Nechipurenko, Inna et al. (2015) International Worm Meeting "A new role for the conserved centrosomal protein Girdin in the regulation of primary cilia biogenesis in C. elegans and mammalian cells."

Blacque, Oliver, Olivier-Mason, Anique, Sengupta, Piali, Nechipurenko, Inna, Kennedy, Julie

[International Worm Meeting, 2015]

Precise regulation of centrosome position is critical for diverse processes including asymmetric cell division, neurogenesis, directed cell migration, and ciliogenesis. Primary cilia, present on the surface of most cell types in metazoans, are key mediators of diverse signaling pathways essential for embryonic development, sensory signal transduction, and adult tissue homeostasis. Cilia from all studied organisms share a conserved core organization comprised of a microtubule-based axoneme anchored by the basal body (BB) and surrounded by a specialized membrane. Primary cilium assembly is a multi-step process that relies on mother centriole-to-BB transition, BB migration and anchoring to membrane, establishment of the 'ciliary gate', and axoneme elongation from the BB via intraflagellar transport of ciliary components. Considerable progress has been made in identifying molecular components of distinct centrosomal and ciliary sub-compartments; however, the mechanisms regulating centrosome positioning during early steps of ciliogenesis are poorly understood. We find that the highly conserved signaling protein Girdin localizes to the BB in C. elegans sensory neurons and mammalian cells. Loss of grdn-1 function in C. elegans results in defective sensory cilia morphology, ultrastructure, and localization of BB, transition zone, and signaling proteins. Similarly, Girdin knockdown in mammalian RPE-1 cells leads to a complete loss of, or shortened cilia, and dissociation of the BB from the nucleus. These findings suggest a conserved function for Girdin in regulating centrosome positioning, and thereby ciliogenesis, in both C. elegans sensory neurons and mammalian cells. Mammalian Girdin has been previously implicated in regulating the actin cytoskeleton, and in coupling multiple classes of surface receptors to downstream signaling events in different contexts (e.g. cell migration, synaptic plasticity, and tumorigenesis). Therefore, Girdin is perfectly positioned to link extracellular cues to the cytoskeleton to ensure correct centrosome/BB positioning during primary cilia formation - a previously uncharacterized role for this protein in any species. We will present our ongoing efforts to further characterize the mechanism, by which Girdin regulates BB positioning and primary cilia biogenesis in C. elegans sensory neurons and mammalian cells.

Huang, Huiyan et al. (2013) International Worm Meeting "A genetic screen for Notch downstream targets regulating C. elegans sleep."

Zhu, Chen-Tsen, Hart, Anne, Huang, Huiyan

[International Worm Meeting, 2013]

The conserved Notch signaling pathway plays well-defined roles in cell fate determination during development. Recently, Notch has also been implicated in non-developmental neuronal processes across species, including invertebrate sleep. In C. elegans, the level of Notch activity affects both quality and quantity of lethargus quiescence, a sleep-like behavior coinciding with larval molts. Also, transiently overexpressing the Notch co-ligand, OSM-11, is sufficient to drive inappropriate quiescence in adult animals, which can be suppressed by loss of either Notch receptor or downstream players in Notch signaling (Singh et al., 2011). To find pertinent transcriptional targets of the Notch pathway and to gain insight into regulation of sleep, we undertook a classical suppressor screen to identify suppressors of inappropriate quiescence in adult hsp::osm-11 animals. In this screen, 2122 mutant lines were assessed; 79 independent isolates suppressed the OSM-11-induced inappropriate quiescence. To exclude mutations with pervasive effects on locomotion, we examined endogenous L4/adult lethargus quiescence in these 79 strains using both an adaption of the Multi-Worm Tracker (Swierczek et al., 2011) and the microfluidic chamber-based assay (Singh et al., 2011). Twenty-seven strains had defects in endogenous lethargus quiescence. We are in the process of identifying the corresponding genes by whole genome sequencing. As Notch signaling also modulates sleep behavior in Drosophila (Seugnet et al., 2011), we are confident that the genes identified in this screen will be conserved regulators of arousal and sleep across species.

Fischer, Christian et al. (2021) International Worm Meeting "MitoSegNet: Easy-to-use Deep Learning Segmentation for Analysing Mitochondrial Morphology"

Fischer, Christian, Haeussler, Simon, Marr, Carsten, Duchen, Michael, Conradt, Barbara, Rolland, Stephane, Singh, Kritarth, Besora-Casals, Laura

[International Worm Meeting, 2021]

While the analysis of mitochondrial morphology has emerged as an important tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a difficult task and bottleneck for statistically robust conclusions. Here, we present the Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology (Fischer, Besora-Casals et al. 2020). The MitoSegNet was generated by training a modified fully convolutional neural network with fluorescent microscopy, maximum-intensity projection images, depicting mitochondria in body wall muscle cells of adult C. elegans worms. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik. MitoSegNet outperformed all other methods in both pixelwise and morphological segmentation accuracy. We successfully applied MitoSegNet to unseen fluorescence microscopy images of mitoGFP expressing mitochondria in wild-type and catp-6ATP13A2 mutant C. elegans adults. Additionally, MitoSegNet was capable of accurately segmenting mitochondria in HeLa cells treated with fragmentation inducing reagents. We provide MitoSegNet for all operating systems as an easy-to-use graphical user interface tool that combines segmentation with morphological analysis. Reference Fischer, C. A., L. Besora-Casals, S. G. Rolland, S. Haeussler, K. Singh, M. Duchen, B. Conradt and C. Marr (2020). "MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology." iScience 23(10).

Bennett, Heather L. et al. (2013) International Worm Meeting "Notch DSL ligand lag-2 is required for C. elegans lethargus quiescence."

Bennett, Heather L., Hart, Anne C., Singh, Komudi, Huang, Huiyan

[International Worm Meeting, 2013]

Sleep is an evolutionarily conserved behavior defined as a rapidly reversible period of inactivity with decreased sensory responsiveness. Despite this knowledge, the genetic underpinnings, mechanisms of sleep regulation, and the specific tissues involved in this behavior remain elusive. C. elegans undergoes sleep-like quiescence during lethargus in coordination with each larval cuticle molt (Raizen et al., 2008). Our laboratory has shown that Notch signaling regulates quiescence, as perturbations in the Notch pathway change the arousal thresholds and amount of quiescence during the last larval lethargus during transition from L4 stage to adult (Singh et al., 2011). We find that decreased function of lag-2, which encodes a Notch DSL ligand, results in increased quiescence and reduced arousal thresholds during the L4 to adult lethargus. However, it remains unclear how specific changes in lag-2 gene function disrupt aspects of this behavior. We are now characterizing the role of lag-2 in regulating behavioral quiescence. The specific cells where LAG-2 functions in this behavior and when lag-2 is required to regulate quiescence is being determined. The knowledge gained from addressing these aims will help us understand the role of Notch signaling, particularly the role of lag-2 in regulating C. elegans quiescence and may illuminate evolutionary conserved mechanisms required to regulate sleep across species.

Jennifer L. Tenor et al. (2007) International Worm Meeting "A conserved Toll-like receptor is required for Caenorhabditis elegans innate immunity."

Jennifer L. Tenor, Alejandro Aballay

[International Worm Meeting, 2007]

Pathogen recognition through Toll-like receptors (TLRs) is the first step required to mount an appropriate immune response against invading pathogens. The only toll-like receptor, tol-1, in Caenorhadbitis elegans was shown to be required for behavioral avoidance to a particular Gram-negative bacterium, Serratia marcescens (1). Although we did not observe significant differences in behavioral avoidance between wild type and the tol-1(nr2033) mutant to various other Gram-negative pathogens, the tol-1 mutant was hypersusceptible to these pathogens suggesting that TOL-1 may provide protection through other mechanisms. Based on survival assays and microscopy, we found that TOL-1 is essential for protecting the pharynx against the enteropathogenic bacterium, Salmonella enterica serovar Typhimurium. Loss of tol-1 not only led to rapid death of C. elegans but also resulted in rapid accumulation of S. enterica in the terminal bulb of the pharynx. We showed that TOL-1 is required for the proper expression of ABF-2 which is a defensin-like molecule expressed in the pharynx (2) and HSP-16.41 which is also expressed in the pharynx and is part of a family of heat shock proteins required for C. elegans immunity (3). These results indicate that TOL-1 plays a direct role in innate immunity. (1) Pujol et al., 2001; (2) Kato, 2002; (3) Singh and Aballay, 2006.

Thomas JH et al. (1996) East Coast Worm Meeting "LINEAGE ANALYSIS OF SYNTHETIC MULTIVULVA MUTANTS"

Horvitz HR, Thomas JH

[East Coast Worm Meeting, 1996]

Synthetic Multivulva (synMuv) genes comprise two distinct classes: A and B. AB double mutants are Muv, whereas AA and BB double mutants, like A and B single mutants, are wildtype for vulval development at the gross anatomical level. These data suggest that the synMuv genes define two functionally redundant pathways that negatively regulate vulval development (Ferguson and Horvitz, 1989, Genetics 123: 109121). If the synMuv genes are completely redundant for the regulation of vulval development, they should exhibit no abnormalities at the cellular level. To test this hypothesis, we analyzed the lineages of selected class A and class B mutants. The lineages of the six Pn.p cells of the vulval equivalence group in animals carrying a strong class A mutation, lin-15(n767), were identical to those of wild-type animals. These Pn.p cell lineages were analyzed in animals carrying a strong class B mutation, lin-36(n766), and were also found to be identical to those of wild-type animals. All six Pn.p cells of the vulval equivalence group in lin-36(n766); lin-15(n767) double mutants adopted vulval like fates. These results suggest that the class A and class B genes are completely redundant at the lineage level, and that in double mutants, all six Pn.p cells are at least partially induced to form vulval tissue.

Bennett, Heather L et al. (2011) International Worm Meeting "A role for heterochronic genes in regulating C.elegans quiescence."

Corkins, Mark E, Anderson, Edward, Singh, Komudi, Hart, Anne C, Bennett, Heather L

[International Worm Meeting, 2011]

Sleep is an essential biological process that is regulated by circadian rhythms, homeostatic regulation and arousal threshold. The nematode Caenorhabitdis elegans undergoes a sleep-like state called quiescence that accompanies each larval molt (Raizen et al., 2008). Many pathways regulate larval development, including heterochronic genes and the Notch signaling pathway. Heterochronic genes control timing of cell differentiation and molting of larval cuticle. In Drosophila, circadian rhythm proteins cycle to regulate sleep behavior. In C.elegans, circadian rhythm homologs in the heterochronic pathway regulate the fusion of hypodermal seam cells during late development (Jeon et al., 1999, Banerjee et al., 2005). Recently, a temporal role in C. elegans molting was suggested for the Notch signaling pathway. Gain of function alleles of a Notch receptor gene, lin-12, result in precocious cell fate changes (Solomon et al., 2007). We have found that Notch also plays a role in the regulation of quiescence. Increased or decreased Notch signaling results in increased quiescence and altered arousal thresholds during the last larval stage (L4) to adult (A) transition (Singh et al., in press). However, the connection between circadian rhythm homologs, heterochronic genes, and Notch in the regulation of quiescence remains unclear. We are assessing gain and loss of function alleles of heterochronic genes acting in the L4/A transition to determine if they play a role in regulating quiescence. Demonstrating a genetic interaction between heterochronic genes, circadian rhythm proteins, and the Notch signaling pathway will be the first step in delineating mechanisms that coordinate cell fate and behavioral changes during development.

Thomas JH et al. (1995) International C. elegans Meeting "THE SYNTHETIC MULTIVULVA GENES MAY ENCODE COMPONENTS OF A CELL SIGNALING SYSTEM"

Thomas JH, Horvitz HR

[International C. elegans Meeting, 1995]

Synthetic Multivulva (synMuv) genes comprise two distinct classes: A and B. AB double mutants are Muv, whereas AA and BB double mutants, like A and B single mutants, are wild-type for vulval development. These data suggest that the synMuv genes define two functionally redundant pathways that negatively regulate vulval development. To isolate new synMuv genes, we conducted several screens in which animals carrying a mutation in a gene of one class were mutagenized, and their F2 progeny screened for Muv animals. We isolated 19 new class A mutations after screening 23,000 haploid genomes. We defined two new class A complementation groups: lin(n2402) IIL and lin(n2728) IIC. We isolated 30 new class B mutations after screening 16,000 haploid genomes. We defined three new class B complementation groups: lin(n2231) IVR, lin(n2994) IIC, and lin(n2987). We have also characterized three additional class B complementation groups, lin(n770) IIIL, lin(n771) IIIR, and lin(n833) IR, originally identified by Ferguson and Horvitz (Genetics 123: 109-121, 1989). We cloned lin-36, a class B gene, and showed that it encodes a novel protein. We have determined the DNA sequences of six of seven lin-36 mutant alleles; none is an obvious molecular null mutation. We are now analyzing alleles isolated in a noncomplementation screen and are beginning expression studies. Our mosaic analysis suggests that lin-36 acts cell autonomously. By contrast, lin-15, a synMuv locus with both A and B activities, has a cell-non- autonomous focus (Herman and Hedgecock, Nature 348: 169-171, 1990). Together, these observations suggest that the synMuvs encode components of a cell signaling system.

JC Bettinger et al. (1999) International C. elegans Meeting "Behavioral Effects of Exposure to Ethanol"

SL McIntire, JC Bettinger

[International C. elegans Meeting, 1999]

Exposure to ethanol interferes with complex behaviors in many model systems, but it has been difficult to correlate effects of ethanol on behavior with observations of its effects on specific molecular targets. Recently, studies of Drosophila demonstrated a link between ethanol sensitivity and a learning pathway 1 : A screen for mutations that cause flies to be hypersensitive to the effects of ethanol on postural control yielded an allele of amnesiac , a putative neuropeptide known to be involved in learning 2 . After exposure to ethanol, C. elegans display uncoordinated movement (characterized by a decreased amplitude of the sine waveform and lethargy), and decreased rate of pumping and egg-laying (SLM, unpublished observations). After several hours of exposure, worms develop an acute tolerance to ethanol, and recover to resemble untreated controls. We are interested in determining whether or not exposure to and development of tolerance to ethanol alter any of the more complex behaviors exhibited by worms, including chemotaxis. Our preliminary experiments on the effect of ethanol on chemotaxis suggest that brief or prolonged exposure to moderate concentrations of ethanol (100-200 mM) does not prevent chemotaxis to the volatile odorant benzaldehyde. With extended exposure, worms become insensitive to chemoattractants in a process termed adaptation. Worms that have adapted to a particular chemoattractant will not climb a gradient of that chemoattractant 3 . Given that worms are able to chemotax in the presence of ethanol, we can test the effect of ethanol on adaptation. We are determining whether or not exposure to low-to-moderate concentrations of ethanol interferes with the process of adaptation to benzaldehyde and other odorants. 1 Moore, M.S.; DeZazzo, J.; Luk, A.Y.; Tully, T.; Singh, C.M. and Heberlein, U. (1998). Cell 93: 997-1007. 2 Feany, M.B. and Quinn, W.G. (1995). Science 268: 869-873. 3 Colbert, H.A. and Bargmann, C.I. (1995). Neuron 14: 803-812.

Singh, Komudi et al. (2013) International Worm Meeting "Is sleep conserved? Making the case in D. melanogaster and C. elegans."

Ju, Jennifer Y., Hart, Anne C., Walsh, Melissa B., Singh, Komudi, DiIorio, Michael A.

[International Worm Meeting, 2013]

Sleep is a ubiquitous behavior. However, it is unclear if the genes and pathways regulating this process are conserved across animal species. Limited evidence of conservation exists; Notch, EGF pathways and cGMP kinase regulate sleep across species. To test conservation more broadly, we surveyed the Drosophila literature and shortlisted eighteen genes required for Drosophila sleep-like behavior. Sixteen orthologous C. elegans genes were identified based on similarity. The impact of these sixteen C. elegans genes on molting lethargus quiescence and arousal was tested using previous defined assays (Singh et al., 2011, WBPaper00038400). All of the genes that affected Drosophila sleep also altered C. elegans L4/adult quiescence. In fact, the change in quiescence was precisely what would be predicted from the Drosophila literature, with one exception- suggesting deep conservation of regulatory mechanisms. Next, we aimed to connect these conserved genes in a pathway and establish site of action in the nervous system of C. elegans. The wake-promoting function of D1 dopamine receptor is conserved as animals lacking D1 dopamine receptors had increased sleep-like behavior in Drosophila and C. elegans. We find that the C. elegans Gsa protein is also required for lethargus quiescence. Further, Gsa, cAMP, PKA, and CREB likely all function in D1 dopamine receptor expressing neurons to regulate C. elegans quiescence. Additionally, we find that serotonin regulates quiescence and likely functions through the Goa and the DAG kinase pathway in quiescence, extending previous studies (Yuan et al., 2006, Guo et al., 2011). Analysis of C. elegans orthologs of Drosophila genes expands the list of conserved genes that regulate sleep across species. The striking conservation observed in these two disparate invertebrate animals suggests that these genes and pathways regulate sleep in all species.