Zhang, Yun et al. (2015) International Worm Meeting "Internal structural damage induces an innate immune response in C. elegans epidermis."

Zhang, Yun, Li, Wenna, Zhu, Yi, Zhang, Huimin, Fu, Rong, Li, Linfeng, Li, Yuanbao

[International Worm Meeting, 2015]

Being a first-line defense, the epidermis is in constant confrontation against pathological or mechanical insults that disrupt the epidermal architecture. Yet whether and how the immune system of the epidermal cells recognizes its structural damage as a danger signal to activate self-defense remains unclear. Here we use C. elegans epidermis as the model to address this question. The adult C. elegans epidermis is a single-cell-layered tissue with highly organized 3-D architecture. When subjected to skin-penentrating fungal infection or physical injury, it activates innate defense through p38-MAPK and TGF-beta signaling cassettes and produces antimicrobial peptides (AMPs) such as nlp-29 and cnc-2. By disrupting each epidermal architectural components seperately, we pinpointed a spatially-restricted group of structures at the apical hemidesmosomes capable of activating epidermal immune defense once damaged. By screening most signaling molecules known to control AMP production, we found that the immune defense against epidermal damage depends on STA-2, but not on any other immune-related signaling pathways known to date. The endogenous STA-2 is mostly associated with apical hemidesmosomes in the healthy epidermis. Disruption of apical hemidesmosomes leads to increased nuclear localization of STA-2 and results in AMP up-regulation. The cooperation between hemidesmosomes and STA-2 enables the epidermis to bypass p38-MAPK and TGF-beta signaling pathways and directly turn on immune effector genes when subjected to severe architectural damage by internal or external causes. Together, our findings uncovered a cell-autonomous surveilance program installed within the stable attachment structures of the epidermal cells, and a novel mechanism for the epidermis to detect danger and activate self-defense. These results may also provide mechanistic insights into the pathogenesis of inflammatory diseases involving epidermal damage. .

Yun Zhang et al. (2003) International Worm Meeting "Olfactory learning in response to pathogenic bacteria."

Cornelia I Bargmann, Yun Zhang

[International Worm Meeting, 2003]

As free soil-living animals, C. elegans encounters a wide variety of bacterial species. Soil bacteria represent a rich pool of foods and pathogens, so the animal's response to different bacteria can have large effects on its survival. We examined C. elegans's ability to grow on ten soil-isolated gram-negative bacterial strains and tested the animals preference for these bacteria in olfactory chemotaxis assays either after growth on OP50 or after growth on the soil bacteria. Two bacteria strains pathogenic to C. elegans, Pseudomonas aeruginosa1 and Serratia marcescens2, show an interesting effect on olfactory preference. Naive worms prefer these two bacteria strains to the E. coli strain OP50 in olfactory choice assays, but animals that were previously exposed to S. marcescens or P. aeruginosa avoid the toxic bacteria, a learning behavior similar to taste-aversion. A similar behavior has previously been observed for accumulation on S. marcescens2. Our result indicates that this represents a stable change of olfactory preference. We hypothesize that this behavior represents a form of olfactory learning in which the toxicity of pathogenic bacteria changes the animals' behaviors towards them from attraction to avoidance.We used sensory mutants to characterize requirements for olfactory learning in response to toxic bacteria. Analyses of cell fate mutants suggested that the AWB and AWC olfactory neurons are required for learning. AWA olfactory neurons are not essential. AWB neurons sense repulsive odors and AWC neurons sense attractive odors, so these neurons have the potential to shift olfactory preferences in either direction or modify chemosensory sensitivity.We also examined mutants with neurotransmitter defects. cat-4(e1141) and tph-1(mg280) mutants did not have a robust shift in olfactory preference after encountering toxic bacteria, suggesting that serotonin may have a role in learning. Ca2+-signaling pathways affect learning and memory in many animals. We found that mutations in the calcium-calmodulin dependent kinase CAMKII unc-43 abolished learning, whereas lesions in the neuronal calcium sensor ncs-1 caused no obvious defect. We are currently conducting cell-specific rescue experiments to refine our cellular and molecular understanding of this learning assay.Reference: 1. M.W. Tan and F.M. Ausubel, Curr. Opin. Microbiol. 3 (2000), 29-34. 2. N. Pujol, et al., Curr. Biol. 11 (2001), 809-821.

Yun Zhang et al. (2004) West Coast Worm Meeting "Olfactory learning of C. elegans in response to pathogenic bacteria"

Yun Zhang, Cornelia I Bargmann, Hang Lu

[West Coast Worm Meeting, 2004]

As a free soil-living animal, C. elegans encounters many species of soil bacteria that represent potential foods and pathogens. The nematode's response to these bacteria can have large effects on its survival, so we asked how different bacteria affect the behaviors of C. elegans. We used an olfactory assay to examine C. elegans ' preferences for the odors of ten gram-negative soil bacterial strains either after growth on OP50 or after growth on the tested bacteria. Two bacterial strains pathogenic to C. elegans , Pseudomonas aeruginosa 1 and Serratia marcescens 2 , showed an interesting effect on olfactory preferences: animals that were previously exposed to both OP50 and S. marcescens or P. aeruginosa avoided the toxic bacteria when it was presented in a choice assay with OP50. This difference between naïve and experienced animals appears to represent a form of learning similar to taste aversion. A previous observation showed that C. elegans does not accumulate on a lawn of S. marcescens 2 . Our results indicate that this behavior arises from a stable change of olfactory preferences induced by exposure to pathogens. By using a four-choice maze assay, we demonstrated that trained animals developed both an increased aversion to the pathogenic bacteria and an enhanced preference for the nonpathogenic bacteria that they were raised on, compared to control pathogenic and nonpathogenic bacteria. Analyses of cell fate mutants suggest that AWC olfactory neurons, which sense attractive odors, are required for this learning ability. AWA olfactory neurons are not essential. We hypothesize that upon training on pathogenic bacteria, C. elegans shifts its olfactory preferences or modifies chemosensory sensitivity of AWC neurons to minimize exposure to toxic food. We also examined mutants defective in neurotransmitter biosynthesis in this paradigm. By testing cat-2(e1112) and tph-1(mg280) mutants, which are defective in dopamine and serotonin synthesis, respectively, we found that serotonin is required for both attractive and aversive learning, whereas dopamine is only involved in attractive learning. We are now conducting cell-specific rescue experiments to refine our cellular and molecular understanding of this learning behavior. Reference: 1. M.W. Tan and F.M. Ausubel, Curr. Opin. Microbiol. 3 (2000), 29-34. 2. N. Pujol, et al. , Curr. Biol. 11 (2001), 809-821.

Yun Zhang et al. (2005) International Worm Meeting "Enhanced signaling of a serotonergic circuit regulates aversive olfactory learning of C. elegans"

Cornelia Bargmann, Hang Lu, Yun Zhang

[International Worm Meeting, 2005]

An animals ability to avoid food with harmful effects is essential for its survival. C. elegans feeds on bacteria in soil, potentially including pathogenic bacteria. For example, two common gram-negative soil bacteria, Pseudomonas aeruginosa (1) and Serratia marcescens (2), can proliferate in C. elegans intestine and cause death. Therefore, learning to avoid toxic food like pathogens could help C. elegans to survive in its natural habitat. Using an olfactory choice assay we found that animals raised on S. marcescens or P. aeruginosa supplemented with a small amount of OP50 avoided the pathogen when it was presented in an olfactory choice assay with OP50, while animals raised on OP50 without being exposed to pathogens did not show strong preferences between the pathogen and OP50. This experience-based change in olfactory preferences appears to represent a form of associative learning similar to taste aversion, a form of learning found in many animals.Adult animals exposed to pathogens acquire an aversion for it in a few hours, indicating that olfactory learning is an acute modification of animals innate preferences. Different pathogens induce olfactory changes specific to animals experiences and learning to avoid pathogens is distinct from adaptation. Using a multiple-choice assay, we found that trained animals acquired both an aversion to the pathogen and an increased attraction for the nonpathogenic bacteria that they were raised on.To identify modulatory pathways in olfactory learning to pathogens, we tested tph-1 mutants, which are selectively defective in serotonin synthesis. tph-1 animals are deficient in both attractive and aversive learning in multiple-choice assays, indicating a regulatory role of serotonin in the learning. Increased serotonin contents are detected by HPLC in animals exposed to pathogens; enhanced serotonin immunoreactivity is specifically detected in one pair of serotonergic neurons, ADF. Expression of tph-1 cDNA in ADF neurons fully rescues aversive learning of tph-1 mutants. Downstream of ADF, we identified MOD-1, a serotonin-gated chloride channel, as a receptor that functions in interneurons to promote aversive learning. Our results indicate that exposure to pathogenic bacteria enhances serotonergic signaling in ADF neurons, stimulating aversive olfactory learning to pathogens through MOD-1.Reference: (1). M.W. Tan, et al., Proc Natl Acad Sci U S A 96 (1999), 715-20. (2). N. Pujol, et al., Curr Biol 11 (2001), 809-821.

Yun Zhang et al. (2001) International C. elegans Meeting "Generation of cell-type-specific RNA and gene expression profiles for Mechanosensation Neurons in C. elegans"

Yun Zhang, Thomas Delohery, Marty Chalfie, Charles Ma

[International C. elegans Meeting, 2001]

One of the great advantages of C. elegans as an experimental system is the ability to characterize mutant phenotypes at the level of single type of cells. With the sequencing of the genome, the use of DNA microarrays offers the opportunity to look at the genome-wide transcriptional activity under different experimental conditions. Unfortunately, array experiments using whole worm RNA do not afford the resolution needed to look, for example, at the differences in expression between specific wild type and mutant nerve cells. We have developed a technique to generate cell-type-specific RNA to use with cDNA microarrays to study gene expression profiles for a single type of C. elegans cells and have applied it to study the differentiation and function of the embryonic touch receptor neurons (ALM and PLM). While saturation mutagenesis for touch insensitive mutants have identified several genes needed for touch cell development and function, those screens could not have identified redundant genes, pleiotropic genes or genes that mutate to a touch super-sensitive phenotype. DNA array data should reveal such genes as well as provide candidates for as yet uncloned mec genes. To identify mec-3 -dependent genes expressed in the touch cells, we collected late-stage embryos from strains with integrated GFP arrays: either P mec-18 gfp in a wild-type background for differentiated touch cells or P mec-3 gfp in a mec-3 (e1338) background for mec-3 mutant cells that should have become touch cells. Cells from dissociated embryos were cultured overnight (many extended processes in culture) and the GFP-positive cells were enriched 100-fold by fluorescence-activated cell sorting. Typical post-sort values from cell sorting are 4x10 6 cells with 40-50% being GFP positive. mRNAs from these cells were linearly amplified 10 6 -fold using a modified Eberwine's method, labeled and applied to cDNA microarrays (in Stuart Kim's lab) representing virtually all of the C. elegans genes. The reliability and sensitivity of the expression profiles is suggested by the finding that 6 of the 10 known mec-3 -dependent genes were among the top 15 genes and 8 out of 10 known mec-3 -dependent genes (including mec-3 itself) had ratios above 4.0 (the two other genes had ratios higher than 2.0). In addition, the profile also provides several groups of interesting candidates besides unknown function genes with ratios equal to or higher than ratios of known mec-3 -dependent genes: 1) the gene with the highest ratio maps to the same position as the previously uncloned mec-17 gene, which is needed for the maintenance of touch cell fate (we have confirmed that this gene is expressed exclusively in the touch cells and have identified two missense mutations in mec-17 (u265) ), 2) two other genes map to the area of mec-15 and a single mutation on X that disrupts touch cell microtubules, 3) several genes that contribute to the T-complex chaperonins, which are needed for the formation of tubulins, 4) several channel genes in addition to mec-4 and mec-10 and 5) genes for G-protein coupled receptors and transcription factors. We are also using RNAs from sorted and unsorted cells to identify touch-cell-enriched genes that are not mec-3 -dependent.

Shen, Yu et al. (2013) International Worm Meeting "eol-1, the homolog of mammalian Dom3z, is a novel genetic regulator of C. elegans olfactory learning."

Zhang, Jiangwen, Zhang, Yun, Calarco, John, Shen, Yu

[International Worm Meeting, 2013]

Neural plasticity, a remarkable feature of the nervous system, allows animals to adjust their behavior based on previous experience. C. elegans is able to modify its olfactory preference to avoid the odor of pathogenic bacteria after ingesting the pathogens [1]; this learning is regulated by a recently identified neural circuit as well as multiple signaling pathways [2-4]. The genetic accessibility and well-characterized nervous system of C. elegans provide a unique opportunity to study the molecular and cellular mechanisms of olfactory learning, which are often conserved from the nematode to higher organisms. To characterize novel molecular regulators of C. elegans olfactory learning, we conducted a forward genetic screen for mutants with altered learning abilities. Using a high-throughput behavioral assay, we have identified several candidate mutants, among which one recessive allele displays significantly enhanced olfactory learning. This phenotype is not due to any detectable deficiency in innate immunity or olfactory preference under the naive condition. By whole genome sequencing and trangenic rescue, we have mapped the gene of interest eol-1 (enhanced olfactory learning-1) to a predicted protein-coding sequence on Chromosome V. GFP reporter driven by the endogenous promoter of eol-1 is expressed in the reproductive system and several identified neurons; neuron-specific expression of eol-1 in the URX sensory neuron restores the learning phenotype in the mutant background. The protein encoded by eol-1 is conserved in different Caenorhabditis species and other eukaryotes; its yeast ortholog Rai1 is involved in RNA metabolism, whereas the function of the mammalian ortholog DOM3Z remains unclear. Our ongoing work to identify genetic interaction of eol-1 and to evaluate its function in C. elegans may contribute to better understanding the function of this conserved gene in more complex systems. References: 1. Zhang et al. Nature 438, 179-184. 2. Ha et. al. Neuron 68, 1173-86. 3. Zhang and Zhang, Proc Natl Acad Sci U S A 109(42):17081-6. 4. Chen et. al. Neuron 77, 572-585.

Zhang, Xiaodong et al. (2011) International Worm Meeting "The function of TGF-b signaling in olfactory learning of C. elegans."

Zhang, Yun, Zhang, Xiaodong

[International Worm Meeting, 2011]

Recent studies have started to reveal that many molecules essential for early development also play important roles in adult neuronal plasticity. Here we investigate the function of the transforming growth factor-b (TGF-b) signaling pathway in animal learning. TGF-b signal transduction is critical in diverse physiological processes such as embryonic development, tissue homeostasis and immune responses. Although a couple of studies have implicated TGF-b in neural plasticity, the function of TGF-b in learning was never directly tested and the underlying cellular and physiological mechanism is essentially unknown. Utilizing a behavioral assay that measures olfactory learning in adult animals, we have found that the Sma/Mab TGF-b signaling pathway is required for aversive olfactory learning of C. elegans. Mutations in the genes of this pathway, including the dbl-1 ligand, sma-6 and daf-4 receptors, and sma-3 Smad, abolish the learning ability of adult animals. We further explored the cellular mechanisms of this pathway in learning through expression analysis, cell/tissue-specific rescues, calcium imaging and behavioral characterization. We identified a set of interneurons that are critical for olfactory sensorimotor response as the essential release site of DBL-1 ligand in regulating learning. The neuronal activity of the interneurons is repressed by training, accompanied by an increase in the expression and secretion of DBL-1 from the interneurons. We also found that downstream Sma/Mab signaling components act in hypodermis and/or sensory neurons to promote learning. In addition, we present evidence that the function of TGF-b signaling in aversive learning of adult animals can be separated from its roles in body size determination and innate immunity based on different signaling strength as well as temporal and spatial requirements. Our results support a model in which training induced changes in neuronal activity lead to alteration of the Sma/Mab TGF-b signaling, which in turn modulates the olfactory response of adult animals. Because TGF-b superfamily is conserved throughout metazoans, we expect our study to shed light on the function of TGF-b signaling in adult behavioral plasticity in other organisms as well.

Harris, Gareth et al. (2015) International Worm Meeting "How do worms choose the right foods? Genes and circuits that drive olfactory dependent sensori-motor behaviors."

Zhang, Yun, Harris, Gareth

[International Worm Meeting, 2015]

Odor sensation, discrimination and preference are critical components of human behavior. The ability to recognize and discriminate between odors, and generate preference for individual scents is associated with many aspects of behavior. An array of neurological diseases share significant deficits in olfactory-based behavior at the level of odor processing, discrimination and plasticity. Using C. elegans, we have previously demonstrated that the worm can recognize and generate olfactory preference among foods, such as E. coli OP50 and PA14. We previously identified distinct but overlapping signaling pathways and neural circuits regulating olfactory sensation versus preference when comparing different foods. The pathways require canonical sensory transduction pathways and glutamate for olfactory recognition, and specific peptide pathways for preference. We have focused on the later pathway, which will allow us to understand not only how a preference is generated, but also how it can be modulated by experience. We have now identified distinct interneurons that mediate olfactory recognition versus preference. Furthermore, we have begun to examine the role of various C. elegans genes whose human homologs are associated with neurodegenerative diseases in regulating olfactory recognition and preference. We have found that the presenilin-encoding gene plays an important role in mediating sensori-motor response to complex olfactory cues. We are further dissecting the functional sites of presenilin in the neural circuits underlying food odor recognition and preference. Various neurological diseases disrupt cognitive functions, including learning and memory. Our study will potentially provide insights into the underlying mechanisms.

Yu, Jingyi et al. (2013) International Worm Meeting "Investigating the neural mechanisms underlying a hypertonic response in Caenorhabditis elegans."

Yu, Jingyi, Zhang, Yun

[International Worm Meeting, 2013]

Environmental osmotic changes can interfere with molecular and cellular functions by influencing cytoplasmic osmolarity. Preventing this damage is an important survival skill for animals that live in environments with unstable osmolarity. It is known that in addition to hormonal responses, mammals also use behavioral strategies to maintain a stable cellular osmolarity , such as water-seeking behaviors driven by thirst. However, little is known about the molecular mechanisms or neural circuits underlying these behavioral responses. Previously, it has been shown that Caenorhabditis elegans generates an acute aversive response to high osmotic shock (Bargmann et al. 1990). Here, we show that C. elegans also produces an aversive response when exposed to prolonged hypertonic conditions. Compared with the response to high osmotic shock, the hypertonic response is slower, evoked by lower osmolarity, and involves increased turning rate over time. It does not require the same molecular mechanism as the response to high osmotic shock, since osm-9 mutants show a wild-type response in the hypertonic condition (Liedtke et al. 2003). The behavioral response is specific to osmotic stimuli, and common mechanosensory or chemosensory mutants do not show any defect. Currently, we are looking for the genetic and circuit mechanisms that regulate this behavior.

Nguyen, Julia et al. (2013) International Worm Meeting "The effect of sex difference on olfactory learning in Caenorhabditis elegans."

Harris, Gareth, Smith, Shane, Zhang, Yun, Nguyen, Julia

[International Worm Meeting, 2013]

*Authors contributed equally. Olfaction is an important means for animals to communicate with the environment, and experience can profoundly shape the representation of olfactory cues to an animal. Consistent findings from olfactory literature suggest that males and females detect, identify, and discriminate odors differently. However, little is known whether sex difference also influences olfactory learning. The nematode worm, Caenorhabditis elegans, feeds on bacteria in its natural environment but is susceptible to infection after the ingestion of harmful pathogenic bacteria. Because learning to avoid harmful food is essential for C. elegans survival, conditioned avoidance of odors associated with toxicity or infection is a robust form of olfactory learning. Previously, we have shown that naive hermaphrodites prefer the smell of the pathogenic bacteria P. aeruginosa (PA14) in comparison with the smell of the common lab food E. coli (OP50), and brief training with PA14 induces a learned olfactory aversion to the PA14 smell (Zhang et al., 2005, Ha et al., 2010). Here, we ask whether sex difference regulates olfactory learning. To this end, we used a high throughput micro-droplet assay system to measure the ability to learn to avoid the smell of PA14 in individual hermaphrodites or males. Preliminary data from our behavioral analysis has revealed that males exhibit defects in naive food odor preference as well as in learning, suggesting sex difference in olfactory plasticity. Previously, we have mapped a neural network that underlies the aversive olfactory learning (Ha et al., 2010). We plan to interrogate the effect of sex difference in this circuit that leads to the behavioral difference in olfactory learning. We hope that these studies will help further the understanding of how gender differences may influence olfactory behavior and plasticity in organisms such as humans.