Wen, Chentao et al. (2021) International Worm Meeting "3DeeCellTracker, a deep learning-based pipeline for segmenting and tracking cells in 3D time lapse images"

Nemoto, Tomomi, ,, Miura, Takuya, Wen, Chentao, Ishihara, Takeshi, Kimura, Kotaro, Hillman, Elizabeth

[International Worm Meeting, 2021]

Optical monitoring of cell movements and activities in three-dimensional space over time (3D + T imaging) has become substantially easier due to recent advances in microscopy technology. However, the development of software for segregating cell regions from the background and for tracking their dynamic positions remains a bottleneck in the field. Individual laboratories still need to develop their own software to extract important features from 3D + T images obtained using different optical systems and/or imaging conditions. Moreover, even when identical optical systems are used, optimization of many parameters is often required for different datasets. We developed a software pipeline, 3DeeCellTracker, by integrating multiple existing and new techniques including deep learning for the first time for tracking. With only one volume of training data, one initial correction, and a few parameter changes, 3DeeCellTracker on a desktop PC with GPU successfully segmented and tracked 100-200 head neurons in both semi-immobilized and "straightened" freely moving worms, ~100 cells in a naturally beating zebrafish heart, and ~1,000 cells in a 3D cultured tumor spheroid. While these datasets were imaged with highly divergent optical systems, such as spinning confocal, SCAPE, and 2-photon microscopes, our method tracked 90-100% of the cells in most cases, which is comparable or superior to previous results, and we extracted complex patterns of calcium dynamics in the worm brain neurons and the rhythmic patterns in synchronization with heart chamber movement in the zebrafish heart cells. Thus, 3DeeCellTracker could pave the way for revealing dynamic cell activities in image datasets that have not been analyzed previously.

Endo, Yuto et al. (2021) International Worm Meeting "Whole-brain imaging with neuronal identities to elucidate the mechanism of a sensory processing"

Wen, Chentao, Endo, Yuto, Kimura, Kotaro, Miyachi, Miyu

[International Worm Meeting, 2021]

A neural circuit in the brain processes sensory stimuli such as an odor to elicit a motor command. To understand the mechanism of such a sensory processing, a number of studies have sought to identify a neural circuit which processes a specific sensory stimulus (functional neural circuit). For identification of a functional neural circuit, it is required to conduct two types of experiments throughout the brain: 1) locating neurons that respond to the stimulus, and 2) identifying their cell types (cell-IDs) to estimate their connectivity based on anatomical connections. Locating stimulus-responsive neurons can be achieved by whole-brain calcium imaging, which record all neuronal activities throughout the brain. However, due to the lack of way to efficiently identifying cell-IDs, it has been difficult to identify a complete functional neural circuit with whole-brain imaging and cell identification. To overcome the limitation, the method for cell identification of all neurons, called NeuroPAL, was developed in C. elegans (Yemini et al., Cell, 2020). Together with the connectome information as well as the records of all neuronal activities, we are now able to estimate a functional neural circuit based on cell-IDs of stimulus-responsive neurons. To identify a complete functional neural circuit by integrating those techniques, we established a whole-brain imaging system combined with efficient cell identification system using NeuroPAL. This system consists of a 3D confocal microscopy, a multi-color imaging system, a microfluidic device, and a pipeline of an image analysis (Chronis et al., Nat. Meth., 2007; Wen et al., eLife, 2021). By using this system, we successfully recorded activities of most head neurons simultaneously with an information of each cell-ID. Now we are identifying a set of odor-responsive neurons using this system with stimulation by a repellent odor 2-nonanone (Kimura et al., J. Neurosci., 2010). This study aims to identify a complete functional neural circuit for specific sensory processing pathways and it may provide clues for how a sensory stimulus is processed in the whole of a neural circuit. We thank Ev Yemini and Oliver Hobert for the help on NeuroPAL.

Antebi A et al. (1996) East Coast Worm Meeting "daf-12 Coordinates Multiple Developmental Events"

Hedgecock EM, Antebi A

[East Coast Worm Meeting, 1996]

In the nematode, developmental events within or between tissues are precisely coordinated. The DAF-12 nuclear hormone receptor plays multiple roles in synchronizing development. As a heterochronic regulator, it instructs stage specific programs in both gonad and soma. In the dauer pathway, it selects dauer or continuous development in response to growth conditions. Gonadal, somatic and dauer functions are genetically separable, suggesting a complex locus, and alleles can be grouped into six idealized classes. In collaboration with Don Riddle, Pam Larsen and Wen Hui Yeh, we have begun a molecular analysis of alleles to correlate structure with function. So far, we have found a sharp clustering of alleles which selectively disrupt dauer formation within the zinc fingers of the receptor. A unifying hypothesis is that a hormone, acting through DAF-12 and related receptors, coordinates events in gonad and some, as well as dauer development. We suggest a simple model in which the commitments or starts to each developmental stage are synchronized by a hormonal pulse available to all tissues. Moreover, we propose that the starts to each developmental stage lie between the molts, as judged by new cuticle synthesis in the hypodermic and transitions in the migratory behavior of gonadal leader cells. These "parastages" may more accurately reflect the actual functional boundaries of developmental stages.

S Saeki et al. (1999) International C. elegans Meeting "Another form of associative learning in chemotactic behavior of C. elegans"

Y Iino, M Yamamoto, S Saeki

[International C. elegans Meeting, 1999]

An associative learning paradigm based on chemotactic behavior has previously been reported by van der Kooy's group (Wen et al ., 1997, Behav. Neurosci. 111:354-368). In this paradigm, chemotaxis to sodium ion and chloride ion were differentially conditioned by paired presentation of one of the ions with food, and the other without food. We have found another form of associative learning in chemotaxis. When worms were starved on plates including NaCl, their chemotaxis to NaCl was dramatically reduced. This conditioning required both presence of NaCl and absence of bacteria, indicating that it is not a mere adaptation or habituation. In contrast, decrease in chemotaxis to the volatile chemoattractant isoamylalcohol after continuous exposure to the same odorant (Colbert and Bargmann, 1995, Neuron. 14:803-812) occurred irrespective of the presence or absence of food. While chemotaxis to isoamylalcohol did not significantly change after conditioning with NaCl, chemotaxis to other water-soluble attractants also decreased. This suggests that altered response of a cell(s) that is specifically involved in chemotaxis to water-soluble chemoattractants is responsible for the behavioral change. Decrease in chemotaxis occurred slowly over 3-4 hours of conditioning and returned quickly to the original level when either of the conditioning stimulus, NaCl or starvation, was removed. Application of serotonin partially blocked this change in chemotaxis, consistent with the proposed function of this neurotransmitter in food signaling. We have isolated several mutants with reduced plasticity as assayed in this paradigm. This form of behavioral plasticity expands the opportunities in which one can study molecular and cellular mechanisms of learning using C. elegans .

de Vries CJ et al. (1998) European Worm Meeting "A cytosolic dynein heavy chain is required for sensory neuron structure and function."

Wicks SR, Plasterk RHA, de Vries CJ

[European Worm Meeting, 1998]

We have developed two new assays of nematode chemotaxis (see abstract Stephen Wicks). The first gene identified and cloned using these assays was a cytosolic isoform (1B subfamily) of the dynein heavy chain. Complementation studies show that this dynein heavy chain is the CHE-3 protein. Previously, this isoform of the dynein heavy chain was shown to be expressed during cilia outgrowth in sea urchins (Gibbons, 1994), and involved in the organisation of the Golgi-apparatus in non-ciliated human cell lines (Vaisberg, 1996). GFP expression studies indicate that che-3 is expressed exclusively in all ciliated sensory neurons in the worm. The highest levels of che-3 expression are correlated with outgrowth of cilia. To visualise the structure of ciliated endings we used two GFP markers of amphid and phasmid integrity (GPA-13::GFP and GPA-15::GFP, as integrated transgenes, gifts of Gert Jansen). These markers indicate that the amphid and phasmid cilia are malformed in the worm, and that the degree of disorganisation appears to increase as the animal ages. These results suggest that dynein heavy chain has a function in cilia outgrowth in C. elegans, but has no role in the organisation of the Golgi apparatus in the worm. Gibbons, B.H., Asai, D.J., Wen-Jing, Y., Hays, T.S., and Gibbons, I.R. (1994). Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins. Mol. Biol. Cell 5, 57-70. Vaisberg, E.A., Grissom, P.M., and McIntosh, J.R. (1996). Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles. J. Cell Biol. 133, 831-842.

Kim, Jinmahn et al. (2019) International Worm Meeting "Decoding head neural circuit underlying rhythmic forward movement in C. elegans."

Kim, Jinmahn, Pyo, Seondong, Kim, Kyuhyung, Yeon, Jihye, Park, Cheon-Gyu, Kim, Jongrae, Suh, Byung-Chang

[International Worm Meeting, 2019]

Forward locomotion of C. elegans is a rhythmic behavior which is initiated by contraction and relaxation of head muscles. However, the neuronal and molecular mechanisms in which to generate and maintain forward movement are not fully understood. Previously, cholinergic B-type and GABAergic D-type motor neurons process proprioceptive signals and regulate activity of body muscles during forward movement (Wen et al., 2012). To investigate the neural circuit underlying head movement, we analyzed neural connectome data and found that the two cholinergic SMB and SMD neurons innervate the head muscles and connect to the RME GABAergic neurons. When we genetically ablated SMB or SMD using recCaspase system, the SMD ablated worms displayed poor forward but normal backward movement. While the SMB ablated worms move normally, they exhibit increased wave width of sinusoidal waveform. We then activated SMD or SMB via optogenetic manipulation and found that transgenic animals expressing ReaChR (red-activatable ChR) in SMD or SMB elicit immediate forward movement or increase of wave width, respectively, upon light exposure. Additionally, to identify the molecules that regulate forward movement, we examined expression pattern of 29 TRP and DEG/ENaC channels and found that unc-8 DEG/ENaC channel gene is expressed in SMB. unc-8 mutant animals fail to maintain wave form during forward movement. Furthermore, expression of UNC-8 in HEK293T cells generates outward currents upon mechanical deformation in a heterologous system. Currently, we are investigating function of the SMB, SMD and RME neurons and their circuit mechanisms in regulation of forward movement.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

Valansi C et al. (1998) European Worm Meeting "DEVELOPMENTAL, GENETIC AND BIOCHEMICAL ANALYSES OF adm-1 AND adm-2 IN C.elegans"

Podbilewicz B, Lapidot Z, Valansi C, Adir V, Maroun M, Broza R, Shemer G

[European Worm Meeting, 1998]

ADM proteins in C. elegans are multidomain membrane receptors that contain A Disintegrin and Metalloprotease domains similar to snake venoms. The ADaM gene family includes fertilin involved in fertilization[1], meltrin-a that appears to be required for myotube formation[2], TACE, a TNF-a converting enzyme[3], KUZ involved in NOTCH signalling in Drosophila[4], and SUP-17 (ADM-3) that is related to KUZ[5]. The mutation sup-17(n316) acts as a suppressor of lin-12d and is the only known mutant of a member of the ADM family in C. elegans. Other members of this family may be involved in different cell-cell and cell-matrix interactions. ADM-1, was the first gene from this family found in C. elegans[6]. ADM-1 expression in cell membranes that participate in epithelial cell fusions and in sperm suggests that it can be involved in cell fusion events of developmental importance. adm-2 was mapped to chromosome X close to sem-1. We found that the cosmid C04A11 containing adm-2 did not rescue sem-1. The cellular localization of adm-1 products together with dsRNA interference experiments suggest that adm-1 and adm-2 can have roles in embryonic development. We are currently exploring the localization of adm-2 using a GFP reporter construct. It is known that the founder members of the ADAMs family (fertilins) form heterodimers after proteolytical processing. Thus, to investigate the processing and the putative biochemical and genetic, interactions between ADM-1, ADM-2 and other proteins we have used a monoclonal antibody against the cytoplasmic domain of ADM-1 to affinity purify and coimmunoprecitate interacting proteins. The processing of ADM-1 from a precursor to a mature form is followed using western blots, the amino termini of different bands will be sequenced and mass spectroscopy will be used to identify interacting proteins. 1.Blobel, C.P., et al. Nature 356, 248-252 (1992). 2.Yagami-Hiromasa, T., et al. Nature 377, 652-656 (1995). 3.Blobel, C. Cell 90, 589-592 (1997). 4.Rooke, J., Pan, D., Xu, T. & Rubin, G.M. Science 273, 1227-1231 (1996). 5.Wen, C., Metzstein, M.M. & Greenwald, I. Development 124, 4759-4767 (1997). 6.Podbilewicz, B. Mol. Biol. Cell 7, 1877-1893 (1996).

Alfred Tamayo et al. (2005) International Worm Meeting "Molecular identification of srf genes suggests a role in protein secretion"

Katie MacFarland, Alfred Tamayo, Patricia Berninsone

[International Worm Meeting, 2005]

In a mutagenic screen designed to select for animals with altered cuticle surface glycosylation, several mutants termed srf (surface) were isolated (1). These loss-of-function mutants display ectopic surface binding to the lectin Wheat Germ Agglutinin. Some of these srf mutants also have gross pleiotropic defects (uncoordinated movement, protruding vulva, abnormal egg laying, aberrant copulatory bursae and malformed gonads) (1). Therefore, the molecular events affected by these genes play important roles not only in expression of cuticle surface glycoconjugates, but also in the normal development of various organs and tissues.Prior genetic mapping located srf-4, srf-8 and srf-9 to specific intervals of LGV (1). We searched these intervals for ORFs encoding products with homology to known components of glycosylation and secretion pathways in other organisms, and tested these candidates by dsRNAi inactivation. Among these candidates, we have identified a family of proteins which when targeted by dsRNAi, generated animals that display phenotypes closely resembling those of the srf mutants. Direct sequencing of the corresponding genes in the srf mutant genomes revealed genetic lesions consistent with strong or complete loss-of function. This protein family has been implicated in ER to Golgi cargo selection, the fidelity of ER sorting mechanisms and the exclusion/inclusion of unfolded proteins in vesicles exiting the ER (2). Our finding supports the hypothesis that the underlying defect in these mutants affects protein secretion.We have found that inactivation of the putative srf genes by dsRNAi resulted in strong induction of hsp-4::gfp, a reporter construct of the mammalian ER chaperone BiP homolog, which is a target of the unfolded protein response (UPR) in yeast and mammals (3, 4). Such RNAi also caused embryonic lethality in a mutant background deficient in an ER molecular chaperone. Furthermore, srf-8 mutants are sensitive to treatments which cause accumulation of unfolded proteins in the ER. Our evidence suggests that mutations in these srf genes cause ER-stress, and that these genes may play a role in the secretion of cellular membrane proteins or extracellular matrix components important for normal development.(1)Link C. et al (1992) Genetics 131: 867-881 (2)Wen and Greenwald (1999) J Cell Biol 145: 1165-1175 (3)Shen X. et al (2001) Cell 107: 893-903 (4)Calfon M. et al (2001) Nature 415: 92-96

Sternberg, Paul et al. (2015) International Worm Meeting "WormBase 2015."

Berriman, Matt, Howe, Kevin, Sternberg, Paul, Stein, Lincoln, Kersey, Paul, Harris, Todd, Schedl, Tim

[International Worm Meeting, 2015]

WormBase has existed for 15 years and has evolved in many ways. The new website is fully operational and has made the process of adding new data types, displays, and tools easier. Behind the scenes we are piloting an overhaul of the underlying database infrastructure to allow us to handle the ever increasing data, have the website perform faster, and allow more frequent updates of information. This is a critical time for the project, as we face considerable pressure from two directions. The first is that our funders really want us to do more with less. We are responding to this by leading the way in making curation (the process of extracting information from papers and data sets into computable form) more efficient using a new version of Textpresso (to be released later this calendar year); by discussing with other model organism information resources ways to work together to be more efficient and inter-connected; and by seeking additional sources of funding. The second, delightful, pressure is an increase in data and results generated by the C. elegans and nematode communities. While we are handling this increase by changes in our software for curation, the database infrastructure, and the website, we do need your help. Many of you have helped us over the last few years to identify data in your papers or by sending us data directly. We now need you to help with a few types of information by submitting the data via specially designed, user-friendly forms that ensure good quality and the use of standard terminology. In particular, we have a large backlog of uncurated information associating alleles with phenotypes. We pledge to make this process as painless as possible, and to improve WormBase's description of phenotypes with your feedback, starting at this meeting at the WormBase booth, workshops and posters. With your help, continual improvement of our efficiency, and additional sources of funding, we are optimistic that we can do much more with even somewhat less effort.Consortium: Paul Davis, Michael Paulini, Gary Williams, Bruce Bolt, Thomas Down, Jane Lomax, Todd Harris, Sibyl Gao, Scott Cain, Xiaodong Wang, Karen Yook, Juancarlos Chan, Wen Chen, Chris Grove, Mary Ann Tuli, Kimberly Van Auken, D. Wang, Ranjana Kishore, Raymond Lee, John DeModena, James Done, Yuling Li, H.-M. Mueller, Cecilia Nakamura, Daniela Raciti, Gary Schindelman.