Schroeder, Nathan et al. (2013) International Worm Meeting "Reversible dendrite arborization in dauers is regulated by KPC-1/furin."

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.

Schroeder, Nathan E et al. (2011) International Worm Meeting "Rapid and reversible arborization of IL2 neurons during dauer development."

Schroeder, Nathan E, Barr, Maureen M, Androwski, Rebecca J

[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.

Schroeder, Nathan E et al. (2009) International Worm Meeting "Why do dauer and non-dauer C. elegans behave differently?"

Schroeder, Nathan E, Barr, Maureen M

[International Worm Meeting, 2009]

The C. elegans dauer larva is an alternative juvenile stage that is formed under conditions of low food and high population density. Dauers differ from well-fed larvae in both altered sensory neuroanatomy and behavioral repertoire. For example, dauers often lie motionless in a quiescent state. However, dauers are also capable of nictation behavior, in which the animal climbs onto projections and stands on its tail. To analyze the causes of dauer-specific behaviors, we are comparing the sensory anatomy and behavior of dauers to well-fed larval and adult stages. To establish a baseline comparison, we are performing a battery of behavioral assays on dauers versus non-dauers. We have found differences in chemotaxis behavior between dauers and well-fed adults in standard chemotaxis assays. Nictation behavior on exhausted culture plates was observed, and may be separated into four components: climbing, where nematodes move up a projection; waving, where the nematode stands on its tail while moving in three-dimensional loops and spirals; standing, where the animal maintains a rigid posture for extended periods of time while standing on its tail; and swarming, in which tens of nematodes aggregate together on top of a single projection. To study the molecular genetics of nictation behavior, we are starting with a candidate gene approach. To examine anatomy, we are using dye-filling. Amphid and phasmid neurons in dauers stained with DiI, DiO, and DiD in a time and compound-dependent manner. IL2 neurons in dauers never filled, confirming previous results that these neurons are less accessible to the environment in the dauer stage (1). Future work includes the continued collection of baseline behavioral data on dauers, an analysis of dauer quiescence, the use of microfluidic substrates to analyze nictation behavior, and a comparison of reporter gene expression in dauer and non-dauer animals. Given the known changes that occur in behavior and anatomy between dauers and well-fed C. elegans, this work may lead to new discoveries in neuroplasticity. Additionally, the homology shown between dauers and the infective stage of many parasitic nematodes enables the use of the well-established tools of C. elegans to better understand the infective process of parasites that are estimated to afflict one-third of the world''s population. 1.Albert and Riddle. 1983. J. Comp. Neur. 219:461-481.

Androwski, Rebecca J et al. (2013) International Worm Meeting "Dendritic arborization in dauer IL2 neurons: Genetic and bioinformatic analyses."

Androwski, Rebecca J, Rashid, Alina, Barr, Maureen M, Schroeder, Nathan E, Ritter, Tom

[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.

Androwski, Rebecca J et al. (2015) International Worm Meeting "Role of autophagy in IL2 dendritic arbors during dauer formation and recovery."

Androwski, Rebecca J, Schroeder, Nathan E, Flatt, Kristen M

[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.

Alicea, Bradly et al. (2015) International Worm Meeting "An experimental evolution model of C. Elegans adaptability."

Schroeder, Nathan, Androwski, Rebecca, Alicea, Bradly

[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).

Qiu, Tian (Autumn) et al. (2021) International Worm Meeting "Bacterial D-alanine metabolism affects C. elegans lifespan under high glucose conditions"

Qiu, Tian (Autumn), Schroeder, Nathan, Sweedler, Jonathan

[International Worm Meeting, 2021]

Microbiome-derived metabolites are able to impact the host's nervous system through the gut-brain axis. D-amino acids (D-AAs), the D-enantiomers of prevalent L-amino acids, have unique functions such as neuromodulators in animals including mollusks, rodents, and primates, while microbiota is a known contributor to the source of D-AAs in animals. Among all D-AAs, D-Ala is a potent agonist of the glycine binding site of N-methyl-D-aspartate (NMDA) glutamate receptors in vitro. Additionally, D-Ala immunoreactivity in pancreatic beta-cells and adrenocorticotropic hormone (ACTH)-secreting cells suggests its involvement in glucose homeostasis. While several researches showed microbiota as a major source of D-Ala in animals, the effect of bacterial D-Ala synthesis and metabolism on host physiology has not been studied. In this study, we fed wild type Caenorhabditis elegans N2 with Escherichia coli mutants with knockout of genes relevant to D-Ala biosynthesis (dadX, alr) and D-Ala metabolism (ddlA), and then examined changes in C. elegans phenotypes with or without the presence of high glucose. All three bacterial mutants displayed similar growth curves in nutrient-rich liquid media, but decreased D-Ala/total alanine ratio compared to the parental E. coli strain. To prevent the introduction of exogenous D-Ala, we cultured C. elegans on peptone-free NGM media. We found no statistically significant differences in life span when fed on the selected E. coli mutants. However, we observed a slight avoidance to all three bacterial mutants compared to the parental strain. When amended with 40 mM glucose, deltaddlA significantly decreased the life span of N2. No food preference was observed on glucose-amended plates. These results indicate that the combination of deficiency in bacterial DdlA activity and high glucose led to a decreased life span in C. elegans.

Qiu, Tian (Autumn) et al. (2021) International Worm Meeting "Chemical profiling of C. elegans single neurons using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)"

Andersen, Peter, Qiu, Tian (Autumn), Schroeder, Nathan, Sweedler, Jonathan

[International Worm Meeting, 2021]

Characterizing single cell heterogeneity helps reveal the chemical and functional complexity in nervous systems and aids in understanding the neurochemical basis of differential neuronal activity. While the nervous system in Caenorhabditis elegans is anatomically and genetically well-defined, the chemical profiles of their single neurons are less well-known. A non-targeted, single-cell chemical profiling of C. elegans neurons of interest is desirable to link the chemical complexity to neuronal activities. Mass spectrometry (MS) has been used to detect proteins, lipids, peptides, and metabolites in single cells. We adapt an optically guided, single cell MS technique, microMS, to profile dissociated and diffusely dispersed C. elegans single neurons. Dissociated single cells from the pan-neuronal RFP reporter rab-3p::NLS::RFP were spread across glass slides, fluorescently imaged, and coated with a chemical matrix. MicroMS identified and converted single cell locations in the fluorescent image into instrument stage positions. The stage positions enable automated single cell sampling with a matrix-assisted laser desorption/ionization MS (MALDI-MS) instrument. We successfully applied lysis and cell dissociation protocols to C. elegans and located individual cells using fluorescent microscopy. Preliminary results showed lipid and peptide-like signals from single neurons and we are currently optimizing the sample preparation to increase mass spectral quality.

Androwski, Rebecca et al. (2019) International Worm Meeting "Dauer IL2 neurons use distinct and shared mechanisms with FLP and PVDs to regulate arborization."

Androwski, Rebecca, Goelzer, Janet, Hofer, Allison, Smith, Cassandra, Schroeder, Nathan

[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.

Ludewig, Andreas H. et al. (2013) International Worm Meeting "Low population density increases lifespan and delays egg laying of C. elegans hermaphrodites."

Doering, Frank, Schroeder, Frank C., Ludewig, Andreas H.

[International Worm Meeting, 2013]

In C. elegans, high population density (overcrowding) has been shown to trigger developmental arrest and dauer formation within early larval stages (Golden and Riddle 1982). The actual population density signal sensed by the worms includes a synergistic mixture of ascaroside pheromones as well as small signalling peptides (Ludewig and Schroeder, 2013; Yamada et al., 2010). However, many aspects of population density-dependent signaling events remain poorly understood. Here, we compared low and intermediate dense C. elegans populations grown under ad libidum feeding conditions. We found that the number of worms per plate strictly determined their average lifespan whereat the number of worms was inverse correlated to mean lifespan. Isolated worms lived around 4 (!) days longer than animals grown up in larger groups. In 1999, Gems and Riddle demonstrated strong dependence of lifespan on population density for males but not for hermaphrodites. However, in those assays worms were placed on experimental plates as L4 larvae, whereas our assays started with eggs, hinting on some defining events during developmental stages L1-L3 in hermaphrodites. We also found that the time point of first egg lay was correlated with the number of worms per plate. Worms that grew up in isolation laid their first egg 9-11 hours later than worms grown up in groups of 25 or more animals per plate. The overall time course of egg laying from isolated worms was shifted towards later time points and the total number of eggs was reduced by 45%. This population dependent early egg laying (= eel) phenotype is completely abolished in daf-16(mu86) mutants and partially abolished in daf-12(rh61; rh411) mutants. Unexpectedly, we also found precocious first egg laying under mild dietary restriction (DR). Evidence from previous studies suggests that small-molecule signalling may trigger DR early egg laying and population dependent longevity (Ludewig and Schroeder, 2013; Yamada et al., 2010; Yzraelit, 2012). To test this hypothesis, we currently examine candidate small molecules for their influence on those phenomena.