adults in tk107. mig-24 encodes a transcription factor of the bHLH family. Genetic mapping experiments assigned tk102 and tk107 to 2473kb and 1853kb overlapping regions of the chromosome III, respectively. Deep sequencing analysis revealed a nonsense mutation in the predicted gene F42H10.5 in the tk107 genome. RNAi knockdown of this gene resulted in an overshoot phenotype similar to that observed in tk107. We also successfully rescued tk107 by a fosmid clone. F42H10.5 appears to encode a Zink-finger DNA-binding protein having weak homology to ZBED4 in mammals. Therefore, it is possible that F42H10.5 is a negative transcriptional regulator of mig-24 which could act in promotion of DTC migration.

Kikuchi, Taisei et al. (2021) International Worm Meeting "An alternative ERGO-1 pathway in a sibling species of C. elegans, C. inopinata"

Sugimoto, Asako, Kikuchi, Taisei, Tanaka, Ryusei, Yoshida, Akemi, Sun, Simo, Hunt, Vicky

[International Worm Meeting, 2021]

The recent discovery of Caenorhabditis inopinata, a sibling species of C. elegans, provides an opportunity to investigate the conservation and diversification of small RNA (sRNA) pathways in two closely related species. We report that the microRNA, PIWI and male-expressed ALG-3/4 siRNA pathways are highly conserved in C. inopinata and C. elegans. However, C. inopinata has lost key components of the female-expressed ERGO-1 siRNA pathway associated with the clearance of transcripts. C. inopinata do not express the ERGO-1 associated 26G primary siRNAs and have similar sRNA expression profiles to C. elegans ergo-1 and eri-6 mutants. However, expression of the downstream secondary 22G siRNAs is retained in C. inopinata. We have identified a class of 22U siRNAs that are candidates for an alternative ERGO-1 siRNA. The 22U loci overlap with 22Gs and target similar genes but, unlike C. elegans 26Gs, are not dicer-processed. We also identified an Argonaute protein with an expression profile similar to the C. elegans ERGO-1, as a candidate for an alternative ERGO-1 Argonaute protein. Our results indicate that there has been a partial loss of the ERGO-1 pathway in C. inopinata and an alternative ERGO-1 pathway has evolved.

Tetsuhiro Kikuchi et al. (2010) East Asia Worm Meeting "Mutations Affecting Cessation of Gonadal Leader Cell Migration in C.elegans"

Kiyoji Nishiwaki, Tetsuhiro Kikuchi

[East Asia Worm Meeting, 2010]

Organs are often formed by elongation and branching of epithelial tubes called branching morphogenesis. Although the mechanisms by which branches are formed and elongated are well studied, those acting in cessation of elongation, which should be essential for generation of proper sized organs, are mostly unexplored. The gonadal development in C.elegans offers a simple model system to study the morphogenesis by epithelial tubes. The migration of the anterior and posterior gonadal leader cells called distal tip cells (DTCs) promote directional elongation of gonad arms during larval development and form a pair of U-shaped gonad arms in rotational symmetry. We placed Venus under the control of the DTC specific mig-24 promoter and fluorescently visualized DTCs. In the wild type adult worms, two DTCs stopped around the dorsal side of the vulva. In this study, we tried to isolate mutants defective in cessation of DTC migration using Ethylmetanesulfonate (EMS) mutagenesis. We successfully identified two mutants, tk102 and tk107. About 80% of DTCs in these mutants overshot the midbody region, but fewer than 10% of DTCs did in the wild type. Both of these mutants are recessive and failed to complement each other, suggesting that they are loss-of-function mutations in the same gene. These mutants seem not to affect the growth rate, animal size and behavior. To understand whether DTCs in tk102 migrate faster than those in the wild type, we analyzed the timing of first and second turns of DTCs relative to the vulval development. We found that DTCs in tk102 make two turns at the same timing as in the wild type. Thus, it is possible that the migration of DTCs in tk102 is affected after the second turn of DTCs. Genetic mapping experiments assigned tk102 and tk107 to 4312 kb and 5101kb overlapping regions of the chromosome III, respectively.

Kikuchi, T. et al. (2017) International Worm Meeting "Evolutionary insights into the C. elegans biology from the morphology, ecology and genome of the sister species Caenorhabditis sp. 34."

Wang, J., Kanzaki, N., Kikuchi, T., Sugimoto, A., Tanaka, R., Berriman, M., Woodruff, G., Tsai, I., Sternberg, P.

[International Worm Meeting, 2017]

One of a few missing pieces of C. elegans to be an exemplary model organism is a 'sister' species which provide deep evolutionary interpretations of identified biological phenomena. C. briggsae is the most widely used satellite model, but at the level of DNA sequence, this species is as divergent from C. elegans as humans are from mice. Other species including C. brenneri and C. remanei have similarly long phylogenetic distances from C. elegans. The gonochoric nematode C. sp. 34, recently isolated from fresh syconia of the fig Ficus septica in Ishigaki Island, Japan, is the long-sought sister species of C. elegans. C. sp. 34 shares typological key characters with other Elegans supergroup species, however the body size is much larger as the adults have nearly twice body length of those of C. elegans. C. sp. 34 occurs mainly in fresh syconia and are vectored by fig wasps. The C. sp. 34 genome assembly was produced using multiple technologies and manually finished into six chromosomes. The 123 Mb assembly is now the second nematode species after the C. elegans for which sequence information comprise solely in chromosomes. By sequencing the males and females of C. sp. 34, we have shown C. sp. 34 possess a XO sexual system. A total of 21,609 gene models were predicted in the nuclear genome and orthologues clustering was inferred between C. sp. 34 and 9 other Caenorhabditis species, confirming its close relationship to C. elegans. Divergence time estimation placed the separation of the two species ~120 million generations ago. LTR retrotransposons are highly expanded in the C. sp. 34 genome, which may be one of driving forces of the drastic evolution of C. sp. 34. On the other hand, gene losses in 7TM GPCR families are clear in the genome, reflecting the characteristic lifecycle. Considering the significant differences in body size, developmental processes, behaviours and lifestyles between the two species, our study on C. sp. 34 provides an outstanding platform to perform comparative evolutionary biological studies.

Sugimoto, Asako et al. (2017) International Worm Meeting "Establishing genetic techniques to study Caenorhabditis sp. 34, a sister species of C. elegans."

Kanzaki, Natsumi, Hoshi, Yuki, Kumagai, Ryohei, Sugimoto, Asako, Kikuchi, Taisei, Namai, Satoshi, Tsuyama, Kenji

[International Worm Meeting, 2017]

Caenorhabditis sp. 34 is a sister species of C. elegans recently isolated from the syconia of the fig Ficus septica on Ishigaki Island, Japan (see abstract by T. Kikuchi, et al.). C. sp. 34 is gonochoric and shares typological key characters with other Elegans supergroup species, but strikingly, adults are nearly twice as long as C. elegans. The optimal culture temperature for C. sp. 34 is significantly higher (27 deg C) than that of C. elegans (20 deg C). Young adult males and females tend to form clumps, and Dauer larvae are rarely observed in laboratory culture conditions. Recently the C. sp. 34 genome assembly was produced into six chromosomes (see abstract by T. Kikuchi, et al.). The marked differences from C. elegans in morphology, behaviors and ecology, and the availability of the complete genome sequence make C. sp. 34 highly attractive for comparative and evolutionary studies. To make C. sp. 34 genetically tractable, we have been developing genetic and molecular techniques and tools. Stable transgenic lines of C. sp.34 could be obtained by microinjecting marker plasmids commonly used in C. elegans, although the efficiency was lower than that in C. elegans. Both soaking and feeding RNAi was as effective as in C. elegans. A panel of antibodies against C. elegans proteins successfully recognized expected structures in C. sp. 34 by immunofluorescence. Thus, many of the rich genetic and molecular resources for C. elegans can be directly used for C. sp. 34 studies. We well present some of the comparative analyses of gene functions regarding the body size, germ cell formation and sex determination.

Stevens, Lewis et al. (2019) International Worm Meeting "Nothing in Caenorhabditis biology makes sense except in the light of evolution: The Caenorhabditis Genomes Project."

Blaxter, Mark, Stevens, Lewis

[International Worm Meeting, 2019]

Caenorhabditis elegans is one of the preeminent model organisms in modern biology, but only recently have we started to understand the details of its natural ecology and evolutionary history. Studying C. elegans alongside its closest relatives provides an important evolutionary context for the origins and constraints that have resulted in the particular instance analyzed in the laboratory. A focussed search for new Caenorhabditis species over the last decade has led to the discovery of over 50 species of Caenorhabditis. As part of an international collaboration, the Caenorhabditis Genomes Project (CGP), we have sequenced the genomes of all Caenorhabditis species currently in culture. We exploit these new genome sequences to perform the most comprehensive reconstruction of the Caenorhabditis phylogeny to date, providing an essential evolutionary framework for downstream analyses. We reveal extensive variation in genome size in the genus, ranging from the 48 Mb genome of C. drosophilae to the 160 Mb genome of C. vivipara. Investigating the origins of this variation, we find that protein-coding gene number is highly correlated with genome size, with C. drosophilae possessing just 13,000 genes due to extensive gene loss. Other genomic features, such as repeat (mobile element) proliferation also contribute to genome size changes. Among the non-coding portions of these genomes, we identified rapid and extensive intron loss across the genus, including in the group that includes C. elegans. We demonstrate the utility of these genome sequences for C. elegans research by analyzing the evolutionary history of key signaling pathways, revealing unexpected complexity. We have made these genomic resources, including genus-wide orthology sets, publicly available via the CGP website (http://www.caenorhabditis.org) which includes a dedicated genome browser and a BLAST server. These new species and their associated genomic resources add to the arsenal of tools available to the C. elegans researcher to interrogate biology of this important nematode. The CGP is a collaboration between the following labs: Blaxter (Edinburgh), Felix (Paris), Ailion (Washington), Andersen (Northwestern), Braendle (Nice), Cutter (Toronto), Fitch (NYU), Fierst (Alabama), Kikuchi (Miyazaki), Rockman (NYU), Schwarz (Cornell), Wang (Academia Sinica).

Mongan NP et al. (1997) International C. elegans Meeting "AN EXTENSIVE NICOTINIC ACETYLCHOLINE RECEPTOR GENE FAMILY IN CAENORHABDITIS ELEGANS."

Baylis HA, Mongan NP, Sattelle DB

[International C. elegans Meeting, 1997]

Although the nicotinic acetylcholine receptors (nAChRs) are the best characterized ionotropic neurotransmitter receptors, the extent of the molecular and functional diversity of the nAChR gene family is not known for a single organism. A number of C. elegans nAChR subunit genes have been identified previously using genetic approaches [lev-1(non-a),unc-38(a),unc-29(non-a),deg-3(a)] and cross-species probing of C. elegans cDNA libraries [acr-2(non-a),acr-3(non-a),and Ce-21(a)]. nAChR a-subunits may be distinguished from all other ionotropic receptor subunits by the presence of adjacent cysteines at positions equivalent to 192,193 in the Torpedo a-subunit. We have applied an RT-PCR strategy to confirm the expression of 8 novel putative nAChR a-subunits predicted by GENEFINDER. Analysis of the regions of the a-subunit considered to (a) contribute to the acetylcholine binding site and (b) the channel lining, has revealed a diversity which may underlie distinct functional roles for members of this multi-gene family.

C Huang et al. (1999) International C. elegans Meeting "Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis"

WG Wadsworth, C Huang, PD Yurchenco

[International C. elegans Meeting, 1999]

Laminin is a heterotrimeric glycoprotein composed of a , b , and g subunits. The genome sequencing project reveals two a , one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin a A and laminin a B, are most similar to the vertebrate a 1/ a 2 and a 5 chains respectively, while the b and g resemble the b 1/ b 2 and g 1 chains. Thus, there are two predicted laminin trimers, a A bg and a B bg . Mutations in epi-1 , which encodes laminin a B, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin a A and a B. By immunostaining, both first appear at gastrulation between germ layers. However, laminin a A is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin a B is more posteriorly localized. Laminin a B becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin a A accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin a A is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3 , which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin a B has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin a A is restricted to specialized membranes and is required for the morphogenesis of specific tissues.

Yanjue Wu et al. (2005) International Worm Meeting "EGb 761 INHIBITS AMYLOID BETA OLIGOMERIZATION AND TOXICITY IN TRANSGENIC CAENORHABDITIS ELEGANS"

Christopher D Link, Yves Christen, Astrid G Zepeda, Yanjue Wu, Zhixin Wu, Yuan Luo

[International Worm Meeting, 2005]

Amyloid peptide aggregation has been postulated to link oxidative stress and neurodegeneration seen in Alzheimer's diesease (AD). We have previously demonstrated that the standardized Ginkgo Biloba extract, EGb 761, inhibits A aggregation in vitro and attenuates the expression of a stress response gene small heat shock protein (hsp 16-2) in Caenorhabditis elegans. In this study, we use EGb 761 as a pharmacological modulator to associate A species with the levels of oxidative free radicals and with A-induced toxicity in an A-expressing transgenic C.elegans model, which can express A in muscle cells after temperature up-shift. We observed that EGb 761 alleviated A-induced toxicity which correlated with its ability to specifically inhibit A oligomerization. Furthermore, EGb 761 reduces intracellular levels of hydrogen peroxide in the transgenic C.elegans. These findings suggest that A oligomers and A-induced oxidative strss are crucial for A toxicity, and EGb 761 has a clear therapeutic potential for prevention and treatment of AD.

Lyashenko, Alexey et al. (2015) International Worm Meeting "Line scanning fluorescent confocal microscope for a 3D multi-neuron imaging of C. elegans."

Polanco, Edward, Lyashenko, Alexey, Arisaka, Katsushi

[International Worm Meeting, 2015]

Conventional confocal microscope uses a physical aperture to reduce the amount of out of focus light to the image sensor. We developed a line scanning confocal microscope that utilizes a software controlled rolling shutter on a CMOS camera for a high-speed 3D volume imaging of dozens of active neurons. The microscope setup allows for a real time worm tracking for freely navigating C. elegans under a localized external stimulation for phototaxis and thermotaxis. An external photo stimulation for optogenetics was also realized.