K Ver Donck et al. (1999) International C. elegans Meeting "'High density C. elegans screening': a drug discovery platform applied to the steerin (unc-53) pathway"

P Van Osta, K Ver Donck, L Bols, I Roelens, T Bogaert, J Geysen, I Mailliet

[International C. elegans Meeting, 1999]

Green fluorescent protein (GFP) technology enables cellular resolution in the living animal. When combined with light sensitive cameras and with up-to-date automated stage driving, image acquisition and image analysis, microscopists can observe, analyse and derive objectively calculated parameters from composite digital images: a novel technology termed 'high density nematode screening'. We developed this technology platform for 96-well plate in vivo nematode phenotype drug screening, using a transformed C. elegans strain, expressing GFP in the vulval myoblasts under the control of the ceh-25 promoter. The vulval myoblasts were previously shown to be dependent on steerin (unc-53) for their correct positioning in larval develpment (Bogaert, unpublished). The screening was run on an inverted fluorescence microscope, equipped with an automated (XYZ) stage and an intensified camera. SCIL-image software (University of Amsterdam) on Silicon Graphics unix was used to write programs for stage driving, automated focussing (patent pending), seamless image grabbing, generation of composite images and semi-automated image picking for visual analysis of the wild type or compound-induced phenotype. Using this screening assay, a lead compound previously shown active in agar plate cultures was confirmed and novel compounds discovered as in vivo inhibitors of the steerin signaling pathway. The activity of this compound has been confirmed using mouse neuroblastoma cells overexpressing nematode steerin. Computer simulations using the crystal structure of GRB-2 C-terminal SH3-domain suggest that this compound may be a diproline mimetic capable of inhibiting SH3-binding to proline-rich helices.

Luth, E.S. et al. (2019) International Worm Meeting "PVF-1/VER signaling regulates GLR-1 glutamate receptor surface levels to control behavior."

Markoja, K., Juo, P., Luth, E.S., Riccio, C., Hofer, J., Hodul, M.

[International Worm Meeting, 2019]

The molecular mechanisms that regulate the formation and function of glutamate synapses in vivo are not well understood. To identify novel genes important for glutamate synapse function, we performed an RNAi screen using a mechanosensory reflex mediated by ASH glutamatergic sensory neurons called the nose touch response1-3. Expression of light-activated channelrhodopsin in ASH4 enabled us to screen populations of worms using ASH-stimulated, glutamate-dependent locomotor reversals as a behavioral readout. RNAi knockdown of ver-1 and ver-4, Vascular Endothelial Growth Factor Receptor homologs2, yielded a specific defect in glutamate-dependent reversal behavior. Loss of function mutants confirmed these RNAi results, and cell-specific rescue experiments showed that both ver-1 and ver-4 act cell-autonomously to regulate nose touch behavior. By imaging GLR-1 tagged with both mCherry and pH-sensitive superecliptic pHluorin (SEP::mCherry::GLR-1)6 we found that ver mutants exhibited a preferential loss of cell surface GLR-1. Additionally, we found that blocking endocytosis with unc-11/AP180 clathrin adaptin mutants, prevents the decrease in cell surface GLR-1 observed in ver-1 mutants, suggesting that VER-1 acts to promotes plasma membrane levels of GLR-1 downstream of its initial insertion and endocytosis. Mutants lacking the VER ligand PVF-17 exhibited similar defects in plasma membrane GLR-1 levels and related reversal behaviors, suggesting that ligand binding may be required for VER-mediated promotion of glutamate signaling. Interestingly, pvf-1 and ver mutants underwent faster habituation to nose-touch, suggesting that VER signaling modulates non-associative learning. Together, our data support a model where VERs and their ligand PVF-1 regulate recycling to promote cell surface levels of GLR-1 and associated behaviors. References: 1. Hart, AC., et al. (1995) Nature 378:6552 2. Maricq, AV., et al. (1995) Nature 378:6552 3. Kaplan, JM and Horvitz, HR. (1993) PNAS 90:6 4. Ezcurra, M., et al. (2011) EMBO J 30:1110 5. Popovici, C., et al. (2002) Neurosci. Lett. 329:116 6. Hoerndli, F. J., et al. (2013) Neuron 80:1421 7. Dalpe, G., et al. (2013) Development 140:4020

Luth, E.S. et al. (2017) International Worm Meeting "The VER-4/VEGF receptor-related protein regulates GLR-1 glutamate receptors and related behaviors."

Luth, E.S., Riccio, C., Juo, P., Markoja, K.

[International Worm Meeting, 2017]

Glutamate synapses are highly dynamic, and their restructuring provides the cellular basis of learning and memory. Failure to properly form or maintain glutamate synapses can lead to a variety of neurological disorders, and controlling their abundance and activity is critical for maximizing functional recovery after stroke. However, the molecular mechanisms that regulate the formation and function of these connections in vivo during normal nervous system development are not well understood. Expression of channelrhodopsin in the glutamatergic sensory neuron ASH1 enabled us to combine optogenetic control of a simple glutamate-dependent behavior, the nose-touch response, with a focused RNAi screen to identify novel genes important for glutamate synapse function. RNAi knockdown of ver-4, a VEGF receptor homolog2, resulted in a strong defect in ASH-stimulated glutamate-dependent locomotor reversals, but did not affect overall glutamate-independent locomotion based on thrashing assays. Analysis of worms with loss of function mutations in ver-4 confirmed these RNAi results. The intensity and density of the presynaptic vesicle marker RAB-3::mCherry, expressed specifically in ASH, was unaltered in ver-4 mutants. In contrast, we observed widespread alterations in the size and intensity of GLR-1::GFP puncta in the nerve ring and ventral nerve cord of ver-4 mutants, suggesting that VER-4 globally affects GLR-1 levels. Confocal imaging of GLR-1 tagged with both mCherry and pH-sensitive superecliptic pHluorin (SEP::mCherry::GLR-1)3 indicated that ver-4 mutants exhibit a preferential loss of cell surface GLR-1. Consistent with this data, ver-4 mutants suppressed the increase in glutamate-dependent spontaneous reversals observed in animals expressing a constitutively-active form of GLR-14. Mutants lacking the VER-4 ligand PVF-15 also exhibited defects in the nose-touch response suggesting that ligand binding may be required for VER-4-mediated promotion of glutamate signaling. Together, our data identify VER-4 and its ligand as novel regulators of GLR-1 trafficking and signaling. Future experiments will identify the cellular and subcellular site of action of VER-4 and the mechanism by which PVF-1 and VER-4 regulate GLR-1 trafficking. References: 1. Ezcurra, M., et al. (2011) EMBO J 30:1110 2. Popovici, C., et al. (2002) Neurosci. Lett. 329:116 3. Hoerndli, F. J., et al. (2013) Neuron 80:1421 4. Zheng, Y., et al. (1999) Neuron 24:347-361 5. Dalpe, G., et al. (2013) Development 140:4020

Carl Procko et al. (2010) East Asia Worm Meeting "C. elegans glia require the thermotaxis gene ttx-1 for dauer-induced neuronal remodeling."

Shai Shaham, Yun Lu, Carl Procko

[East Asia Worm Meeting, 2010]

The shapes of neuronal structures are plastic, and can be modified by an animal's environmental experience. In C. elegans, the ciliated endings of the bilateral AWC sensory neurons are structurally remodeled when the animal enters the dauer stage in response to environmental stressors, including high temperature. How dauer-inducing signals from the environment are integrated into pathways controlling this remodeling is unknown. We found that a pair of glial cells that ensheath the AWC neuronal endings respond to dauer entry and also high temperature by up-regulating expression of the VEGF receptor-like gene ver-1. Surprisingly, up-regulation of ver-1 in glia at high temperature was independent of neuronal input, but did require the Otx transcription factor ttx-1. ttx-1 was previously shown to be required for behavioral responses to temperature and for wild-type morphology of the thermosensory neuron AFD. We now show that ttx-1 is also expressed within glia, and acts cell autonomously within the glia to regulate temperature- and dauer-dependent ver-1 expression by a direct interaction with the ver-1 promoter. Furthermore, when animals entered the dauer stage, glial expression of ver-1 and ttx-1 were required for remodeling and fusion of the two glial cells. Glial fusion in turn facilitated shape changes in the AWC neuronal endings. To find other genes in addition to ver-1 and ttx-1 which may contribute to glial and neuronal remodeling, we have conducted screens for mutants with inappropriate expression of ver-1. We are currently mapping and characterizing these mutant alleles. Together, our results suggest that glia, like neurons, can respond to environmental stressors and that this may facilitate the remodeling of the glia and AWC sensory neurons in dauer animals.

C Buesa et al. (1999) International C. elegans Meeting "Steerins (UNC-53) in health and disease: from C. elegans gene to drug discovery"

M Vandecraen, E Stringham, T Bogaert, M De Raeymaeker, P Verhasselt, K Ver Donck, C Platteeuw, J Geysen, C Buesa, L Maertens

[International C. elegans Meeting, 1999]

Genetic, cellular, biochemical and structural evidence suggested that nematode steerin is a central regulator of 'directional' cell motility, by transducing receptor tyrosine-kinase signals into recruitment of F-actin and microtubuli in the nematode and in mammalian cells; indicating the existance of a conserved pathway in mammals and hence the existance human steerins (see also Vandecraen et al., 1999). A human steerin gene family has been cloned. Steerins comprise a N-terminal F-actin binding domain and a C-terminal predicted nucleotide binding domain and a middle domain of alpha-helical structure comprising a microtubule binding domain, coiled-coils I, II and III and 2 Pro-rich SH3 binding-like signatures. Human steerins display five more highly conserved boxes, discriminating them from nematode steerin. Funcional equivalence with nematode steerin was confirmed for human steerin-1 for activation of the F-actin cytoskeleton and binding to microtubule (+)-ends. Steerin-1 immunoreactivity localised to the microtubule (+)-ends in G361 melanoma cells (polyclonals). Expression of the human steerins is complex, both in terms of variants per tissue and tissue specificity (multiple tissue Northern blots). The chromosomal loci of both steerins are indicative of a putative role in neoplasms. A nematode screening platform was developed to search for inhibitors of the steerin/unc-53 pathway in the nematode. A first compound discovered induces pharmacologically an unc-53 (lf) phenotype in the sex myoblasts of wild type C. elegans (see Ver Donck et al., 1999, Celegans99 meeting abstract). In conclusion, animal steerins emerge as a novel structurally and functionally conserved animal protein family operational in a conserved pathway that transduces positional signals over e.g. receptor tyrosine kinase pathways into recruitment of the F-actin cytoskeleton. Steerins appear to perform this function from the microtubule (+)-end. Indicative evidence exists for a role of steerins in neoplastic disease and amenability of the pathway to pharmacology was shown using the nematode as a drug screening model.

Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[International Worm Meeting, 2007]

Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes egl-1, ced-3 and ced-4 and anti-apoptotic genes ced-9 and icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene eif-3.K. Loss-of-function by mutation or RNAi inactivation in eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of eif-3.K weakly but significantly increased cell corpses. Interestingly, the eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 or ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express eif-3.k in touch neurons, we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human eif-3.K can functionally substitute C. elegans eif-3.K in an eif-3.K mutant, the function of eif-3.K in apoptosis is likely conserved in evolution.

Chun-Yi Huang et al. (2006) East Asia C. elegans Meeting "eif-3.K is a pro-apoptotic gene in C. elegans"

Teng-Wei Huang, Chun-Yi Huang

[East Asia C. elegans Meeting, 2006]

Programmed cell death or apoptosis is an important feature during C. elegans development. The pro-apoptotic genes egl-1, ced-4 and ced-3 are required for the execution of cell death. We have identified and characterized a novel pro-apoptotic gene eif-3.K. A loss-of-function mutation or inactivation by RNA interference in eif-3.K resulted in a reduction of cell corpse number during embryogenesis, whereas heatshock-induced over-expression of eif-3.K weakly but significantly promoted programmed cell death. In addition, eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 and ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the genetic pathway during programmed cell death. Using a cell-specific promoter we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. We generated antibodies against bacterially expressed EIF-3.K protein. The immunostaining result showed that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. To better understand the cell-death defect of eif-3.K mutants, we are currently performing a 4D microscopic analysis of the cell death process in wild-type and eif-3.K mutants.

Procko, Carl et al. (2009) International Worm Meeting "C. elegans glia require the thermotaxis gene ttx-1 for temperature and dauer dependent functions."

Lu, Yun, Procko, Carl, Shaham, Shai

[International Worm Meeting, 2009]

An animal senses stressful stimuli within its environment and responds appropriately. The nematode Caenorhabditis elegans responds to stresses, including high temperature and nutrient availability, by becoming a developmentally-arrested dauer larva. Many of these stresses are detected through a pair of bilateral amphid sensory organs in the head of the animal. Previous studies have revealed roles for the AFD amphid neuron in temperature sensation. Like other amphid neurons, AFD is closely associated with a glial sheath cell that surrounds the specialized dendritic ending of the neuron. It has been shown that in dauer larvae, the bilateral amphid sheath glia expand and fuse with one another at the nose tip. In addition, the dendritic endings of another pair of sensory neurons expand and overlap within the fused glia. To understand how stress signals from the environment regulate sheath glia fusion in dauers, we have characterized a sheath cell-specific GFP reporter for the ver-1 gene, and have shown that it exhibits temperature- and dauer-dependent activity. Surprisingly, activity of ver-1::gfp depends on the LIM homeobox gene ttx-1. ttx-1 has been shown to be required to establish AFD thermosensory identity, and, in a thermal gradient assay, ttx-1 mutants demonstrate a constitutive cryophilic behavior. Ablation of AFD did not, however, affect temperature-dependent ver-1::gfp expression in glia. Using cell-specific promoters we demonstrated that expression of TTX-1 specifically within amphid sheath cells could restore ver-1::gfp reporter activity in ttx-1 mutants. In contrast, AFD neuron-specific expression of TTX-1 restored wild type thermotaxis behavior, suggesting that the glial temperature response does not contribute to this process. Using an electrophoretic mobility shift assay, we have identified a direct TTX-1 binding site within the temperature- and dauer-dependent ver-1 promoter, and used this to find other TTX-1 regulated genes. These results may indicate that within the nervous system, glia as well as neurons can possess sensory functions. As described, high temperature is also one of a few stresses that regulates dauer entry. We further find that in ttx-1 mutants the sheath glia fail to fuse in dauer larvae, resulting in aberrant extension of the dendritic ending of an amphid sensory neuron. Preliminary studies using cell specific promoters to restore TTX-1 function indicate that TTX-1 is required within the glia for this process. Collectively, these results may suggest that amphid sheath glia can respond to environmental stresses independently of sensory neurons, and in turn this may facilitate the remodeling of the sensory neurons and glia in response to stress in the dauer larva.

Roubin R et al. (2000) European Worm Meeting "Expression of VER-1 (Vascular Endothelial growth factor receptor Related) in amphid and phasmid sheath cells"

Popovici C, Roubin R, Birnbaum D

[European Worm Meeting, 2000]

An overall analysis of Tyrosine Kinase Receptors (RTKs) encoded by the C. elegansgenome has unveiled a family of four RTKs structurally related to Vascular Endothelial Growth Factor Receptors (VEGFRs) (Popovici et al, 1999). These genes are carried by the F59F3 and T17A3 cosmids. One EST for the gene F59F3.1 already existed in the Yugi Kohara's collection, and we were able to amplify two others (F59F3.5 and T17A3.1) by RT-PCR. Gene silencing done by RNAi on 2 of them indicated that these genes were not redundant. We focused on the T17A3.1 gene that we tentatively named ver-1 and studied its pattern of expression using a GFP fusion reporter gene construct. Ver-1::gfp expression was found mainly in amphid and phasmid sheath cells which have glial cells features. To overcome the absence of mutants, we made a construct devoid of the transmembrane and tyrosine kinase domains which was expected, when injected into wild type animals, to play a role as a soluble receptor for endogenous ligands. Overexpression of this soluble receptor led to less fertile animals than wild type, laying few eggs which often stop developing as oocytes. Surviving adults exhibited morphologic and functional alterations in their sheath cells and chemosensory neurons. When this construct was coinjected with ver-1::gfp, the sheath cells did not fluoresce anymore and their ability to be stained with ANEPS (a dye which preferentially colours sheath cells) was lost. In addition, the dye-filling of the chemosensory neurons was affected. We observed a posterior displacement of the lipophilic DiO filled amphid neurons and the appearance of additional neurons in the middle of the isthmus capable to be filled with the dye. The lack of ANEPS staining associated with the inability of visualising GFP positive cells are in favour of a loss of the sheath cell integrity in these animals. These features, related to the modifications in the dye-filling of chemosensorial neurons, are in favour of VER-1 playing a role in guidance and/or migration of sensory neurons. Thus, as already described in mammals for another related RTK family with 5 instead of 7 immunoglobulin domains (Platelet Derived Growth Factor Receptors, PDGFRs), VER-1 is produced by glial cells and may play a role in neurogenesis. It is one of the first observations in invertebrates of angiogenesis being related to neurogenesis. Studying VEGFR like in C. elegans, an animal devoid of a circulatory blood system, may help understand VEGFR function in mammals. Popovici C., Roubin R., Coulier F., Pontarotti P., Birnbaum D. (1999) - The Family of C. elegansTyrosine Kinase Receptors: Similarities and differences with Mammalian Receptors. Genome Res 9:1026-1039

Johnson, Christina K. et al. (2019) International Worm Meeting "Requirement of glial Na+/K+-ATPase in touch sensation highlights ionic and metabolic link between glia and touch neurons in C. elegans."

Johnson, Christina K., Bianchi, Laura, Wang, Ying, Fernandez Abascal, Jesus

[International Worm Meeting, 2019]

Isolated microenvironments, such as the tripartite synapse, where the concentration of ions is regulated independently from the surrounding tissues, exist throughout the nervous system, including in mechanoreceptors. Modulation of the ionic composition of these microenvironments has been suggested to be achieved by glia and other accessory cells. However, the molecular mechanisms of ionic regulation and effects on neuronal output and animal behavior are poorly understood. Using the model organism C. elegans, our lab published that Na+ channels of the DEG/ENaC family expressed in glia control neuronal Ca2+ transients and animal behavior in response to sensory stimuli. DEG/ENaC Na+ channels are known to establish a favorable driving force for K+ excretion, which occurs via inward rectifier K+ channels, in epithelial tissues across species. We hypothesized that a similar mechanism exists in the nervous system. Using molecular, genetic, in vivo imaging, and behavioral approaches, we showed that expression in glia of inward rectifier K+ channels and cationic channels rescues the sensory deficits caused by knock-out of glial DEG/ENaCs without disrupting neuronal morphology, supporting our hypothesis. Based on this model, Na+/K+-ATPases are also needed to maintain ionic concentrations following influx of Na+ and excretion of K+. We show here that, in addition to glial Na+ and K+ channels, two specific glial Na+/K+-ATPases, EAT-6 and CATP-1, are needed for touch sensation and that their requirement can be bypassed by a high glucose diet. The effect of glucose is dependent on ATP binding capability of the pump, translation, transcription, and the activity of CATP-2, a third Na+/K+-ATPase ?-subunit. Taken together, our results support metabolic and ionic cooperation between glia and neurons in C. elegans mechanosensors, a mechanism that is essential to regulating neuronal output and may be conserved across species.