Kaplan, Oktay I. et al. (2019) International Worm Meeting "Functional characterisation of Joubert Syndrome associated CEP-41 and ARMC-9."

Kilic, Atiye, Altunkaynak, Betul, Pir, Mustafa S., Pir, Betul, Cevik, Sebiha, Yenisert, Ferhan, Kaplan, Oktay I.

[International Worm Meeting, 2019]

Joubert syndrome is a rare genetic disorder with an occurrence of approximately 1/100 000 live births. Developmental defects in cerebellum and brainstem are observed in patients with JS, and JS is defined as a neurodevelopmental disorder. Up to now 35 different genes have been linked to JS, and the functions of many of these genes including CEP41 and ARMC9 remain uncharacterised. To elucidate the functions of Joubert Syndrome associated CEP-41 (F42G8.19) and ARMC-9 (F59G1.4) genes, we have generated transgenics containing their own promoter driven GFP labelled constructs and generated/obtained null mutants for these genes. CEP-41 was found to be exclusively expressed in the ciliated sensory neurons, and its localisation is restricted to the middle segment of cilia while ARMC-9 localizes to the entire length of the ciliary axonemes. While null mutants for these genes possess no abnormal cilia structures, a number of double mutant combinations of cep-41 with other ciliopathy genes reveal defects in cilia structure in C. elegans.

Cevik, Sebiha et al. (2009) International Worm Meeting "A C. elegans homologue of Joubert syndrome-associated Arl13B associates with the ciliary membrane and is required for proper targeting and motilities of ciliary transmembrane and IFT proteins."

Kaplan, Oktay, Blacque, Oliver, Cottell, David, Toivenon, Tiina, Kida, Katarzyna, Cevik, Sebiha

[International Worm Meeting, 2009]

Joubert syndrome (JS) and JS-related disorders (JSRD) are clinically heterogeneous, characterised by cerebellar malformation and hypervariable phenotypes such as cystic kidneys, retinitis pigmentosa and polydactyly. Emerging evidence indicate that JS/JSRD are caused by defects in primary cilia, which serve diverse sensory and signaling roles during chemo/mechano-sensation, phototransduction, and development. Although multiple JS/JSRD proteins are known, the molecular bases of their functions remain poorly understood. Here, we investigate the molecular functions of C. elegans ARL-13, a homologue of the small Ras G-protein Arl13b, which causes classical JS and is required for vertebrate cilium formation and sonic hedgehog signaling. First, we find that ARL-13 is mostly restricted to proximal (middle) segments of ciliary axonemes, does not undergo intraflagellar transport (IFT), and requires N-terminal palmitoylation lipid modification for its ciliary targeting/retention. Loss of ARL-13 function disrupts cilium chemosensory function, morphology, and ultrastructure, with defects including truncated axonemes, microtubule misplacement/malformation, enlarged middle segments and ciliary accumulation of matrix/membranous material. Consistent with these phenotypes, arl-13mutants are defective in localisation and/or translocation of ciliary proteins, with ciliary transmembrane proteins partially mislocalised and IFT proteins exhibiting anterograde IFT motility defects, indicative of kinesin2 motor uncoupling, differential separation of IFT complex A/B components, and reduced OSM-3 translocation rates. Finally, arl-13 interacts synthetically with bbs-8, dyf-5 and nph-4, with double mutants exhibiting enhanced cilia integrity and IFT defects. Taken together, we propose that ARL-13 associates with the ciliary membrane where it maintains cilium structure/function and the localisation and motilities of ciliary transmembrane and IFT proteins, perhaps by regulating aspects of ciliary membrane biogenesis and/or turnover. We also suggest that defects in this function underlie the molecular basis of Arl13b-associated Joubert syndrome.

Li, yujie et al. (2011) International Worm Meeting "Coordination of Arls in cilia."

zhang, yuxia, Wei, Qing, Hu, Jinghua, Li, yujie

[International Worm Meeting, 2011]

Joubert syndrome (JS) is the most common inherited cerebellar malformation disorder that belonging to a rapidly expanding group of human diseases called ciliopathies. All identified JS loci encode cilia-related proteins, however, with the precise roles enigmatic. Small GTPase Arl13B was identified as one JS locus. Here, we report the function of ARL-13, the sole C. elegans ortholog of human Arl13B, in regulating cilia biogenesis. In C. elegans, ARL-13 specifically localizes to ciliary doublet segment in its proline-rich C-terminus dependent manner. arl-13 animals exhibit shortened cilia with various cilia ultrastructural defects as well as disrupted association between intraflagellar transport (IFT) subcomplex A and B. Intriguingly, depletion of ARL-3, another evolutionarily conserved ciliary small GTPase, can suppress ciliogenesis defects in arl-13(lf) via a microtubule associated deacetylase HDAC-6 dependent pathway by restoring the binding efficiency between IFT subcomplex A and B. BBS proteins are a group of proteins mutated in another human ciliopathy Bardet-Biedl syndrome. It was reported that IFT-A and IFT-B dissociate in several worm bbs mutants. By checking BBS-7 and BBS-8 in arl-13 null worms, we found BBS proteins move with integrate IFT particles, but not with dissociated IFT-A or IFT-B subcomplex. These observations suggest the gene products encoded by different ciliopathy loci can have a functional crosstalk in same cellular process involved in ciliogenesis.

Corey L. Williams et al. (2007) International Worm Meeting "Delineating the role of basal body protein complexes in mediating cilia-specific signaling events: common mechanisms disrupted in multiple cystic kidney diseases."

Marlene E. Winkelbauer, Bradley K. Yoder, Corey L. Williams

[International Worm Meeting, 2007]

Nephronophthisis (NPH), Joubert Syndrome Type B (JS), and Meckel-Gruber Sydrome (MKS) are three clinical disorders that exhibit renal cystic dysplasia. Patients diagnosed with JS have been found to have mutations in genes that are also linked to NPH and MKS, suggesting that these diseases may represent different spectra of a common underlying disorder. Many proteins related to cystic kidney disease have been identified in cilia and/or basal bodies including NPH proteins nephrocystin-1 and nephrocystin-4 as well as MKS proteins MKS1 and mecklin. Studies in Caenorhabditis elegans have provided insight into the connection between cilia and cystic kidney disease. With this in mind, we have sought to identify genes that are regulated by the ciliogenic transcription factor DAF-19 and are expressed in ciliated sensory neurons that may encode components of the basal body complex. C. elegans homologs of nephrocystin-1 (nph-1) and nephrocystin-4 (nph-4) protein products NPH-1 and NPH-4 localize to the transition zone at the base of cilia and are required for proper sensory transduction of signals originating in cilia. We have identified additional proteins that also localize to the transition zone and may be part of the same complex as NPH-1 and NPH-4. Continuing identification and characterization of the proteins that interact with NPH-1 and NPH-4 at the base of cilia to mediate signaling events is necessary to further elucidate the mechanism of kidney cyst formation in mammalian systems.

Li Y et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Small GTPases ARL-13 and ARL-3 coordinate IFT integrity and ciliogenesis"

Wei Q, Hu J, Li Y, Zhang Y

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Intraflagellar transport (IFT) machinery mediates the bidirectional movement of IFT cargos that are required for the assembly and maintenance of cilia. However, little is known about how IFT process is regulated in vivo. Here, we showed that a small GTPase ARL-13, encoded by the C. elegans homolog of human Joubert Syndrome (JS) causal gene Arl13B, localizes exclusively to the doublet segment of the cilium in C-terminal proline-rich domain dependent manner. arl-13 mutants exhibit shortened cilia with various ultrastructural deformations as well as disrupted association between IFT subcomplex A and B. Intriguingly, depletion of ARL-3, another ciliary small GTPase, partly suppresses ciliogenesis defects in arl-13 mutants by indirectly restoring the binding efficiency between IFT subcomplex A and B, and this function is through a deacetylase HDAC-6 dependent pathway. Thus, we propose that two conserved small GTPases, ARL-13 and ARL-3, coordinate to regulate IFT integrity and that perturbing this balance results in cilia deformations.

Beck CDO et al. (1991) International C. elegans Meeting "LIFESPAN CHANGES IN LEARNING AND MEMORY IN C. ELEGANS."

Cerniuk SR, Beck CDO, Rankin CH

[International C. elegans Meeting, 1991]

At adulthood, C. elegans is capable of simple non-associative forms of learning : habituation, dishabituation and sensitization as well as short and long-term memory for habituation training (Rankin, Beck & Chiba, Behav. Brain Res., 3 7 :89, 1990). We examined habituation, dishabituation and recovery from habituation during the larval and postreproductive periods of the lifespan of C. elegans for age-related changes in these forms of behavioral plasticity. Developmental studies of larval worms show that both habituation and dishabituation are present at all stages of development in reversal and acceleration response to tap. In postreproduction development, animals 7 and 12 days old habituated,and dishabituated as well as 4 day old adults. However 12 day old animals did not recover from habituation. This failure to recover in old animals is probably not due to fatigue from habituation training, as animals this age can show dishabituation after the same habituation training. Furthermore, using a response competition protocol, habituation training produced the same facilitation of response by release from inhibition at all ages. Thus there appears to be a specific loss of function in recovery from habituation at old age in C. elegans which suggests functional changes in the aging nervous system. These studies of learning and memory throughout the lifespan of C. elegans will allow a characterization of some of the properties of the development when postembryonic neurons are arising and forming connections and during aging when the nervous system may be degenerating functionally.

Kiyoshi Kunoh et al. (2001) International C. elegans Meeting "GENETIC AND PHENOTYPIC ANALYSES OF HABITUATION ABNORMAL MUTANTS"

Kiyoshi Kunoh, Xizhen Xu, Ryuji Hosono, Toshihiro Sassa

[International C. elegans Meeting, 2001]

C. elegans shows habituation response when given repeated mild mechanical stimuli such as touch or tap. The behavior was originally noticed and extensively characterized by C. Rankin. (1 ) However, genes contributing to the behavior have not been considered. We undertook genetic analyses of the habituation behavior abnormal mutants that were isolated more than ten years ago. The habituation behaviors of these mutants were classified into three groups based on the response patterns: slow ( hab-1, hab-3 ), rapid( hab-2, hab-5 ) and normal but incomplete( hab-4 ) habituations. These mutants are also different in patterns of recovery from the habituation . We focused here on analyses of hab-1 . The hab-1 mutant is normal in chemotaxis and adaptation response to volatile chemicals tested. The mutants normally respond to the single tap stimulus. However, the mutant is slowly habituated and rapidly recovered from the habituated state. To identify neurons in which the hab-1 gene functions, we ablated neurons constituting neural circuit for the mechanical response with a laser microbeam and tested habituation response. From these results we conclude that the gene hab-1 does not function at the specific neuron. The gene was positioned on the linkage group I. From the three- and two- factor crosses, the gene is located to the right of aex-6 and left of lev-10 . By more detailed three-factor crosses with SNP markers the hab-1 gene is located between 22.3 and 22.7 map units*. We are now sequencing the chromosomal region to identify the mutation site of hab-1 ( cn308 ). * Informations on SNPs between N2 and CB4856 were provided by S.R.Wicks (Dept, Mol, Biol, Netherland Cancer Institude) and Genome Sequence Center (Washington University). 1. Rankin,C.H., Beck,C.D.O., Chiba,C.M.(1990) Caenorhabditis elegans : a new model system for the study of learning and memory. Behav. Brain. Res. 37, 89 - 92

Lees, Hayley et al. (2013) International Worm Meeting "Investigating the role of the wrn-1 helicase in the nematode worm, C. elegans."

Cox, Lynne, Lees, Hayley, Woollard, Alison

[International Worm Meeting, 2013]

In order to ameliorate the deleterious consequences of old age, the biology of human ageing must be understood. The study of human progeroid disorders which recapitulate many of the features of normal ageing have helped to contribute to our understanding of normal human ageing. Werner syndrome is a canonical progeroid disorder, caused by mutation of the WRN gene. WRN encodes both RecQ helicase and exonuclease activities, and is known to participate in DNA replication, repair, recombination and telomere maintenance. In addition, although many interacting partners have been identified, the exact molecular functions of the WRN gene remain largely unknown. In order to dissect the roles of WRN helicase in ageing, we use mutants of the WRN homologue and interacting partners in the nematode worm, C. elegans. Reduction of function by RNA interference of the C. elegans WRN homologue wrn-1 leads to ageing phenotypes and shortened lifespan 1. We have shown that mutation of wrn-1 leads to genomic instability: interestingly, this phenotype is enhanced in a mutant cep-1 background (the C. elegans p53 homologue). Notably, lifespan also shows significant modulation, while brood size remains unchanged from that of either single mutant. Therefore we suggest that cep-1 status has a significant effect upon the role of wrn-1 helicase in longevity and germline maintenance in worms. References 1.Lee, SJ; Yook, JS; Han, SM; Koo, HS. A Werner syndrome protein homolog affects C. elegans development, growth rate, life span and sensitivity to DNA damage by acting at a DNA damage checkpoint. Development, 2004. 131(11): p. 2565-2575.

Keith A Boroevich et al. (2004) West Coast Worm Meeting "Identification of Regulatory Elements in Caenorhabditis elegans using Orthology Biased Gibbs Sampling"

Steven JM Jones, David L Baillie, Keith A Boroevich

[West Coast Worm Meeting, 2004]

Much attention has recently been placed on the problem of identification of gene regulatory sequences. Two main computational approaches exist: 1) identification of sequences that share similarity to known regulatory elements, and 2) de novo identification of common motifs in a set of co-regulated genes. Orthology Biased Gibbs Sampling (OrBS) applies a modification of the Gibbs Sampler put forth by Lawrence, Altschult et al . 1 in a attempt to solve this problem. Additional features include analysis of negative strand, multiple (or no) occurrences of a motif in any given sequence and identification of multiple unique motifs, all of which have appeared in previous incarnations of the Gibbs Sampler. The unique feature of OrBS is the use of comparative genomics as an informative prior and in the calculation of motif probability. OrBS will be applied to the identification of regulatory elements C. elegans . Clustering of spatially co-regulated genes as defined by the C. elegans Gene Expression Project (see Johnsen et al . poster). This data is more amenable to regulatory element detection than the more common microarray data because the sequence in which a cis -acting element may reside is strictly defined. Interspecies sequence conservation will be identified through alignment of the C. elegans gene promoter region to the promoter region of the C. briggsae ortholog using LAGAN 2 . Predicted regulatory elements will be verified in vivo using site directed mutagenesis and promoter::GFP fusions. References: 1 Lawrence CE, Altschul SF, Boguski MS, Liu JS, Neuwald AF, Wootton JC. (1993). Science 262: 208-214. 2 Brudno M, Do C, Cooper G, Kim MF, Davydov E, Green ED, Sidow A, Batzoglou S. (2003). Genome Research 13: 721-731.

Yukari Suzuki et al. (2005) International Worm Meeting "Both cec-1 and gtl-1 are essential for long-term memory"

Yasushi Kawahara, Yukari Suzuki, Ryuji Hosono, Toshihiro Sassa, Yuji KOHARA, Yu Suzuki

[International Worm Meeting, 2005]

Learning is one of the most complicated behavior. The habituation learning accompanied by harmless tap stimuli in C. elegans was originally noticed by C. Rankin(1). She established the desirable assay methods and proved that the behavior is suitable for the analysis of the nonassociative learning. We mainly undertook genetic analyses to clarify the behavior and isolated many mutants showing abnormal behaviors on the habituation learning(2). In parallel with genetic analyses, we applied the cDNA microarray technique for further identification of the related gene to the behavior. We observed that many gene expressions are altered during tap stimuli. From these identified genes, we found that C08F11.1, gtl-1 and cec-1 are essential for habituation learning(1). In this report, we focus our attention to the latter two genes. The gtl-1 gene encodes the TRP channel, one of a TRPM subfamily member. The mammalian GTL-1 homologue RRDC7 is highly expressed in brain.However, its role is not yet cleared.The gtl-1 gene is expressed in ASK and ASI amphidal neurons. Cec-1 is a chromobox-contains gene encoding a potential transcriptional suppressor. cec-1(RNAi) animals show similar phenotype as gtl-1(RNAi) animals on habituation behavior. Wild-type animals show chemotaxis toward NaCl but avoid NaCl after conditioning with NaCl in the absence of food. Both RNAi animals are also abnormal in associative learning with the NaCl/starvation paradigm. We then examined the learning behaviors of gtl-1(ok375) and cec-1(ok1005) animals, We found that the two learning phenotypes in ok375 and ok1005 animals are similar to phenotypes in each RNAi animals. That is, habituation behaviors in both mutants are normal when given short interval tap stimuli, but abnormal when given long interval tap stimuli. To further inquire into the contribution of both genes to LTM, channel properties of GTL-1 and target genes of CEC-1 are searching.(1)Rankin H, Beck D, Chiba M (1990) Behav Brain Res 37:89-92(2)Xu et al., (2002) J.Neurogenetics 16:1-16(3)Suzuki Y et al.,(2005) Identification of genes contributing to learning in C.elegans using cDNA microarrays( submitted to press )