Salome-Correa, Jose et al. (2021) International Worm Meeting "Genomic landscape of the obligately outcrossing Caenorhabditis becei"

Salome-Correa, Jose, Noble, Luke, Sloat, Solomon, Rockman, Matthew

[International Worm Meeting, 2021]

Breeding systems determine the way genes are transmitted to the next generation, consequently driving genetic variation and genome evolution. Breeding systems may also play a role in the activity and prevalence of transposable elements. Selfing should increase the selection efficacy against TEs as a result of high homozygosity, with the majority of TEs present having reached fixation through genetic drift. Conversely, outcrossing should facilitate the spread of TE insertions throughout populations, and result in a higher number of moderately deleterious mutations. In the Caenorhabditis group Elegans, there doesn't appear to be a clear pattern of TE dynamics across partially-selfing species and obligately outcrossing species. Most species in the Elegans group, appear to have chromosomal heterogeneity in repeat density, with chromosome arms being more repeat rich, and gene poor, than the centers. This association is not always consistent, as is the case for the outcrossing species C. inopinata and C. bovis, where some, but not all, repeat types have a more uniform distribution across the chromosome, with little divergence between copies, which may indicate recent TE activity. It is currently unknown whether these observations are also found across species from the obligately outcrossing Caenorhabditis group Japonica. Here we present the first chromosome-scale assembly for a species belonging to the Japonica group, Caenorhabditis becei. This high-quality assembly was generated from PacBio long read data, Hi-C data, and a genetic map of an advanced intercross panel. The gene annotation for this genome was generated using Iso-Seq data and RNA-seq data from C. becei, as well as orthologous protein-coding and protein sequences from other Caenorhabditis species. We then characterized features such as synteny to other Caenorhabditis species, genome size, recombination rate domain structure, gene content, orthology, and distribution. We created a classified library of repetitive elements identified through de novo approaches. We generated TE annotations for the C. becei genome, and we reveal the patterns of repeat density, prevalence, distribution, and divergence.

Gallotta I et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Cell Specific Knock-down of Gene Function in Targeted C. elegans Neurons"

D'Orta M, Vitale M, Castro S, Esposito G, Hilliard M A, Di Schiavi E, Gallotta I

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

An important aspect in the study of gene function is the possibility to dissect the role exerted by a gene of interest selectively in specific cells or groups of cells. In C. elegans the available approaches are time consuming and largely restricted to non-essential genes for which mutants are available. We have developed a simple reverse genetics approach to reduce the function of specific genes in chosen C. elegans neurons (Esposito et al., 2007). By expressing sense and antisense RNA corresponding to a gene of interest under cell-specific promoters, we obtain an efficient, heritable and cell autonomous knock-down of the targeted gene function in the specific neurons. This strategy has now been used by several other labs, with a variety of genes and on different neurons (Harris et al. 2009; Yamada et al. 2009; Jose et al. 2009; Ezak et al. 2010). To optimize the efficiency of this approach we have tested the influence of genetic backgrounds that affect RNA interference. We show that in a rrf-3(pk1426) background silencing is more efficient. Although this result indicates the involvement of RNAi pathways in the silencing mechanism we have shown that there is no spreading of the silencing effect at least among amphidial chemosensory neurons and within the motorneurons of the ventral cord. We are now targeting chemosensory and mechanosensory neurons and two classes of motorneurons (GABAergic and HSN) using a variety of different promoters. We are testing the effect of silencing several essential genes on the development, morphology and survival of these neurons as well as on the behavior that they control.

Collins, Kevin M. et al. (2013) International Worm Meeting "Understanding how neurotransmitter signaling drives two-state activity of the C. elegans egg-laying behavior circuit."

Collins, Kevin M., Koelle, Michael R.

[International Worm Meeting, 2013]

We are interested in understanding how neurotransmitter signaling allows a neural circuit to execute distinct behavior states. C. elegans regulates egg laying by alternating between an inactive phase and a ~3 minute "active" state during which clusters of 3-6 eggs are laid. Six VC motor neurons release acetylcholine to excite the vulval muscles, and two HSN motor neurons release serotonin which signals through vulval muscle receptors to promote the active phase. Four uv1 cells release tyramine to inhibit egg laying. We are using GCaMP calcium imaging in behaving animals to understand how the signaling events in the circuit produce its two state behavior. We recently reported that the vulval muscles are rhythmically excited during each locomotor body bend (1). We found the UNC-103 K+ channel depresses muscle excitability below the threshold that drives calcium transients and contraction during the inactive phase, while still allowing signals above threshold during the active phase. To understand how the HSN, VC, and uv1 neurons regulate vulval muscle excitability, we are manipulating neurotransmitter release from these cells and using GCaMP to record changes in neuron and vulval muscle activity. Activation of serotonin release from the HSNs using Channelrhodopsin induces rhythmic vulval muscle twitching and egg-laying behavior-hallmarks of the active phase. We find that VC neuron activity increases during the active phase, and preliminary results show that egg-laying events mechanically distort the uv1 cells and trigger calcium transients. We have previously shown that TRPV channels, which can be mechanically gated, are required for uv1 to inhibit egg laying (2). We propose that successive egg-laying events increase uv1 calcium signaling, promoting release of tyramine and neuropeptides which terminate the active phase of egg laying. (1) Collins and Koelle (2013). J. Neurosci. 33, 761-775. (2) Jose et al. (2007). Genetics 175, 93-105.

Shu Liu et al. (2008) European Worm Meeting "Towards the search for a thermonociceptor"

Shu Liu, Maren Hertweck, Ralf Baumeister

[European Worm Meeting, 2008]

Upon exposure to noxious temperature, Caenorhabditis elegans executes an. escape reflex (Tav response) similar to the response to body touch. We had. previously shown that sensory neurons in the head and tail, but not in the. midbody region, are involved in the preception of heat. In vertebrates, the. capsaicin-sensitive vanilloid receptor VR1 of the TRP family has been shown. to be involved in the perception to heat, capsaicin, and low pH, and. several attempts have been made in other labs to relate TRP channels to the. sensation of external stimuli. We have begun a systematic phenotypic. analysis of available TRP channel mutants. Of 11 TRP mutants tested, only. osm-9 ocr-2 double mutants exhibited a severe Tav response defect. OSM-9. and OCR-2 have previously been demonstrated to function coordinately in C.. elegans olfaction and nose-touch responses (Tobin et al., 2002), and our. data support a similar model of coordinated sensory transduction in. thermonociception. OCR-2 and OSM-9 expression overlaps only in a few head. and tail neurons (Jose et al., 2007), suggesting that these could in. principle include candidates for thermonociceptors. We next tested a. variety of mutants whose genes have been reported to be expressed in. subsets of these neurons. sem-4 and unc-86 indeed displayed a reduced Tav. response in the tail, similar to the osm-9 ocr-2 double mutants'' defect. In. addition, one receptor mutant expressed in several classes of head neurons. also resulted in a mild yet significant anterior Tav defect, similar to. that seen in eat-4 and osm-9ocr-2 (79% Tav response). Taken together, we. are narrowing down our search for (a) sensory neuron(s) involved in the. perception of thermonoxious stimuli. We plan to carry out neuron ablations. to verify our findings.

Johnson, Matthew et al. (2013) International Worm Meeting "Sexually dimorphic synaptic connectivity in the C. elegans tail."

Ryan, Deborah, Portman, Douglas, Johnson, Matthew

[International Worm Meeting, 2013]

Sex-specific wiring of neural circuits is likely to have important roles in behavior. However, little is known about how sexual cues might influence synapse formation or stability. Interestingly, the recently described connectome of the C. elegans male tail (Jarrell et al., 2012, Science 337:437) has revealed several instances of sex-specific synaptic connections between non-sex-specific ("shared") neurons. In particular, the architecture of phasmid and posterior touch sensory circuitry appears to to be significantly different in the adult male compared to the adult hermaphrodite, perhaps to enable efficient male copulatory behavior. Using GRASP and other fluorescent markers, we are asking several questions about these sexually dimorphic connections. (1) Are sex differences in connectivity established in the embryo, or do these arise later, as a re-wiring process that occurs in parallel with male-specific tail neurogenesis and circuit formation? (2) What determines the sex-specificity of these connections? Is the genetic sex of the pre- and/or post-synaptic cells important, or do non-autonomous sexual cues have a role? (3) How do these sex differences in connectivity alter neural circuit function and behavior? Our preliminary results indicate that one putative hermaphrodite-specific connection, PHB-AVA, is indeed present in larval males. Unexpectedly, we can also often detect strong PHB-AVA GRASP signal in adult males. One possibility is that PHB-AVA synapses are removed as part of male tail re-wiring in L4, but that GRASP is unable to detect (or perhaps even disrupts) this removal. We are currently using GRASP to label other putative sex-specific connections to explore this further. The unique tools available in C. elegans should offer the opportunity to understand how genetic sex regulates modulators of synapse specification and maintenance to bring about sex differences in circuit connectivity. We are grateful to S. Emmons (Albert Einstein) for sharing unpublished data on male tail connectivity and to M. VanHoven (San Jose State) for sharing the wyIs157 (PHB-AVA GRASP) transgene.

Sean M O'Rourke et al. (2003) International Worm Meeting "Exploring the centrosome with proteomics and functional genomics."

Sean M O'Rourke, Bruce Bowerman

[International Worm Meeting, 2003]

The centrosome is a large eukaryotic organelle that organizes microtubules for cellular functions including setting up the mitotic spindle. Despite the important role of the centrosome, relatively little is known about its composition and regulatory mechanisms in metazoans. SPD-5 is a component of C. elegans centrosomes and contains multiple coiled-coil domains that may interact with other proteins thereby governing centrosomal structure (1). Because SPD-5 is thought to be a central component of centrosomes, it represents an excellent handle to pursue other genes and proteins that are also involved in centrosome function. We are taking two experimental routes to uncover additional centrosomal components and regulators.First, SPD-5 antibodies are being used to isolate protein complexes from embryonic extracts with the goal of purifying SPD-5-interacting proteins. Such proteins will be identified by mass spectrometry. To date, we have prepared embryonic extracts from N2 animals and we have successfully immunoprecipitated SPD-5 protein. Interestingly, two SPD-5 isoforms are apparent in our extracts, the significance of which remains to be determined. We are currently optimizing the purification procedure to obtain larger quantities of SPD-5 and interacting proteins. The interacting proteins we identify will be initially characterized by localization and RNAi inhibition studies.In the second route to understanding centrosome function, we are employing genome interaction screens to identify genes which, when knocked-down by feeding RNAi, alter the phenotypes of temperature-sensitive mutants. Building on the work of Jose Eduardo Gomes (see Gomes and Bowerman abstract), we have begun to use multiple-well plates for culturing worms and a robot to easily and accurately dispense the dsRNA-producing E. coli. We have performed a pilot semi-automated screen using the chromosome I library (2). Current results will be presented. In addition to examining spd-5, we are planning to perform genome-wide interaction RNAi feeding screens with 20 different temperature-sensitive mutants defective in several different processes. We envision generating a genetic interaction map that will be useful for determining how many different processes require any given gene. We anticipate that our use of many different sensitized genetic backgrounds will enable us to identify gene requirements missed in previous classical and genomic screens.(1) Hamill, D. R. et al. Developmental Cell 3, 673-684 (2002).(2) Fraser, A. G. et al. Nature 408, 325-330 (2000).