De Vore, Deanna Michele et al. (2013) International Worm Meeting "Exploring the role of mechanosensory extracellular matrix components in the structure and function of primary cilia."

Knobel, Karla, De Vore, Deanna Michele, Barr, Maureen

[International Worm Meeting, 2013]

PKD2 encodes a transient receptor potential polycystin (TRPP) channel receptor protein found in non-motile, primary cilia of mammalian cells 1. In humans, PKD2 mutations result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). In the nematode Caenorhabditis elegans, the polycystin-2 homolog, PKD-2, localizes to the primary cilia of male-specific sensory neurons (CEMs and RnBs in the head and tail respectively) where it is required for male mating behaviors. Given the ancient and evolutionarily conserved role for polycystin-2 in cilia, I am using C. elegans as a model to identify new genes required for ciliary receptor localization. Our laboratory performed a forward genetic screen for PKD-2::GFP ciliary localization (Cil) defective mutants (Bae et al 2008). The cil-2(my2) mutants exhibit excess unusual PKD-2 accumulation at the ciliary base, cilium proper, and around the distal dendrite. We performed three-factor and deficiency mapping, whole genome sequencing, and single gene rescue experiments and determined that the cil-2(my2) allele is a mutation in the gene, mec-5. MEC-5 is a unique collagen protein found in the extracellular matrix (ECM) surrounding touch receptor neurons (Du et al 1996). In touch receptor neurons (TRNs), MEC-1, MEC-5, and MEC-9 are found in the ECM, play a role in mechanosensation, and may localize degenerin/epithelial sodium channels (DEG/ENaCs) along the TRN processes (Du et al 1996, Emtage et al 2004). Our preliminary data indicates that mec-1 and mec-9 regulate PKD-2::GFP localization in male-specific sensory neurons. We are currently determining how these ECM proteins regulate localization and channel properties of the PKD-2 TRP polycystin channel. These studies will shed light on how sensory receptors like PKD-2 are targeted to cilia, and may advance the understanding and treatment of ADPKD. References: 1) Bae YK et al, Dev Dyn. 2008 Aug;237(8):2021-9. 2) Du H. et al, Neuron. 1996 Jan;16(1):183-94. 3) Emtage L et al, Neuron. 2004 Dec 2;44(5):795-807.

Dao, Chris et al. (2017) International Worm Meeting "Development of a high-statistics, three dimensional imaging system for C. elegans under magnetic and chemical stimuli."

Arisaka, Katushi, David, Angela, Vincent, Nitin, Thatcher, Joseph, Chow, Aaron, Wang, Charles, Dao, Chris, Du, Angela, Alberto, Jasmine, Li, Lina

[International Worm Meeting, 2017]

In contrast to previous behavioral experiments limited to two dimensional surfaces, we extended the study of C. elegans into three dimensional environments to observe the resulting unconstrained motion. While research has been initiated towards single worm, high magnification tracking, this has limited practicality in terms of gathering a large number of statistics with bulk behavior of many worms simultaneously. Incorporating stimuli allows for a new, more thorough examination of motion and environmental interaction, extending inquiry beyond simple free motion. Previously performed, two dimensional results suggest hypotheses, which may serve as limiting-case projections of greater trends in three dimensional space. A novel, cost effective system was developed to image C elegans' behavior over a period of 30 to 60 minutes using time lapse acquisition. The setup consists of three, perpendicularly oriented DSLR cameras, synchronized via the open source, MIT developed DigiCam control software and illuminated with lowly-invasive, conical, red LED light sources. Samples consisted of N2 type worms embedded in the center of 2% porcine gelatin (4 to 5 cm) quartz cuvettes. Each camera had an affixed aperture to elongate its depth of field, promoting focus throughout the volume of the cubes. In addition to free motion studies, three sets of Helmholtz coils (with axes coinciding with the cameras' optical axes) were utilized to generate a uniform magnetic field at the location of the sample. NaCl concentration gradients throughout the gelatin were also simulated and created for three dimensional chemotaxis experiments. Numerous free motion trials have been conducted alongside magnetic field experiments of field magnitude ranging from 0 Gauss (cancelling the geomagnetic field) to 10 Gauss. Using this data, analysis software tools were developed in MATLAB in order to extract the center of mass trajectory of worms navigating the gelatin, enabling further quantification of motional trends such as the worms' velocities and distributions of initial and final positions. While preliminary results for free motion (with chemical attractants) appear to reflect standard biased random motion, positive verification of magnetic field trials remains elusive.

Xu, Yichi et al. (2017) International Worm Meeting "Systematic decoding of cell movement in C. elegans early embryogenesis."

Xu, Yichi, Bao, Zhirong

[International Worm Meeting, 2017]

Morphogenesis is the fundamental process in biology that describes the formation of a tissue or organism. It encompasses multiple layers of controlling upon molecular basis (Turing, 1952) and cellular basis. Taking advantage of the invariant cell lineage in C. elegans (Sulston, 1983), we asked how cells move to correct destinations. Previous studies proposed cell movement is guided by local cell-cell interaction (Schnabel, 2006), which were supported by evidences in early-stage embryos and limited types of mutants. Here, we hypothesized that, upon cell-cell interaction, differential affinities between neighboring cells drive their next moves. With our collection of all cell tracks up to 350-cell stage in hundreds of wild-type and perturbed embryos (Du, 2015), we devised a computational model to optimize cellular configuration over time and infer cell-cell affinities for individual embryos. The comparison between wild-type and perturbed embryos demonstrated those affinities were specified by cell fate rather than cell lineage. This study provided a basis to systematically elucidate how affinity network can serve as a new paradigm to positional information and orchestration in complex tissues.

Duggan A et al. (1997) International C. elegans Meeting "CLONING OF THE EGL-46 GENE"

Duggan A, Chalfie M

[International C. elegans Meeting, 1997]

The egl-46 gene is required for the correct differentiation of the HSN neurons, a pair of motor neurons necessary for egg-laying (1,2). In addition, an extra pair of touch receptor-like cells is present in egl-46 mutant animals (3). This finding led to the hypothesis that egl-46 represses touch cell fate in these cells. We have cloned the egl-46 gene using transformation rescue and we have confirmed the assignment by identifying the mutations present in the four known egl-46 alleles. A 5.5kb fragment of CO9E6 partially rescues the Egl phenotype of egl-46 animals. The egl-46 gene (K11G9.4) encodes a C2H2 zinc finger protein that contains three putative zinc fingers. We have confirmed the exon-intron structure of egl-46 by sequencing a cDNA and by 5' RACE analysis. egl-46::lacZ and egl-46::GFP reporter constructs are expressed in a complex pattern which includes cells in the head, tail and ventral cord. We are now trying to determine the identity of the staining cells. Future work will address the mechanism by which the egl-46 gene controls neuronal fate and/or differentiation. (1) Desai, C., Garriga, G., McIntire, S.L. and H.R.Horvitz (1988) Nature 336, 638-646. (2) Desai, C. and H.R.Horvitz (1989) Genetics 121, 703-721. (3) Mitani, S., Du, H., Hall, D.H., Driscoll, M. and M. Chalfie (1993) Development 119, 773-783.

Materi WP et al. (1998) West Coast Worm Meeting "THE ROLE OF UNC-119 IN AXONOGENESIS IN C. ELEGANS"

Pilgrim DB, Maduro MF, Materi WP

[West Coast Worm Meeting, 1998]

UNC-119 is the first example of a novel class of protein found in both the developing and adult nervous systems of the nematode, the fly, the zebrafish (see poster by Angela Manning) and in mammals. While UNC-119 is found throughout the C. elegans nervous system, beginning around the 60-cell stage, the human and rat homologs (HRG4 and RRG4, respectively) have only been found in retinal tissue, beginning shortly after birth. The fly homolog (DmUnc-119) is expressed throughout the larval central nervous system. In the worm, four unc-119 null alleles have been characterized in addition to the wild type. Apart from the canonical nearly paralyzed, uncoordinated phenotype, these mutants also exhibit constitutive feeding behavior even in the absence of food and are unable to enter into the dauer larva stage when subjected to overcrowding and starvation. Both the behavioral and morphological phenotypes suggest that UNC-119 plays a role in growth cone guidance in the developing nervous system. An UNC-119::GFP fusion protein is found in the cytoplasm of all cells of the nervous system. In an unc-119 mutant the ventral nerve cord (VNC) is defasciculated and the lateral commisures, which normally project from the VNC dorsolaterally to join the dorsal nerve cord have an abnormal, highly branched structure. We have integrated GFP constructs, driven by promoters specific to subsets of neurons, into both wild-type and unc-119 mutant strains and compared neural structures at high magnification using confocal microscopy. The projections of many neurons are unaffected by the unc-119 mutation while abnormally long axons, abnormally branched axons and defasciculated axons are associated with neurons affected by the mutation. We have also conducted a yeast 2-hybrid screen using an unc-119 bait construct and have found several positive interactions that are selective for UNC-119 versus a SNF-4 bait. These preliminary results are presently being confirmed using the human homolog, HRG4, as bait in a 2-hybrid experiment. Co-immunoprecipitation experiments are also underway.

Laurent, P. et al. (2017) International Worm Meeting "A genetic analysis of the dense core vesicle biology in C. elegans neurons."

Jospin, M, Laurent, P., Chen, C.C., De Bono, M, Ch'ng, Q.

[International Worm Meeting, 2017]

Although both neurotransmitters and neuropeptides are released in response to calcium entry, they are not packed into the same organelles. The Dense Core Vesicles (DCVs) typically mediate the secretion of neuropeptides. Analyses in mammalian (neuro)endocrine cells link DCVs to multiple pathophysiologies. However, the cell biology of the DCV is poorly understood, particularly in neurons: e.g. how their biogenesis, trafficking and secretion is coupled to neuronal activity. We generated a new model to study the DCV biology in the well-characterised neuron PQR whose activity can easily be manipulated by the external [O2]1. Fluorescently tagged pro-neuropeptides have proven reliable reporters of DCV in other models. INS-1-VENUS expressed in PQR accumulated in its cell body and in puncta along the length of the PQR axon. Two other tagged-neuropeptides behaved similarly to INS-1 and all colocalised largely with IDA-1, a specific membrane marker for DCV. Using this assay, we revaluate the role of multiple genes proposed to modulate neuropeptide secretion in C. elegans and other models. Specific phenotypic signatures based on DCV distribution in the neuron and neuropeptide secretion are revealed for mutations known to alter DCV maturation, anterograde and retrograde trafficking and exocytosis. We performed a candidate gene screen for new mutants potentially affecting DCV biology, and classified each mutant in those phenotypic categories. We explored the new genes with biogenesis mutants-like phenotypic signatures. Together with previously published work (2,3,4,5) 3 new genes identified during this screen suggest new models for DCV biogenesis in neurons. 1. Busch, K. E. et al. Nat. Neurosci. 15, 581-591 (2012). 2. Paquin, N. et al. Curr. Biol. 26, 862-871 (2016). 3. Topalidou, I. et al. PLoS Genet. 4. Ailion, M. et al. Neuron 82, 167-180 (2014). 5. Du, W. et al. Elife 5, (2016).

Antonio, Melissa et al. (2017) International Worm Meeting "Effects of engineered nanomaterials on the behavior and embryonic development of the nematode, Caenorhabditis elegans."

Hartoonian, A., Antonio, Melissa, Hibbard, S., Constante, J.

[International Worm Meeting, 2017]

Since C. elegans are soil-dwelling organisms in the environment we are interested in probing the question of how the exposure of specific engineered nanomaterials (ENMs), namely molybdenum disulfide and graphene oxide, may pose a threat to living organisms. Although the use of ENMs is increasing, concern of adverse effects on soil communities is also rising. Not only are ENMs toxic to various organisms in soil, "but can bioaccumulate, trophically transfer and even biomagnify in some systems" (1). Molybdenum, for example, is a metal that is continuously used for plant growth and an essential component for nitrogen fixation and nitrate reduction (2). In addition, graphene, a carbon nanomaterial, has been widely used in various ways, such as pollution prevention and remediation. However, as the production and application amount is increasing, it is also released into the environment resulting in potential risks of ecological environment and human health (3). Of course, the use of an ENM such as molybdenum and graphene may provide various benefits, but little is known on the potentially negative effects they may have on the environment. Therefore, it is crucial that the behavior of ENMs in soils, and ultimately in organisms that thrive in the soil such as C. elegans, is well studied and understood to prevent negative impacts to the environment. The data we have collected thus far consists of preliminary findings testing the affects of heavy metals, such as CdCl2, CuSO4, and ZnSO4, on the mobility/behavior of toxicity mutants and wild type N2 strains. We will soon proceed with exposing both strains to the ENMs mentioned above and observe potential affects on behavior and embryonic development. 1. McKee MS, Filser J. Impacts of metal-based engineered nanomaterials on soil communities. Environ Sci: Nano. 2016;3(3):506-533. 2. Molybdenum in plants and soils [Internet]. [cited 2016 Nov 23]. Available from: http://www.imoa.info/HSE/environmental_data/biology/plants_soils.php 3. Du J, Hu X, Zhou Q. Graphene oxide regulates the bacterial community and exhibits property changes in soil. RSC Adv. 2015;5(34):27009-27017.

Veigl, Sophie (2021) International Worm Meeting "Extending immunity through small RNA inheritance"

Veigl, Sophie

[International Worm Meeting, 2021]

Invertebrate immune systems are most often characterized by a "lack of x" compared to mammalian immune systems (Pradeu & Du Pasquier, 2018). "Lacking" processes akin to clonal selection and expansion of B- and T-cells, C. elegans is believed not to display an adaptive immune response. Ideas and concepts guiding the description of immunity-related phenomena, such as self/nonself, biotic/abiotic, and innate/adaptive, are developed from a mammalian perspective. This perspective is, however, epistemically pernicious, as it excludes other possible instantiations of immune systems. Ideally, a framework that resists biases is also a framework that describes the processes studied more accurately (Longino, 2008). I will give a conceptual talk, discussing the possibility of reinterpreting C. elegans small RNA inheritance as a transgenerational adaptive immune system. Upon viral infection, C. elegans produces virus-derived RNAs (viRNAs), which immunize offspring against repeated infection (Rechavi et al., 2011). Similar heritable responses are launched against abiotic stressors (e.g., Houri-Ze'evi et al., 2016). I will discuss C. elegans small RNA inheritance as an intersection of genetic- and immune systems. Considering small RNA inheritance as part of the C. elegans immune system requires redefining immune systems in several ways. An immune system is, first and foremost, a system that immunizes. Any step further needs to avoid presupposing particularities based on what works in mammals. On the one hand, the differentiation between biotic and abiotic stressors needs to break down. On the other hand, the idea of self/nonself, organism/environment, needs to be reconsidered: Apart from small RNAs recognizing what is "foreign," other small RNAs transmit information on what is "self" (Rechavi, 2014). Inheriting self- and nonself-recognizing small RNAs results in an intricate equilibrium, shifted by environmental stimuli (Ishidate et al., 2018), and thus, what the organism will recognize as "self" and "nonself." The environment continuously "blends into" the organism. In conclusion, I will not only show how examining C. elegans small RNA inheritance helps overcome biased accounts of immune systems but also how it contributes to addressing current theoretical questions in immunology that debate the adequacy of self-nonself-, danger-, and continuity theory (Matzinger, 1994; Pradeu, 2012) as overarching theoretical frameworks.

Minevich, G. et al. (2011) International Worm Meeting "Identifying Genes that turn Skin Cells into Neurons."

Hobert, O., Korswagen, H., Doitsidou, M., Gowtham, S., Minevich, G.

[International Worm Meeting, 2011]

Under specific circumstances, skin cells can change their differentiated state from a skin to a neuronal state. This is observed in several systems, from worms to flies to vertebrates. For example, in the L2 stage of C. elegans, the differentiated skin cell V5 generates a neuroblast that then forms two mechanoreceptor neurons, PVD and PDE. Emmons and co-workers previously described that a loss of the conserved bHLH transcription factor lin- 32 (Atoh1 in mouse) results in a failure of this skin-to-neuron transformation (Zhao, et al., 1995); instead, the neuroblast converts into a skin cell, like all other V cells do. Using forward genetic screens, we have identified additional genes required in this skin-to-neuron transformation. Our mutant collection contains a subset of conserved transcription factors that may have similar roles in vertebrates. As proof of principle, we have isolated several lin-32 alleles: one of which is a regulatory mutation in a region that is completely conserved between 5 species of nematode and contains a C2H2-type zinc finger binding site. In another mutant, dopy-1, lin-32::gfp expression fails to be induced in the V5 lineage, suggesting that we isolated a regulator of lin-32 expression. dopy-1 is a C2H2 zinc finger transcription factor and while it may control lin-32 directly, it is broadly expressed, suggesting that other activators of lin-32 remain to be identified. Another gene we have implicated in the skin-to-neuron transition is hlh-2, a gene encoding a highly conserved protein known to heterodimerize with LIN-32 and play a role in male ray neurogenesis (Portman, et al., 2000). The allele of hlh-2 that we retrieved also appears to be a regulatory allele that deletes upstream promoter elements. Using a parallel candidate gene approach, we identified vab-15 as another mutant where the skin-to-neuron transformation fails to occur. vab-15 was previously found to encode the C. elegans homolog of the vertebrate Msx homeobox genes (Du, et al., 2001). We find that vab-15 also controls lin-32 expression. From a collection of over 30 mutant alleles in which neurons in the postdeirid lineage fail to be induced, we expect to find more genes that act to induce lin-32 expression and/or cooperate with lin-32 to control induction of the postdeirid lineage. We will present our analysis of these additional mutant loci.

Benavidez, J. et al. (2019) International Worm Meeting "Transcriptional and post-translational mechanisms regulate HLH-2 to specify the anchor cell during C. elegans gonadogenesis."

Benavidez, J., Greenwald, I.

[International Worm Meeting, 2019]

A fundamental question of developmental biology is how initially equivalent cells specify into the many cell types present in multicellular organisms. We study four cells that initially have the potential to be the Anchor Cell (AC) of the C. elegans gonad (Seydoux et al. 1990): two proximal "alpha" cells (Z1.ppp and Z4.aaa) flanked by their distal sisters, the "beta" cells (Z1.ppa and Z4.aap). The beta cells lose AC competence soon after they are born and commit to becoming Ventral Uterine precursor cells (VUs). Their fate is influenced by, but does not require, lin-12/Notch activity. The fates of the alpha cells are naturally variable (Kimble & Hirsh 1979); they signal to each other via the LIN-12/Notch signaling pathway to determine which becomes the AC and which becomes a VU. Unlike the beta cells, the alpha cells require lin-12/Notchactivity to adopt the VU fate (Greenwald et al. 1983; Seydoux & Greenwald 1989; Sallee et al. 2015). The transcription factor HLH-2 is crucial to AC specification-it is required to confer AC competence to the alpha and beta cells, to drive transcription of lag-2 (a LIN-12 ligand) during the AC/VU decision, and for the differentiated functions of the AC (Karp & Greenwald 2003 & 2004). HLH-2 is initially present in the four alpha and beta cells, but is post-translationally degraded in the three VUs by a proteasome-dependent mechanism (Sallee & Greenwald 2015). In addition to the established post-translational regulation of HLH-2, we have now observed that hlh-2 is transcriptionally downregulated specifically in beta cells, potentially accounting for their early adoption of the VU fate. In contrast, we hypothesize that the post-translational mechanism is necessary for an alpha cell to adopt the VU fate. To elucidate this mechanism, we used CRISPR/Cas9 to engineer an allele of hlh-2 that encodes GFP-HLH-2 and conducted an RNAi screen of 175 conserved ubiquitin-related genes (Du et al. 2015; Kim et al. 2018). We identified 10 E3 ubiquitin ligases that caused ectopic stabilization of GFP-HLH-2: 4 of these are generally associated with cullin-containing complexes, 3 are monomeric HECT-domain ligases, and 3 are RING-finger ligases. We are currently analyzing these genes for their roles in gonadogenesis and regulation by/of lin-12.