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[
International Worm Meeting,
2015]
When grown under conditions of low food, high temperature and high population density, C. elegans larvae will develop through an alternative, adaptive stage called dauer. While in dauer the larva show changes in behavior and morphology. For example, we have shown six Inner Labial 2 (IL2) neurons undergo dramatic reorganization during dauer1. IL2 quadrant (IL2Q) neurons arborize extensively during dauer and resorb their branches upon recovery from dauer. Moreover animals which have passed through dauer as part of development show several differences during adulthood in comparison to adults which have not experienced dauer. We show the IL2Q neurons resorb the bulk of their branches but remnant branches remain in post-dauer adults. This unique feature of IL2Q branching provides us with a model for testing proteins that help the cell recover from stress. We address the question of how the IL2Q neurons show a three-fold increase in dendritic length, which is maintained throughout dauer, when the worm is trying to conserve energy. We are examining the role of autophagy in IL2 branching. Autophagy which is upregulated during dauer, is a mechanism through which the cell degrades intracellular components and is widely conserved across species. Autophagy is an adaptive stress response that promotes survival under adverse conditions. C. elegans orthologues to yeast autophagy proteins are critical for both C.elegans autophagy and dauer formation.2 The basic steps of autophagy include vesicle nucleation, vesicle elongation, docking with the lysosome, and vesicle breakdown and degradation. We found that LGG-1::gfp, a widely used marker for autophagy, is expressed in the IL2s during dauer . We can use the temporal expression of LGG-1 to determine at which time points during dauer formation and recovery autophagy is upregulated in the IL2 neurons. Additionally we are assessing
unc-51 mutants for IL2 branching and branch recovery defects; UNC-51 is involved in induction of autophagy.
unc-51 animals have been described as forming 70% abnormal dauers2 - to address differential dauer formation, we are examining IL2 arbors in both abnormal and normally formed dauers. 1)Schroeder NE, Androwski RJ, Rashid A, Lee H, Lee J, Barr MM. Curr Biol. 2013 Aug 19;23(16):1527-35.2)Melendez A1, Talloczy Z, Seaman M, Eskelinen EL, Hall DH, Levine B. Science. 2003 Sep 5;301(5638):1387-91.
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[
International Worm Meeting,
2013]
Under adverse environmental conditions, C. elegans enters a stress resistant dauer stage. We discovered a wild-type dauer-specific phenotype wherein six inner labial sensory neurons (IL2) undergo dramatic reorganization including extensive arborization of the IL2 quadrant (IL2Q) neurons (see N. Schroeder et al abstract). To identify genes involved in dauer-specific IL2 remodeling , we used a candidate gene approach. Representative genes were examined from several molecular pathways: dauer formation, ciliogenesis and intraflagellar transport, Notch signaling, cell fate and axon guidance. For example, both UNC-86 and LIN-32 are transcription factors necessary for IL2 cell fate. Most
unc-86 alleles are defective in IL2 formation. However,
unc-86(
n848) mutants show normal IL2 morphology in non-dauers, but defects in dauer-specific IL2 arbors. Interestingly,
lin-32 mutants that form IL2 neurons show no obvious defects in arborization. The RFX-transcription factor DAF-19, is a master regulator of ciliogenesis.
daf-19(
m86) mutants lack all sensory cilia, but show extranumerary branching in the lateral IL2s suggesting a role for cilia in inhibiting arborization.
Through a forward genetic screen we identified
kpc-1, a
kex2-like proprotein convertase and furin homolog, as required for organized arborization in both dauer IL2 neurons (See N Schroeder et al abstract) and adult PVD and FLP multidendritic neurons (see Rashid et al abstract). To identify potential KPC-1 substrates, we cross referenced genes upregulated in PVDs (1), genes upregulated during dauer (2), and genes containing putative proprotein cleavage sites (3). We have assembled a list of 159 potential regulators of dendritic branching which we are beginning to characterize.
1.Smith et al. Dev Bio. 345 (2010) 18-33.
2.Jeong et al. PLoS One. (2009 Jan) 4(1)
e4162.
3.Duckert et al. Protein Eng Des Sel. 17 (2004) 107-112.
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[
International Worm Meeting,
2019]
Environmental stress can significantly influence the shape of dendrites and dendrite morphology plays a key role in proper neural signaling. We aimed to determine the underlying molecules that regulate stress-induced dendritic arborization. When C. elegans are grown under well-fed conditions, the two FLP neurons located in the head begin to arborize rapidly during L4, these arbors persist through adulthood. During the stress-induced dauer stage, four quadrant IL2 neurons arborize similarly to the FLPs, extending arbors out to the body wall and covering the head of the worm. We observed that the FLPs maintain an unbranched state during dauer. Using a forward genetic screen, we identified the membrane-bound receptor DMA-1, which was previously shown to regulate FLP branching, as essential for dauer-specific IL2 arborization. We found that the IL2s use identical DMA-1 binding partners during IL2 dauer arborization. In adult animals a DMA-1::GFP translational reporter clearly localizes to the FLP. Using this same reporter, we observed DMA-1 localization in the IL2 dendrite during dauer, though not during other developmental stages. However, overexpression in the IL2s did not induce branching, suggesting that additional dauer-specific components regulate IL2 arborization during dauer and that the IL2s are inhibited from branching in the adult animal. Intracellularly, DMA-1 enables actin polymerization through interactions with the HPO-30 and TIAM-1. Our mutant analysis shows that HPO-30 and TIAM-1 are necessary for IL2 arborization. While the DMA-1 complex is needed we've found that several of the regulators of the complex in FLP/PVD are dispensable in the IL2s. For example, the unfolded protein response protein IRE-1 is required for proper DMA-1 localization in the PVD/FLPs but is dispensable in dauer IL2s. However, the FOXO transcription factor, DAF-16 can compensate for a blocked UPR. We found that
daf-16;
daf-7 partial dauers are defective for IL2 arborization while the FLPs remain unaffected. Altogether our results show that DMA-1 functions during dauer to mediate stress-induced dendrite plasticity in the IL2 neurons. The role of DMA-1 during dauer mirrors its function in the adult FLPs. Both extracellular and intracellular DMA-1 binding partners are necessary for IL2 arborization. However, DMA-1 is regulated uniquely during dauer.
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Barr, Maureen, Lee, Junho, Schroeder, Nathan, Lee, Harksun, Androwski, Rebecca, Rashid, Alina
[
International Worm Meeting,
2013]
Neuroplasticity in response to adverse environmental conditions can entail both hypertrophy and resorption of dendrites. How dendrite remodeling occurs in response to unfavorable environmental conditions is unclear. We discovered that the six IL2 sensory neurons undergo dendrite remodeling during development of the stress-resistant dauer stage. Based on our findings we divide the IL2 neurons into two separate anatomical classes. The four IL2Q (quadrant) neurons undergo extensive dendritic arborization and a shift from bipolar to multipolar neurons during dauer formation. The two IL2Ls (lateral) extend only a single additional process during dauer formation. During dauer recovery, the IL2 arbor retracts, leaving behind remnant branches in post-dauer L4 and adult animals. We isolated a mutation in
kpc-1 (
kex2/subtilisin-like proprotein convertase), from a forward genetic screen, which results in disorganized and truncated IL2Q arbors. In mammals, the KPC-1 homolog furin is responsible for the cleavage and activation of numerous proproteins associated with various pathologies including neurodegenerative diseases. While broadly expressed in C. elegans,
kpc-1 is upregulated in dauer IL2 neurons and acts cell autonomously in the regulation of dauer-specific arborization. The IL2s are required for nictation behavior. We found that
kpc-1 mutant dauers are defective for nictation.
kpc-1 is also required for multidendritic neuron morphology and behavior during non-dauer stages (See Rashid et al. abstract) suggesting that, similar to furin, KPC-1 plays multiple roles in C. elegans. We are currently searching for potential substrates of KPC-1 that affect dauer-specific IL2 remodeling (See Androwski et al. abstract). The C. elegans IL2 sensory neurons provide a paradigm to study stress-induced reversible neuroplasticity, and the role of environmental and developmental cues in this process. Our discovery of KPC-1 as required for dendrite morphogenesis provides insight into the role of proprotein convertases in nervous system development.
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[
International Worm Meeting,
2015]
While the genetic architecture of C. elegans is well-studied, the adaptability of various strains and their reproductive fitness is less understood. To address this, an experimental-based model will allow for characterization of C. elegans adaptability amongst strains which may exhibit low reproductive potential and/or survivability under selection. Two experimental approaches representing within- and between-generation survivability (preconditioning and experimental evolution, respectively) will allow survivability to be assessed. Preconditioning [1] assesses adaptability within a generation using environmental selection, while experimental evolution [2] assesses adaptability via cross-generational artificial selection. For a given strain, populations grown from single founder individuals are repeatedly subject to genetic drift and then selected for fecundity (largest brood size). These results demonstrate how specific genotypes exhibit robustness in the face of adaptive constraints.Biological adaptability involves both survivability and reproductive fitness. Survivability is the degree to which a single founder worm can survive and produce viable clones (e.g. the number of generations constituting a single genealogy). Reproductive fitness is the mean population size for a given genealogy. In strains with high survivability, founder populations with high reproductive fitness should also generate subsequent founder populations with a high degree of survivability. Strains with high reproductive fitness but low overall progeny sizes may also exhibit high adaptability if selection results enhances adaptation to varying environmental conditions. While this model does not control for stochastic effects and demographic constraints [3], it does allow for genealogies with consistently high survivability [4].1. Calabrese, E.J. et.al, Tox App Pharm, 222(1), 122-128 (2007).2. Kawecki, T.J. et.al, Trends Ecol Evol, 27(10), 547-560 (2012).3. Szendro, I.G. et.al, PNAS, 110(2), 571-576 (2012).4. Wahl, L.M., Krakauer, D.C., Genetics, 156(3), 1437-1448 (2000).
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[
International Worm Meeting,
2011]
Dendrite morphology and plasticity profoundly affect neuronal signaling and behavioral outputs (1). However, little is known regarding the molecular signals governing morphogenesis of dendrite structure. During non-dauer stages of C. elegans development the inner labial (IL2) neurons, a set of six ciliated putative chemosensory neurons, display a bipolar morphology with an unbranched dendrite and axon. Under adverse environmental conditions, C. elegans can develop into a dauer larva, an alternative juvenile stage with altered morphology and behavior. We found that during the dauer stage, the IL2 neurons exhibit hierarchical dendritic branching and a switch from a bipolar to multipolar morphology.
During dauer formation the ventral and dorsal IL2 primary dendrites establish branching and extend de novo processes from the cell bodies which undergo additional branching. The lateral IL2 neurons branch exclusively at the distal dendrites, forming a circular "crown" extending around the circumference of the head. Using time-lapse imaging, we found that plasticity in the IL2s begins with the onset of the dauer molt. Following the cessation of pharyngeal pumping puncta begin forming and resorbing in a dynamic fashion along the primary dendrite for several hours. Rapid and dynamic branch formation with periodic pruning events occur during a 3-4 hour period preceding radial shrinkage. Following recovery from dauer, the branches are incompletely resorbed, leaving behind occasional remnant secondary branches.
Using a forward genetics screen, we isolated 28 candidate mutants with branching defects. Variations in defects include ectopic branches, disorganized branching or an incomplete crown. Several mutants were backcrossed and are being identified using traditional mapping and whole genome sequencing. We are currently using laser ablation to examine tiling and possible roles of surrounding tissue on IL2 branching. Additionally, we are testing candidate genes that may play a role in the IL2 dauer branching phenotype. Various developmental disorders are associated with defects in dendrite structure (1). IL2 branching in dauers may serve as a new and rapid model to understand the molecular basis of arborization and dendritic pruning that underlie these disorders.
1. Jan and Jan. 2010. Nat. Rev. Neurosci. 11:316-328.
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[
European Worm Meeting,
2006]
Rebecca Hall, Peter Klappa and Fritz A. Mhlschlegel. Human pathogenic nematodes infect over 160 million people per year. Brugia malayi and Onchocerca volvulus are the causative agents of elephantiasis and river blindness, respectively. Treatment options are restricted and the most commonly used drug, Ivermection, is stage specific and has significant side effects. Furthermore, treatment failures due to drug resistance have been reported.. Brugia malayi and O. volvulus exhibit an indirect life cycle where an incubation period within an intermediate host vector is essential for development of the infectious stage. During host-vector transition pathogenic nematodes are exposed to extreme environmental changes including variations in pH. We decided to exploit the different environmental pH conditions encountered, in order to identify new therapeutic targets. Using C. elegans as a model we designed a survival assay and were able to show that the nematodes are extremely resistant to environmental pH changes. This result was shown to be independent of the cuticle by the use of collagen cuticle mutants. Microarray experiments identified a set of pH-regulated genes. These genes were up regulated at pH9, an environment encountered by pathogenic nematodes residing in the vector gut. The genes were silenced using RNA mediated interference to establish their requirement for survival. There were no significant phenotypic changes observed after five days of treatment, and exposure of the silenced nematodes to acidic and alkaline environments did not significantly reduce survival. However, in vivo studies are currently being carried out using carbonic anhydrase inhibitors to identify whether the candidate genes have potential for drug targeting.
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[
Worm Breeder's Gazette,
1975]
As most people are probably aware by now, I first started by collecting a large number of temperature-sensitive mutants that are blocked in the reproductive life cycle. These mutants were then placed into six phenotypic categories. The zygote-defective mutants are those that lay fertilized eggs that fail to hatch. Gonadogenesis- defective mutants are those that when reared at restrictive temperature produce neither fertilized eggs nor progeny. Spermatogenesis mutants are those that when reared at restrictive temperature, can be rescued for progeny production by mating with wild type males at restrictive temperature. The accumulators are those that grow to an intermediate larval stage and either stop growing or die at an immature stage. The abnormal F1 are mutants that when placed at restrictive temperature grow up to be adults, produce progeny, but those progeny grow up to be sterile adults. In addition, a few temperature sensitive morphological mutants were isolated. Rebecca Vanderslice and I originally studied in greater detail three particular mutants in the zygote-defective category. They were interesting in two respects. (1) Not only did they show zygote- defective phenotype when the adults were placed in restrictive temperature, they also showed gonadogenesis-defective phenotype if they had been reared from L1 onward at high temperature. (2) Each of the three mutants is a maternal-effect mutant. At the present time, Becky Vanderslice and Bill Wood are re-examining the zygote-defective class and the gonadogenesis-defective class to find out which are and which are not maternal-effect mutants. One of the central questions we are asking is, what is the distribution of critical times of temperature sensitivity among the mutants and which of these are maternal effect mutants? We're asking how far into development do the mutants go with maternal contributions.
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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,
2015]
The IL2 neurons in C. elegans, are a set of 6 sensory neurons located posteriorly to the metacorpus. Throughout most of the animals' life cycle, the IL2s maintain themselves as cell bodies with a single dendrite extending to the nose and an axon extending into the nerve ring. When exposed to environmental stress, the animals will enter the dauer-larval stage in which they exhibit many morphological changes. Included amongst these changes is the remodeling of the IL2 neurons. During the molt into dauer, the IL2 neurons undergo an extensive arborization phenomenon, in which the neurons will show a three-fold increase in dendritic length. During this stage-specific arborization, the dorsal and ventral dendrites extend processes up to the midline, anteriorly and posteriorly along the midline and down the body walls, and the cell bodies also form additional dauer-specific primary dendrites from the cell body1. The mechanisms by which these neurons and their newly formed arbors are maintained is a question that we are currently addressing. A screen of IL2 branching phenotype candidates revealed DYF-7 as a gene of interest. DYF-7 encodes a transmembrane protein required to anchor dendritic tips during retrograde extension2. Though there were no obvious IL2 branching phenotypes associated with this mutant, we did observe dauer-specific defects in cell-body maintenance. In
dyf-7 dauer mutants, the cell bodies were observed to be unorganized and scattered throughout the anterior portion of the animal. However, cell-body maintenance assays of L1 larvae and young-adult animals that had not gone through dauer showed no statistical differences between mutant and wild-type animals. This suggests that DYF-7 is required for maintenance of cell body position during dauer formation. Ongoing experiments seek to determine the cause of this maintenance defect, and attempt to determine the role that DYF-7 plays in cell-body stabilization during dauer formation. In addition, we have begun exploring transmission electron microscopy as a tool to allow us to observe these neurons at the substructural level.1) Schroeder NE, Androwski RJ, Rashid A, Lee H, Lee J, et al. (2013) Dauer-specific dendrite arborization in C. elegans is regulated by KPC-1/Furin. Curr Biol 23: 1527-1535. doi: 10.1016/j.cub.2013.06.0582) Heiman MG, Shaham S (2009) DEX-1 and DYF-7 establish sensory dendrite length by anchoring dendritic tips during cell migration. Cell 137: 344-355 doi: 10.1016/j.cell.2009.01.05719344940.