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[
International C. elegans Meeting,
1997]
Previous studies of morphogenesis of the male tail tip by MH27 staining and Nomarski have revealed dramatic retraction events during fan formation, but were insufficient to explain the cellular events involved. Eleven late larval animals have been examined in serial thin sections by EM, 9 males at early phases of tail tip retraction and 2 hermpahrodites. Preservation of tissues was often excellent. At their birth, all Rn daughters (Rn.xx) begin as part of a single epidermal layer. Proximal neurites immediately begin growing toward the preanal ganglion and dendritic tips adhere to the cuticle almost as quickly. Retraction events overlap in time, and may be triggered by separate, discontinuous signals, since their absolute order varies. 1. Gap junctions connect posterior hyp cells 8, 9, 10 and 11 prior to any cell movements or syncytial formation; they may allow cell-cell signaling. 2. "Vacuoles" form in hyp cells 8-11. 3. Breakage of cell membranes leads to fusion of hyp 8-11. 4. Extracellular space increases, perhaps due to release of intracellular vacuoles by the hypodermis. 5. Many cells and nuclei begin to migrate anteriorward. 6. "New" cuticle appears on the surface and in central pockets of hyp 8-11; new secretion and involution may both be involved. Eventually, retraction pulls most tissues and cell bodies forward past the anus leaving only the sensory rays stretched across the fan.
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[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
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[
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.
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[
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.
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[
International Worm Meeting,
2017]
Cells release extracellular vesicles (EVs) that serve as nano-sized packages allowing for exchange of protein and genetic content. Cilia are hair-like projections that play important roles in development and signaling. The cilium both releases and binds to EVs. EVs play a role in cell signaling in health and pathologies, and may carry beneficial or toxic cargo. An understanding of the biogenesis, release, uptake, and signaling of ciliary EVs is lacking. Using C. elegans as a model, we aim to identify the molecules and mechanisms involved in EV biology. A subset of the ciliated neurons of C. elegans release EVs containing cargo that include the polycystins LOV-1 and PKD-2 and a myristoylated protein CIL-7 (Wang et al. Current Biology 2014; Maguire et al. MBoC 2015). Transcriptional profiling of the EV releasing neurons (EVNs) revealed candidates that could play a role in EV biogenesis and/or release (Wang et al. Current Biology 2015).
rab-28 is expressed in all ciliated neurons of C.elegans including the EVNs.
rab-28 encodes a small RAB GTPase. RAB-28 is important for amphid ciliary ultrastructure, amphid glial sheath cell volumes, and amphid-mediated sensory behaviors (Jensen. et al. PLoS Genetics 2016). Here, we explore the role of RAB-28 in EVNs. We investigated the role of RAB-28 in EV biology. We examined shedding and release of GFP-tagged EV cargoes using live imaging of
rab-28(
tm2636) mutants.
rab-28 mutants display altered localization of ciliary EV cargoes PKD-2 and CIL-7. Defects in the release of EV cargo from the tips of EVNs and/or alterations in the ciliary localization of GFP-tagged EV cargo in the EVNs may indicate defects in ciliary trafficking or defects in EV biogenesis and/or release. We are examining ciliary ultrastructure and EVs in
rab-28 mutant males using transmission electron microscopy (TEM). The EV releasing CEM cilia have a unique ciliary ultrastructure and are housed within the cephalic sensillum (Silva et al. Current Biology 2017). The male cephalic sensillum is comprised of CEM and CEP neurons, and glial support and socket cells, the latter create a lumenal space that contains EVs. We are determining whether
rab-28 regulates CEM ciliogenesis, the integrity of the cephalic sensillum, or EV biogenesis. Our work could shed light on the contribution of ciliary ultrastructure and cilia-glia interactions to EV biology.
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[
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.
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[
Biology of the C. elegans Male, Madison, WI,
2010]
Having completed the connectivity of the posterior nervous system of the C. elegans male, we are now pursuing reconstruction of the C. elegans male head. Unlike the posterior nervous system, which contains 85 male-specific neurons, 55 common neurons whose cell bodies are in the posterior and 18 processes of common neurons running into the tail from the anterior, the head is occupied by 200 common neurons and four male-specific neurons as well as 4 processes of male-specific neurons running through from the posterior to the anterior. Our present connectivity results demonstrated that some common neurons have sexually dimorphic wiring. They establish input and output synapses with male-specific neurons in the tail and also may be connected differently to each other. Therefore, we suppose that common neurons in the head may also display different wiring in the male compared to the hermaphrodite. Male-specific neuron processes which run into the head will establish further differences. In addition to copulation, male behavior differs from that of the hermaphrodite in several general ways, such as locomotion, chemotaxis, and attraction to food and mates. To compare male connectivity in the head to that of the hermaphrodite, we have begun to collect EM images of the male head. We chose males which were capable of mating and used traditional methods to fix and section them. We have 5000 serial sections of three animals' heads. We are collecting EM data from two available electron microscopes in Albert Einstein College of Medicine. Digital images are computationally aligned (see abstract by Xu et al) and neurons are traced using our reconstruction platform Elegance. If everything is on schedule, we can reconstruct the C. elegans male head within this year.
-
[
International C. elegans Meeting,
2001]
Electrophysiological properties of striated muscle cells were investigated with the patch clamp technique in the Nematode C elegans . Worms were immobilised with cyanoacrylate glue and longitudinally incised using a tungsten rod sharpened by electrolysis. Recording pipettes were sealed on GFP-expressing body wall muscle cells. In the whole cell configuration, under current clamp conditions, in the presence of Ascaris medium in the bath and K-rich solution in the pipette, no action potential could be induced in response to current injection. Under voltage clamp control and in the same ionic conditions, depolarizations above -30 mV from a holding potential of -70 mV gave rise to outward K currents. Outward K currents resulted from two components, one fast inactivating component blocked by 4-aminopyridine, one delayed sustained component blocked by tetraethylammonium. In the presence of both blockers, an inward Ca current was revealed and inhibited by cadmium. Single channel recording using the inside-out configuration revealed the existence of a Ca-activated Cl channel and a Ca-activated K channel. Single channel experiments are currently performed to characterise voltage-gated conductances at the unitary level.
-
Emmons, S, Wang, Y, Nguyen, K, Jarrell, T, Cook, S, Yakovlev, M, Hall, D
[
International Worm Meeting,
2015]
We have assembled for the first time complete wiring diagrams of the entire nervous systems of C. elegans adults of both sexes. For the hermaphrodite, we reanalyzed the electron micrographs that served as the basis for the 1986 publication "The Mind of a Worm" (White et al, 1986). For the male, we combined the posterior connectivity we published previously (Jarrell et al, 2012), obtained by analyzing Cambridge electron micrographs (Sulston et al, 1970), with data from our own series through a male head. For the pharynx we reanalyzed the micrographs analyzed previously by Donna Albertson (Albertson and Thomson, 1976). For some neurons, we added data from new electron micrographs of legacy grids. Notably, we added connectivity of SAB motorneurons to body wall muscles in the head (muscles 1-7); these neurons contribute 37% of the total input to these muscles. Finally, significant gaps still remained: to our knowledge, a region posterior of the vulva (posterior of the N2U series, anterior of the male N2Y series) has never been examined by electron microscopy. To fill in these areas, we added connections to complete the chains of motor neurons and muscles by assuming a regular structure. Connectivity matrices, neuron maps, and synapse lists are available on our website: WormWiring.org. Analysis of the complete, quantitative datasets by graph layout algorithms yields biologically meaningful displays. When an algorithm is applied that utilizes the "spring-electric" approach, in which nodes repel each other uniformly and attract according to the strength of their connectivity (Allegro Layout, allegroviva.com), neurons of similar or related function are grouped together. At the next higher level, clear modules are separated. When the arrangement is graphically illustrated using the display tools of Cytoscape, the functional pathways and hierarchical arrangement of the entire worm neuromuscular system are revealed-the layout closely matches the worm's anatomy. The overall arrangement is the same in both sexes, with the addition in the hermaphrodite of the vulva muscles and circuits and to the male "tail" of the enormous copulatory circuits. Along with the sensory pathways for head contact and for odorants and chemicals, pathways leading from pheromone sensing neurons reveal a sexual circuit in the head, elements of which (AVF, PVQ, and RIM interneurons) are shared by both sexes, along with, in the hermaphrodite, the HSN neurons, and in the male, the CEM sensory neurons and the EF and MCM interneurons (for the new MCM interneurons, see the Abstract by Sammut et al).
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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
Invertebrate axons and those of the mammalian peripheral nervous system are able to regenerate in the adult. Functional recovery takes place when a damaged axon regains connection with its target tissues. In C. elegans, following laser axotomy, the regrowing axon still attached to cell body (proximal) is able to reconnect with its separated distal segment through unknown mechanisms. Using the mechanosensory neurons ALM and PLM as a model system, we have found that during axonal regeneration reconnection between the proximal and distal axonal fragments occurs through a mechanism of axonal fusion, with reestablishment of cytoplasmic and membrane continuity. We found that when axonal fusion does not occur the distal fragment inevitably undergoes Wallerian degeneration and the original axonal tract cannot be restored. Through the use of dual colour labeling of adjacent axonal pairs, we found a high level of specific recognition occurring between a proximal re-growing axon and its own separated distal fragment, revealing possible cross talk between the two processes. Finally, from a candidate mutant approach, we have identified a molecule with homology to a human protein implicated in axonal degeneration, as being necessary for successful regeneration and specifically involved in the process of axonal fusion. We anticipate that a similar mechanism of axonal regeneration could be exploited to improve the outcome of axonal regeneration following injury in mammalian systems.