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[
International C. elegans Meeting,
2001]
Filarial parasites of the genus Brugia live in mammals (including humans) and are transmitted between hosts by the bite of a blood-feeding mosquito vector. The infective form for the mosquito is the L1 or microfilariae (Mf) a life cycle stage that is highly adapted for life in the bloodstream of the mammal. Mf are developmentally arrested and undergo no further development until ingested by a mosquito. The link between the progression of the developmental cycle and the transition between hosts implies that the Mf has a mechanism by which it can sense its changing environment. Results in other systems have shown that heat shock factor (HSF) can act as a cellular thermometer directly monitoring temperature and oxidative state. As temperature is one of the major differences between the mosquito and mammalian hosts we are interested in investigating the role of HSF in developmental progression. As the tools for functional genomics do not exist for Brugia, we are using C. elegans as a model system in which to define the role of HSF in a nematode. ACeDB identifies a single HSF-like gene with a highly conserved DNA binding domain. We have used an RNAi feeding vector to determine the likely function of HSF in the nematode. A number of different phenotypes were apparent at 20oC including: slow growth, scrawny appearance, thermo-sensitivity, decreased fertility and egg-laying defective. At 25oC these phenotypes were more pronounced. In an
hsp-16/GFP reporter background, RNAi abolished GFP expression in the intestine but not in the pharynx or nerve ring. These results are consistent with a requirement for HSF function in the gut. Ongoing studies are focused on determining the spatial and temporal expression pattern of hsf throughout development. We aim to determine the pathways in which HSF is active under normal developmental conditions and to identify down-stream targets of HSF.
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[
West Coast Worm Meeting
]
Are there multi-neuron computational modules in the C. elegans nervous system? We attempt to answer this question by applying a systematic statistical approach to the C. elegans wiring diagram (White et al. 1976). Our approach is to identify multi-neuron inter-connectivity patterns that are significantly over-represented in the actual wiring diagram compared to the randomized wiring diagram, which preserves the number of synapses per neuron but not the identity of connections. To do this we compute the numbers of occurrences of all n-neuron (n=2...5) inter-connectivity patterns in the actual and randomized wiring diagrams. This statistical approach confirms previous reports of the over-abundance of reciprocal connections and triangular connectivity patterns (White et al. 1976). Moreover, we discover several new four-neuron and five-neuron inter-connectivity patterns that appear significantly more often in C. elegans than in randomized wiring diagrams. We suggest that these inter-connectivity patterns may serve as computational modules that perform stereotypical functions.
-
[
West Coast Worm Meeting,
2002]
Are there multi-neuron computational modules in the C. elegans nervous system? We attempt to answer this question by applying a systematic statistical approach to the C. elegans wiring diagram (White et al. 1976). Our approach is to identify multi-neuron inter-connectivity patterns that are significantly over-represented in the actual wiring diagram compared to the randomized wiring diagram, which preserves the number of synapses per neuron but not the identity of connections. To do this we compute the numbers of occurrences of all n-neuron (n=2...5) inter-connectivity patterns in the actual and randomized wiring diagrams. This statistical approach confirms previous reports of the over-abundance of reciprocal connections and triangular connectivity patterns (White et al. 1976). Moreover, we discover several new four-neuron and five-neuron inter-connectivity patterns that appear significantly more often in C. elegans than in randomized wiring diagrams. We suggest that these inter-connectivity patterns may serve as computational modules that perform stereotypical functions.
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[
European Worm Meeting,
2002]
Filarial parasites of the genus Brugia live in mammals (including humans) and are transmitted between hosts by the bite of a blood-feeding mosquito vector. The infective form for the mosquito is the L1 or microfilariae (Mf) a life cycle stage that is highly adapted for life in the bloodstream of the mammal. Mf are developmentally arrested and undergo no further development until ingested by a mosquito. The link between the progression of the developmental cycle and the transition between hosts implies that the Mf has a mechanism by which it can sense its changing environment. Results in other systems have shown that heat shock factor (HSF) can act as a cellular thermometer directly monitoring temperature and oxidative state. As temperature is one of the major differences between the mosquito and mammalian hosts we are interested in investigating the role of HSF in developmental progression. As the tools for functional genomics do not exist for Brugia, we are using C. elegans as a model system in which to define the role of HSF in a nematode. ACeDB identifies a single HSF-like gene with a highly conserved DNA binding domain. Using an RNAi feeding vector containing fragments of different lengths of C.elegans HSF, we have defined a number of phenotypes. The penetrance of these phenotypes increases with the size of the RNAi fragment and higher growth temperatures. RNAi treated worms have defects in thermotolerance, lifespan, fertility and egg-laying. In addition treated worms are significantly smaller and have a scrawny appearance. In an hsp- 16/GFP reporter background, RNAi abolished GFP expression in the intestine but not in the pharynx or nerve ring. The decrease in HSP-16 levels has been confirmed by immunoblotting. Using a yolk protein/GFP reporter, flourescence in the oocytes is significantly reduced in RNAi treated worms. These results may relate to defects in gut function in RNAi treated worms, and this is consistent with a decreased gut function as defined by feeding assays. Ongoing studies are focused on determining the spatial and temporal expression pattern of hsf throughout development. Our aims are to determine the pathways in which HSF is active under normal developmental conditions and to identify down-stream targets of HSF.
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[
International Worm Meeting,
2015]
A connectome is a comprehensive map of all neural connections in an organism's nervous system. The first connectome was published almost 30 years ago by White et al. (1986) and described the structure of the nervous system of the nematode C. elegans adult hermaphrodite. Subsequent network analyses of this data have focused only on the synaptic connectivity of the nervous system, while neglecting much of the spatial information in the data. Initial spatial analyses of the C. elegans connectome reported in (White et al., 1983; Durbin, 1987) used only a sparse sampling of physical neuron contacts. Using the original electron micrographs from (White et al., 1986), we have extended this analysis by performing a 3D reconstruction of every neuron in the C. elegans nerve ring in both the L4 and adult. This represents the first complete volumetric reconstruction of the main neuropil of any animal from multiple developmental stages. With this enriched data set, we have been able to do a comparative analysis of synaptic connectivity, characterize the spatial distribution of synapses for each neuron and analyse the relationship between neuron contact and synapse formation in the C. elegans nerve ring. Similar to (White et al., 1983), we found that ~40% of all possible physical contacts result in a synapse or gap junction. We also found a positive correlation between the frequency of synapse formation and the amount of physical contact between neurons. Specifically, the frequency of synapse formation between two neurons approaches ~0.7 as the amount of physical contact approaches 10% of a neuron's total measured surface area. However, like (Durbin, 1987), we find that synapse probability and synapse number between any pair neurons does not depend strongly on the amount of shared physical contact. Furthermore, synapse volumes appear to be conserved between the L4 and adult, while the number of synapses between any two neurons appear to be, on average, greater in the adult. This could suggest that during late nervous system development, synaptic partnerships are reinforced by creating additional small synapses between neurons rather than enlarging the volume of current synapses. .
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[
Japanese Worm Meeting,
2002]
The synaptic connectivity of C. elegans is well known from observations of the somatic system by White et al. and those of the pharyngeal system by Albertson et al. So far, three databases were constructed for computational usage by Achacoso et al. and Durbin, and recently in WormBase. However, they lack some data such as those in tables of White's paper and those in figures of Albertson's book. Our database (K. Oshio, S. Morita, Y. Osana and K. Oka: Technical Report of CCEP, Keio Future No.1, 1998) includes all data described in White's paper and Albertson's book. Unfortunately, some mistakes were found in the database through private communications with John White who is the author of White's paper and with the users of the database. Thus we have been proceeding with the revision to make it perfect one. We are planning to complete the revision in September 2002. The database should be worthwhile not only for neurophysiological studies, but also for post-genomic interests mediating genomes and behavior.
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[
International Worm Meeting,
2015]
In an RNAi-based modifier screen we identified 3 intermediate filaments (IFs: IFA-4, IFB-1, IFC-2) that enhanced the cystic ERM-1-overexpression excretory canal (EC) phenotype. Although IFB-1's function in EC morphogenesis has been noted, it remains unclear how IFs contribute to this process. Here we compare IFs' role in EC tubulogenesis with the roles of microfilaments (MFs; previously analyzed) and microtubules (MTs). IFs were found to build an intracellular perilumenal lattice, and to be non-redundantly required for lumen extension. Severe interference with IFs causes cell-body close cysts and loss of canal extension, but does not abort lumen formation. In contrast, severe interference with MFs via ERM-1/ACT-5, that we think expand the lumenal membrane via vesicle fusion, abolishes lumen formation. Selective IF removal during larval EC extension generates a "multiple lumen varicosity" phenotype with collapsed lumens between varicosities (periodic structures along extending canals that serve as osmotically sensitive "fueling stations" for wild-type growth). In contrast, larval MF removal produces short, thin lumens with small vacuoles at canal tips. IF removal generates true ectopic "lumens" (vacuoles lined with an apical membrane and cytoskeleton) not seen with MF removal. This scenario could suggest that IFs function by laterally integrating vacuoles into a lumenal membrane that has already acquired an actin coat, as opposed to MF-dependent membrane expansion via vesicle fusion at the tip that concomitantly assembles actin. A TBB-2/tubulin::GFP fusion localizes to the EC cytoplasm, but also to strands on the perilumenal IF lattice that in turn resides on perilumenal MFs. Colocalization and loss-of-function studies during EC development suggest that ERM-1 indirectly recruits lumenal IFs, whereas perilumenal lattice formation may depend on MTs. Interference with MTs copies the IF phenotypes and, unlike interference with MFs, enhances it. Thus, MTs like IFs support single lumen maintenance and lateral vacuole fusion, suggesting that one of MTs' roles in lumen extension is mediated by IFs. Effects of the 3 cytoskeletal components on endosomal and canalicular vesicles will be presented. Our findings suggest that EC lumen extension relies on tip and lateral growth and requires a resilient perilumenal IF matrix that allows vacuoles to dock at the lateral lumen (conspicuous in varicosities), thereby shaping the lumen's cylindrical tunnel structure and transmitting fluid pressure between varicosities that maintains lumen diameter and supports its anterior-posterior extension. .
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[
International Worm Meeting,
2007]
Caenorhabditis elegans embryos undergo stereotypic cell division patterns. Because the cleavage plane for each cell division is defined by the orientation of the mitotic spindle, positioning of spindle poles, or centrosomes, is crucial to regulate cell division patterns. Previous reports revealed that centrosomes are positioned by interactions between microtubules and the particular region of the cell cortex (1-3). However, a detailed mechanistic understanding of centrosome movement is still limited. As a step to better understand the regulation of centrosome movement in early cell divisions, we are developing a quantitative approach to analyze dynamic movement of centrosomes at high spatial and temporal resolution. 4D images of embryos expressing GFP markers visualizing centrosomes and cell membrane are acquired using a spinning-disc confocal microscope, and positions of individual centrosomes are extracted from each image. Using these positional data, direction and speed of each centrosome movement, and rotation angle and distance of each centrosome pair are quantitatively and statistically analyzed in a 3-dimensional space. We will present our progress on the analysis of wild-type and mutant embryos having spindle orientation defects. 1.Hyman and White (1987) J. Cell Biol. 105:2123-2135. 2.Hyman (1989) J. Cell Biol. 109:1185-1193. 3.Keating and White (1998) J. Cell Sci. 111:3027-3033. .
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[
East Coast Worm Meeting,
1998]
Uncoordinated movement in
unc-37 adults is superficially similar to the impairment observed in
unc-4 animals (1). In
unc-4, loss of coordination results from a specific wiring deficit between interneurons and motorneurons in the ventral nerve cord (3,4). We are currently analyzing the detailed synaptic pattern in an adult
unc-37 nerve cord from serial thin sections. Preliminary data were discussed previously (2). We have now identified the major interneurons and 20 motorneurons in the anterior ventral nerve cord, and have determined their synaptic interactions. The morphological phenotype of
unc-37 is not as limited as the specific wiring defect in
unc-4. Many neurons show minor changes in branching or axon caliber, and there is a wider variety of wiring changes. However, in both mutations the AVB interneurons form inappropriate gap junctions onto class A targets, while AVAs fail to make normal junctions onto these targets. This specific change may explain the similarity in their gross behavioral phenotypes. 1. Pflugrad et al. (1997) Development 124:1699-1709. 2. Hall, German and Miller (1997) 11th Annual C. elegans meeting. 3. J.G. White et al. (1986) Phil. Trans. R. Soc. Lond 314:1-340. 4. J.G. White et al. (1992) Nature 355:838-41.
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[
International C. elegans Meeting,
1999]
From the detailed report of White et al. and Albertson et al. , we have almost complete knowledge about the synaptic connectivity of C. elegans with the type of synapse (electric junction or chemical synapse). However, the type of each chemical synapse (excitatory or inhibitory one) is not described. Conventional electrophysiological methods for C. elegans is difficult because the size of the neurons is too small to penetrate the intracellular electrode. On the other hand, computational studies of neuronal circuit are now possible by virtue of the above mentioned elaborate experimental studies of neuroanatomists. We have built a new data base of the whole neuronal circuit including pharyngeal neurons only from the article of White et al. and Albertson et al. There exist two other data bases to the knowledge of the present authors. The first data base was constructed by Achacoso and Yamamoto who also analyzed the properties of the network. Another data base was constructed from the article of White et al. by Durbin, which is available on the internet homepage. To begin understanding signal processing on the nervous system, we have investigated the neuronal connectivity by putting random walkers on certain neurons of the network. Here we ignore internal structure of neurons. Random walker is a particle which moves among neurons randomly, and can be considered to be transmitted signal. In our simulation, random walkers are put on certain sensory neurons at each time step, this corresponds to stimulation which sensory neurons accept. We removed random walkers at certain motor neurons, this means, for instance, signals are transmitted to muscles which cause normal action. As a result, we have found that the degree of relation of each neuron for input neurons can be known from the probability to find random walker, although the difference between excitatory and inhibitory of chemical synapses is not taken into account. The simulational results will be shown comparing with real function such as the touch sensitivity. E-mail: oshio@future.st.keio.ac.jp