H Wang (1999) International C. elegans Meeting "Expression of amyloid precursor and related protein in C. elegans"

H Wang

[International C. elegans Meeting, 1999]

The major component of senile plaques in the brains of Alzheimer's disease patients is the amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Early-onset familial Alzheimer's disease has been shown to correlate with mutations in three genes, including the APP gene. We are studying a APP -related gene, apl-1 , in C . elegans . To identify genes that interact with apl-1 , transgenic lines in which an apl-1 cDNA is under the control of a neural-specific promoter (VAMP) were generated. These transgenic strains were confirmed by Western blot analysis to overexpress APL-1. The transgenic animals have several phenotypes, including sluggish movement and a flattened waveform. To identify genes that interact with apl-1 , we mutagenized transgenic animals with EMS, and looked for reversion of the sluggish phenotype. After screening about 3,000 haploid genomes, two strains that suppress the sluggish phenotype were isolated. The yn8 strain shows wild-type movement, while the yn9 strain shows hyperactive movement. These possible suppressor genes are currently being mapped. The putative suppressor mutation in the yn9 strain does not map to chromosome V or X. APL-1 does not contain the amyloid peptide. To determine whether production of amyloid peptide has any effect in C . elegans , transgenic strains containing a mouse APP cDNA under the control of the VAMP promoter were generated. The mAPP transgene is transcribed in transgenic animals as assayed by RT-PCR. Western blot analysis to determine whether the amyloid peptide is produced is underway. Several phenotypes have been characterized. The transgenic animals have an egg-laying defect, decrease in movement, and a longer defecation cycle. The huAPP cDNA was obtained recently, and similar studies will be performed.

Wang, H. et al. (2017) International Worm Meeting "cGAL and split cGAL for precise transgene control in C. elegans."

Yuet, K.P., Wang, H., Sternberg, P.W., Hill, A., Schwarz, E.M., Gharib, S., Chai, C.M., Liu, J., Pokola, N.

[International Worm Meeting, 2017]

Genetic tools that allow tight control of gene expression in desired spatiotemporal patterns are critical for dissecting neural circuits and elucidating gene function. The bipartite GAL4-UAS system, in which a transcriptional activator protein (Gal4p) is driven in specific cells to bind its target sequence (UAS) upstream of an effector and drives expression of the effector gene, has become a powerful tool for manipulating gene expression in several model organisms. However, this system has not been successfully adopted for C. elegans research, due to its weak performance at C. elegans cultivation temperatures (15-25 deg C). By systematically engineering the three major components, we have established cGAL, a robust GAL4-UAS system for C. elegans. First, we replace the endogenous Galp4 transcriptional activation domain with a synthetic strong activator VP64; second, to improve the binding of Gal4p to the effector construct, we put 15 copies of UAS sites upstream of the effector gene; lastly, to enhance the performance of GAL4-UAS system at low temperatures, we adopt a Gal4p DNA-binding domain (DBD) from a cryophilic yeast strain S. kudriavzevii. Unlike S. cerevisiae (grown best at 30-34 deg C) from which the canonical Gal4p DBD is derived, S. kudriavzevii has the optimal growth temperature at 23-24 deg C, much closer to C. elegans cultivation temperatures. The optimized cGAL system displays temperature robustness of driving GFP effector across the 15-25 deg C range. We further demonstrate the utility of the cGAL system using reporter expression in multiple tissues, site-of-action experiments, and gain-of-function channelrhodopsin experiments. We have also built a basic cGAL toolkit with cGAL strains and plasmids for genetic labeling, cell ablation, visualization and manipulation of neural activity. However, for many cell types, especially neurons, there are not known DNA regions that direct expression only in that cell type. We thus develop a novel split cGAL system using intein, in which VP64 and Gal4p DBD are driven by two distinct promoters. The two components are reconstitute into a functional transcriptional activator to trigger the expression of the effector gene only in those cells where both promoters are active. We expect that cGAL and split cGAL will significantly aid C. elegans research by providing precise transgene control.

Wang H et al. (1998) East Coast Worm Meeting "Expression of amyloid precursor and related proteins in C. elegans"

Wang H, Li C

[East Coast Worm Meeting, 1998]

The major component of senile plaques found in the brains of Alzheimer!s disease patients is the b-amyloid peptide, which is derived from a larger amyloid precursor protein (bAPP). Early-onset familial Alzheimer!s disease has been shown to correlate with mutations in four genes, including the bAPP gene. We are studying an bAPP-related gene, apl-1, in C. elegans. To identify genes that interact with apl-1, transgenic lines in which an apl-1 cDNA is under the control of a neural specific promoter (VAMP) were generated. These transgenic strains, NY2012, NY2013, and NY2014, were confirmed by Western blot analysis to overexpress APL-1. The transgenic animals have defects in movement; specifically, they move slowly and are sluggish. To look for genes that interact with apl-1, we mutagenized transgenic animals with EMS and looked for reversion of the sluggish phenotype. After screening about 1,500 F1 animals, we found two strains that suppress the sluggish phenotype. One strain shows a hyperactive phenotype, while the other strain shows wild-type movement. We are currently mapping these possible suppressor genes. APL-1 does not contain the b-amyloid peptide. To determine whether production of b-amyloid peptide has deleterious effects in C. elegans, we generated transgenic strains containing a mouse APP cDNA under the control of the VAMP promoter; the transgene was integrated by UV irradiation. The strains were backcrossed into a wild-type background to remove unlinked mutations. We are currently characterizing the phenotype of these transgenic lines, and will determine whether the b-amyloid peptide is produced.

Wang, H. et al. (2017) International Worm Meeting "Regulation of locomotion quiescence during stress-induced sleep by FMRFamide-like peptide FLP-24."

Gharib, S, Wang, H., Liu, P., Wang, Z.W., Sternberg, P.W.

[International Worm Meeting, 2017]

The ALA neuron is the master control center for stress-induced sleep in C. elegans. The molecular and cellular mechanisms underlying ALA-mediated sleep are not clear. Our previous work suggests that multiple neuropeptides from the ALA neuron work collectively to regulate sleep, each of which primarily regulates particular aspects of sleep that include pumping quiescence, locomotion quiescence, and sensory depression. Here, we focus on the FMRFamide-like peptide FLP-24. We found that flp-24 is expressed in the ALA neuron and a few other neurons. Constitutive overexpression (OE) of flp-24 specifically led to a lethargic phenotype, characterized by the lack of movement. Preliminary electrophysiological data showed that flp-24(OE) worms have normal evoked neurotransmitter release at the neuromuscular junction and normal muscle sensitivity to exogenous levamisole, nicotine and GABA, but may have defects in muscle excitability. From a forward genetic screen, we identified multiple strong suppressors, all of which define the same gene, encoding a G protein-coupled receptor, temporarily called FLIPPER. Transcriptional reporter analysis of flipper revealed that it is strongly expressed in body wall muscles and a handful of neurons. Tissue-specific rescue experiments showed that flipper is likely to function in body wall muscle. We also found that flp-24 is sufficient to inhibit sensory function when its putative receptor flipper is ectopically expressed in sensory neurons. Based on these results, we propose that FLP-24 controls locomotion quiescence during stress-induced sleep in C. elegans primarily by reducing the excitability of muscles.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

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.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Wendy Hanna-Rose, Rasika Kalamegham

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Schwartz, Hillel et al. (2013) International Worm Meeting "Developing Heterorhabditis nematodes as an experimental system for the study of mutualistic symbiosis."

Schwartz, Hillel, Sternberg, Paul

[International Worm Meeting, 2013]

Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora. Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility. In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.

Schwartz H et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Towards a SNP map for Heterorhabditis bacteriophora"

Sternberg P, Schwartz H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Heterorhabditis bacteriophora is a species of insect-parasitic nematode that lives in mutually beneficial symbiosis with pathogenic Photorhabdus luminescens bacteria. P. luminescens bacteria are lethal to insects and to other nematodes, including the soil nematode Caenorhabditis elegans, but are required for H. bacteriophora growth. The symbiosis between H. bacteriophora and P. luminescens therefore offers the potential to study the molecular genetic basis of their cooperative relationship. We are interested in developing tools to make such studies more feasible; in particular, we propose to generate tools for genetic mapping in H. bacteriophora. We will test independent isolates of H. bacteriophora to ensure that the isolates are cross-fertile. We will then examine the abilities of these isolates to grow on and to become infected with different wild-type and mutant Photorhabdus bacteria. From these tests we will select an isolate on which we will use next-generation high-throughput sequencing technology for the purpose of refining the existing draft genome sequence and for the creation of a SNP map. We anticipate that this SNP map will enable us and the wider insect-parasitic nematode community to identify induced mutations and natural variations affecting the interactions between H. bacteriophora nematodes and pathogenic Photorhabdus bacteria.