Rashid, A. et al. (2013) International Worm Meeting "Cutting Edge: Expression and function of KPC-1/furin in C. elegans."

Rashid, A., Schroeder, N., Barr, M., Androwski, R.

[International Worm Meeting, 2013]

Proprotein convertases (PCs) are responsible for the cleavage of proproteins into their biologically active forms. The human PC family contains seven proteins that cut at dibasic cleavage sites while there are four PCs in C. elegans (KPC-1, BLI-4, AEX-5, and EGL-3). KPC-1 is homologous to furin, a PC essential for mammalian development and associated with numerous pathologies. However, C. elegans kpc-1 mutants are viable allowing us to study the function of this gene in living animals. We found that kpc-1 is required for dauer-specific dendrite arborization in four IL2 quadrant (IL2Q) neurons (See N. Schroeder et al. abstract). In C. elegans there are three classes of multidendritic neurons: the IL2Qs in dauer and adult PVD and FLP neurons. We examined the PVD and FLP neurons in kpc-1 adults. Similar to the IL2 neurons in dauers, kpc-1 mutants show highly disorganized and truncated PVD and FLP neurons, demonstrating that KPC-1 is a general regulator of dendrite arborization. We are currently determining whether kpc-1 is required for PVD and FLP mediated behaviors such as proprioception and harsh touch response. kpc-1 expression showed strong temporal control. However, consistent expression was observed in the ventral nerve cord.To assess the effect of kpc-1 on movement we used both standard body bend assays and thrashing assays. We found that kpc-1 mutants are sluggish compared with wild-type animals. kpc-1 expression was also observed in eight vulval precursor cells during the "Christmas tree" stage of vulval development. However, we observed no obvious defects in vulva morphology in kpc-1 mutants. Finally, we observed temporal expression of kpc-1 during embryogenesis and a reduced brood size in kpc-1(gk8) mutants, suggesting that kpc-1 plays a role in early development. We conclude that kpc-1 plays a multifaceted role in development, dendritic branching, and nervous system function.

Rashid, Alina et al. (2021) International Worm Meeting "Y-to-PDA transdifferentiation occurs through an epithelial cell intermediate and requires ngn-1, hlh-16, unc-44, unc-119, and unc-33"

Rashid, Alina, Tevlin, Maya, Shaham, Shai

[International Worm Meeting, 2021]

In C. elegans, the rectal epithelial Y cell transdifferentiates into the PDA motor neuron (Jarriault et al., 2008). Transdifferentiation (Td) has been suggested to occur in discrete steps: the Y cell loses rectal contacts and de-differentiates, migrates away from the rectum, and re-differentiates into a motoneuron. Using a membrane-tagged fluorescent reporter, we now observe a more complex pattern of events. While the cell body migrates away from the rectal slit, the Y cell maintains rectal tube contacts, marked with the apical junction protein AJM-1. From the rectal contact site, the cell then extends a process that matures into the PDA axon. Rectal apical junctions are lost later. Previous studies revealed that SEM-4 and EGL-5 transcription factors are required for erasure of Y cell epithelial identity and acquisition of neuronal identity. We identified two novel Td transcriptional regulators, the bHLH proteins NGN-1 and HLH-16, also required for these steps. ngn-1 expression increases as the Y cell migrates away from the rectum and changes cellular morphology. From reporter fusion studies, we found that SEM-4 represses ngn-1 expression, whereas EGL-5 and HLH-16 promote ngn-1 expression. Although ngn-1 induction correlates with the timing of Y cell migration, precocious expression of ngn-1 in sem-4 mutants does not change migration timing, suggesting involvement of other regulators. We also identified three cytoskeletal organizing proteins required for acquisition of neuronal identity: unc-44, unc-119, and unc-33. These proteins appear to act downstream of NGN-1 and HLH-16 and are required for PDA axon extension. Y-to-PDA Td bears striking morphological and molecular similarities to vertebrate spinal-cord pMN-domain motoneuron formation, and to islet cell formation in the pancreas. During development, motoneuron and pancreatic beta cell precursors originate from an epithelium. These precursors then lose their junctions and delaminate from the epithelium. As they migrate and mature, the cells initiate transcription of motoneuron-associated genes, become innervated, and secrete neurotransmitters or insulin, respectively. In both the spinal cord and pancreas, NGN-1 and HLH-16 homologs (Ngn2/Olig2 and Ngn3/bHLH4, respectively) are required for epithelial identity erasure. Furthermore, unc-44/Ankyrin promotes neuronal and beta cell maturation. Thus, our findings may provide mechanistic insight into the differentiation of these important vertebrate cell types.

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.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[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.

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.

Flatt, K. M. et al. (2015) International Worm Meeting "Role of DYF-7 in IL2 cell-body maintenance during dauer formation."

Flatt, K. M., Schroeder, N. E.

[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.

Ghose, P. et al. (2019) International Worm Meeting "One cell, many deaths: the orchestrated demise of the C. elegans tail-spike cell."

Pena, S., Shaham, S., Ghose, P.

[International Worm Meeting, 2019]

Programmed cell death (PCD) is vital to development and homeostasis. Many of our cells, including melanocytes, glia and neurons are morphologically complex, bearing long processes. How different subcellular compartments communicate with eachother in these cells, and how cell function is coordinated between these compartments is an interesting yet poorly understood problem. This is especially relevant in the context of PCD, where the entire cell needs to die and be removed efficiently to prevent aberrent cell activity and autoimmune consequences. The C. elegans tail-spike cell (TSC) dies during embryonic development in a fascinating way: it segments into three parts, each degenerating differently (Ghose et al.,2018). The soma rounds up like a simple apoptotic cell. The proximal process segment forms beads, much as in Wallerian degneration of axons. The distal process retracts (resembling axon retraction following nutrient deprivation) into a central swelling. Importantly, an identical pattern of degeneration is seen in the CEM neurons, which die sex specifically in the embryo, suggesting that TSC-like death may be a universal mode of eliminating complex cells. How is cell death inititated and coordinated in the TSC? Our findings show that death requires the caspase CED-3, acting independently in the process and soma. Preliminary studies suggest an intriguing role for CED-3 in mitochondrial trafficking. Death also requires the transcriptional factor BLMP-1/BLIMP-1, which appears to coordinate the elimination of TSC compartments. Canonical apoptosis engulfment genes are not required for TSC process removal. A forward genetic screen and subsequent studies reveal that the cell-cell fusogen EFF-1 is required for phagosome sealing around the distal process fragment following retraction (Ghose et al., 2018). EFF-1 functions in the surrounding hyp10 cell and is localized to phagosome arm tips. Importantly, EFF-1 appears to be the first direct regulator identified of phagosome sealing, a poorly understood step of phagocytosis. To identify additional genes promoting TSC death and degradation, we isolated mutants with defects in various aspects of TSC death and clearance following EMS mutagenesis. These include a mutant for the process retraction step and another phagosome sealing mutant, which may represent a regulator of EFF-1. Our mutant collection, together with our previous studies, make the TSC a powerful model to study novel aspects of PCD and removal in the context of morphologically complex cells. References: Ghose, P., Rashid, A., Insley, P., Trivedi, M., Singhal, A., Shah, P., Lu, Y., Bao, Z., Shaham, S. (2018). EFF-1 Fusogen Promotes Phagosome Sealing During Cell Process Clearance in Caenorhabditis elegans. Nat Cell Biol. doi:101038/s41556-018-0068-5.

Mongan NP et al. (1997) International C. elegans Meeting "AN EXTENSIVE NICOTINIC ACETYLCHOLINE RECEPTOR GENE FAMILY IN CAENORHABDITIS ELEGANS."

Baylis HA, Mongan NP, Sattelle DB

[International C. elegans Meeting, 1997]

Although the nicotinic acetylcholine receptors (nAChRs) are the best characterized ionotropic neurotransmitter receptors, the extent of the molecular and functional diversity of the nAChR gene family is not known for a single organism. A number of C. elegans nAChR subunit genes have been identified previously using genetic approaches [lev-1(non-a),unc-38(a),unc-29(non-a),deg-3(a)] and cross-species probing of C. elegans cDNA libraries [acr-2(non-a),acr-3(non-a),and Ce-21(a)]. nAChR a-subunits may be distinguished from all other ionotropic receptor subunits by the presence of adjacent cysteines at positions equivalent to 192,193 in the Torpedo a-subunit. We have applied an RT-PCR strategy to confirm the expression of 8 novel putative nAChR a-subunits predicted by GENEFINDER. Analysis of the regions of the a-subunit considered to (a) contribute to the acetylcholine binding site and (b) the channel lining, has revealed a diversity which may underlie distinct functional roles for members of this multi-gene family.

C Huang et al. (1999) International C. elegans Meeting "Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis"

WG Wadsworth, C Huang, PD Yurchenco

[International C. elegans Meeting, 1999]

Laminin is a heterotrimeric glycoprotein composed of a , b , and g subunits. The genome sequencing project reveals two a , one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin a A and laminin a B, are most similar to the vertebrate a 1/ a 2 and a 5 chains respectively, while the b and g resemble the b 1/ b 2 and g 1 chains. Thus, there are two predicted laminin trimers, a A bg and a B bg . Mutations in epi-1 , which encodes laminin a B, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin a A and a B. By immunostaining, both first appear at gastrulation between germ layers. However, laminin a A is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin a B is more posteriorly localized. Laminin a B becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin a A accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin a A is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3 , which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin a B has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin a A is restricted to specialized membranes and is required for the morphogenesis of specific tissues.