Fernandez, R.W. et al. (2017) International Worm Meeting "An atlas of G protein-coupled neurotransmitter receptor expression patterns in C. elegans."

Wang, E., Pepper, J., Fernandez, R.W., Koelle, M.R.

[International Worm Meeting, 2017]

Neurotransmitters signal by activating ion channels or G protein-coupled receptors (GPCRs), and C. elegans provides a model for understanding signaling by these highly conserved molecules. Neurotransmitters can diffuse from their synaptic release sites to activate GPCRs on distant neurons. Thus, although the ~5,000 synapses between the 302 neurons of C. elegans have been mapped, and the neurons that release each of the C. elegans' seven small-molecule neurotransmitters have also been mapped, it remains unclear what target neurons respond to these neurotransmitters. We are creating an atlas of which cells in C. elegans express each of its 27 neurotransmitter GPCRs. To identify each GPCR's cellular expression pattern, we are making chromosomally-integrated fosmid-based GPCR::GFP reporter transgenes and crossing them into red fluorescent protein marker strains. Some GPCRs have published expression patterns, but in many cases these remain incomplete and contain inaccurate cell identifications. Our results show that a typical neurotransmitter GPCR is expressed in a few dozen cells, mostly neurons and muscles, but also some glial and epithelial cells. For example, the serotonin GPCR SER-4 is expressed in 35 neurons, four muscle cells, and two epithelial cells. Some of the SER-4 expressing neurons receive direct synapses from serotonin-releasing cells, but most do not, and SER-4 on these latter neurons could mediate extrasynaptic serotonin signaling. Interestingly, some SER-4-expressing neurons receive direct synapses from cells releasing other biogenic amines similar in structure to serotonin, suggesting these lower-affinity SER-4 ligands, may activate SER-4 on these cells. The Atlas can be used to determine what GPCRs are expressed in any neural circuit. For example, in the egg-laying circuit, our data confirm several published findings, correct the cells that express the GAR-2 acetylcholine receptor, and identify additional acetylcholine and octopamine GPCRs expressed in this circuit. Completion of our Atlas, integrated with the synaptic wiring and neurotransmitter maps, will provide a path to understand neurotransmitter signaling through GPCRs in each individual neural circuit and every behavior carried out in C. elegans. It should generate insights into how neural GPCR signaling is organized that generalize beyond C. elegans.

Fernandez, R.W. et al. (2019) International Worm Meeting "An Atlas of G protein-coupled Neurotransmitter Receptor Expression Patterns in the C. elegans egg-laying circuit."

Mikalauskaite, D., Wang, E., Fernandez, R.W., Kim, S., Koelle, M.R., Pepper, J., Olson, A., Wei, K., Navodiya, N.

[International Worm Meeting, 2019]

Neurotransmitters signal by activating G protein-coupled receptors (GPCRs). C. elegans has seven neurotransmitters and 27 neurotransmitter GPCRs that are conserved with those in the human brain, yet its simpler anatomy provides a system for studying how neural GPCR signaling is organized to help the nervous system carry out its functions. The ~5,000 synaptic connections between the 302 neurons of C. elegans have been completely mapped, and the individual neurons that express each of the seven neurotransmitters have been catalogued. We are adding an additional layer to this nervous system signaling map by creating an Atlas of which cells express each of C. elegans' 27 GPCRs for small-molecule neurotransmitters. For each GPCR, we made a chromosomally-integrated transgene in which the GPCR gene is fused to the GFP coding sequences, so that individual cells expressing the GPCR are fluorescent and can be identified by confocal microscopy. We tested the utility of this Atlas in the egg-laying circuit of C. elegans, since the role of neurons and neurotransmitters involved in modulating this circuit have been well-studied. Some key results from our work: 1) We found 15 of the 27 neurotransmitter GPCRs are strongly expressed in this circuit, while an additional three receptors are expressed weakly at levels just above background; 2) The average egg-laying neuron strongly expresses seven neurotransmitter GPCRs and weakly expresses an additional nine; 3) The egg-laying muscles strongly express nine neurotransmitter GPCRs weakly express an additional one; 4) The GABA GPCRs, GBB-1 and GBB-2, which are thought to form an obligate heterodimer, are not always expressed in the same cells; 5) There may be extensive extrasynaptic signaling that regulates egg laying, as several neurotransmitter GPCRs are expressed in cells that do not receive direct synapses from corresponding neurotransmitter-releasing neurons. We analyzed knock outs and overexpressors of all 27 GPCRS for changes in egg-laying behavior. We also tested how the several octopamine GPCRs are expressed in the C. elegans egg-laying circuit mediate inhibition of egg laying by exogenous octopamine. Our results provide the first comprehensive analysis of neurotransmitter GPCR signaling for a neural circuit. The tools we develop allow such an analysis of every neural circuit in C. elegans, and we expect such analyses should generate insights that generalize beyond C. elegans into how neural GPCR signaling is organized.

Fernandez RW et al. (2020) J Neurosci "Cellular Expression and Functional Roles of All 26 Neurotransmitter GPCRs in the C. elegans Egg-Laying Circuit."

Koelle MR, Kim S, Weissenborn S, Wang EY, Fernandez RW, Wei K, Olson A, Sarov M, Mikalauskaite D, Christie N, Pepper J

[J Neurosci, 2020]

Maps of the synapses made and neurotransmitters released by all neurons in model systems such as <i>C. elegans</i> have left still unresolved how neural circuits integrate and respond to neurotransmitter signals. Using the egg-laying circuit of <i>C. elegans</i> as a model, we mapped which cells express each of the 26 neurotransmitter G protein coupled receptors (GPCRs) of this organism and also genetically analyzed the functions of all 26 GPCRs. We found that individual neurons express many distinct receptors, epithelial cells often express neurotransmitter receptors, and receptors are often positioned to receive extrasynaptic signals. Receptor knockouts reveal few egg-laying defects under standard lab conditions, suggesting the receptors function redundantly or regulate egg-laying only in specific conditions; however, increasing receptor signaling through overexpression more efficiently reveals receptor functions. This map of neurotransmitter GPCR expression and function in the egg-laying circuit provides a model for understanding GPCR signaling in other neural circuits.<b>SIGNIFICANCE STATEMENT</b>Neurotransmitters signal through G protein coupled receptors (GPCRs) to modulate activity of neurons, and changes in such signaling can underlie conditions such as depression and Parkinson's disease. To determine how neurotransmitter GPCRs together help regulate function of a neural circuit, we analyzed the simple egg-laying circuit in the model organism <i>C. elegans.</i> We identified all the cells that express every neurotransmitter GPCR and genetically analyzed how each GPCR affects the behavior the circuit produces. We found that many neurotransmitter GPCRs are expressed in each neuron, that neurons also appear to use these receptors to communicate with other cell types, and that GPCRs appear to often act redundantly or only under specific conditions to regulate circuit function.

Wei, K. et al. (2019) International Worm Meeting "The Role of Gao-coupled Neurotransmitter GPCRs in the C. elegans Egg-Laying Circuit."

Fernandez, R.W., Wei, K., Koelle, M.R.

[International Worm Meeting, 2019]

The C. elegans egg-laying circuit is among the best-studied model neural circuits, as the function of each cell and the neurotransmitters it releases have been characterized. Our work has shown there are 20 neurotransmitter GPCRs detectably expressed in this circuit (see abstract by Fernandez et al.), yet their functions remain largely unstudied. We screened knockout mutants for every one of these neurotransmitter GPCRs to look for egg-laying defects. The single receptor knockouts showed at most mild egg-laying defects. We hypothesized that the weakness of these defects could be due to functional redundancy among the receptors. For example, knocking out GaO, a G protein known to inhibit neurotransmitter release in neurons of the egg-laying circuit, causes a very strong hyperactive egg-laying phenotype, and we found seven GaO- coupled neurotransmitter GPCRs are expressed in the egg-laying circuit that might redundantly activate this G protein. To test this idea, we generated various knockout combinations of five of the GaO-coupled neurotransmitter GPCRs. However, our results showed that even the quintuple knockout resulted in only a weak egg-laying defect. As a strategy to identify the functions of these GPCRs despite their apparent very high functional redundancy, we examined transgenic overexpressors of the GPCRs, as overexpression of this type of receptor can result in a gain-of-function effect. We found that the GABA receptor GPCR, GBB-1, when overexpressed from a high-copy transgene, showed a strong hyperactive egg laying phenotype. GBB-1 is thought to form a functional GABA receptor by forming an obligate heterodimer with a second GPCR called GBB-2. Knocking out gbb-2 suppressed most but not all of the effect of the GBB-1 overexpressor on egg laying. The residual effect of GBB-1 overexpression in the absence of GBB-2 suggests that GBB-1 may have additional dimerization partners besides GBB-2. These other partners may be other members of the family of class C GPCRs, which are known to form heterodimers with each other.

Gandhi, Jay et al. (2021) MicroPubl Biol

Fernandez, Anita G., Gandhi, Jay, Crosio, Giulia

[MicroPubl Biol, 2021]

Most cellular processes require the coordinate activities of multiple gene products. These collaborations vary with different contexts. mel-28 encodes a conserved multi-functional nucleoporin required for nuclear envelope integrity, the post-mitotic rebuilding of the nuclear pore, and the proper segregation of chromosomes during mitosis (Galy et al. 2006; Fernandez and Piano 2006). Loss of mel-28 results in strict maternal-effect embryonic lethality. Thus homozygous mutant embryos produced from heterozygous mothers show typical wild-type development until they become adults and produce normal-sized broods of inviable embryos. This suggests that mel-28 is required for embryonic development but is dispensable for post-embryonic development and adult processes such as fertility. To identify genes that function collaboratively with mel-28, we did an RNAi screen to find genes that cause fertility defects in mel-28 mutant animals but not wild-type animals (Fernandez et al. 2014). Among the genes identified were those encoding components of the minus-end-directed microtubule motor dynein.

Jun Liang et al. (2002) East Coast Worm Meeting "sma-9 functions in TGF-beta /Sma/Mab pathway and encodes a zinc finger protein homologous to Drosophila Schnurri"

Cathy Savage-Dunn, Ling Yu, Jun Liang

[East Coast Worm Meeting, 2002]

The Sma/Mab pathway, a TGF- related pathway, regulates body length and male tail pattern in C. elegans. Mutants of dbl-1 ligand, daf-4 type II receptor, sma-6 type I receptor, and sma-2, sma-3, sma-4 Smads display similar small body length and abnormal male tail sensory ray fusion. Although the major components were identified, we expected the existence of additional cofactors that are critical for the signaling. In a genetic screen in R.W Padgett's lab, one of the new genes identified was sma-9. sma-9 mutants show similar body length and abnormal male tail as the other identified sma mutants.

Wang L et al. (2023) STAR Protoc "Protocols for treating C. elegans with pharmacological agents, osmoles, and salts for imaging and behavioral assays."

Wang L, Bianchi L, Graziano B

[STAR Protoc, 2023]

Research rigor can be enhanced by pairing genetic tools with pharmacology and manipulations of solutes or ions. Here, we present a protocol for treating C.&#xa0;elegans with pharmacological agents, osmoles, and salts. We describe steps for agar plate supplementation, addition of the compound to the polymerized plates, and using liquid culture for exposure to the chemical. Treatment type depends on the stability and solubility of each compound. This protocol is applicable to both behavioral and in&#xa0;vivo imaging experiments. For complete details on the use and execution of this protocol, please refer to&#xa0;Wang et&#xa0;al. (2022),¹ Fernandez-Abascal et&#xa0;al. (2022),² and Johnson et&#xa0;al. (2020).³.

McGhee JD et al. (2000) West Coast Worm Meeting "Looking for synergy with PHA-4 on the myo-2 promoter"

Okkema PG, McGhee JD, Kalb JM

[West Coast Worm Meeting, 2000]

PHA-4 is a fork head transcription factor that is essential for pharyngeal development. Forced expression of PHA-4 outside of the pharynx can activate pharynx-specific genes. For example, it has been previously shown that PHA-4 activates myo-2, pharyngeal myosin, through the C-subelement in the myo-2 promoter. Ectopic PHA-4 expression leads to strong ectopic expression of a C-subelement regulated reporter gene. However, it only weakly activates endogenous myo-2; with ectopic expression of PHA-4 throughout the embryo, ectopic expression of myo-2 is only detected in body wall muscles, showing factors in addition to PHA-4 are necessary for myo-2 expression. Thus, we suggest that PHA-4 works with co-factors to activate gene transcription. One likely candidate to be a co-factor with PHA-4 is PEB-1. PEB-1 is a novel DNA binding protein expressed in the pharynx and was identified by its ability to bind to the C-subelement in the myo-2 promoter (Thatcher, Fernandez, Beaster-Jones, Haun and Okkema). The binding sites for PHA-4 and PEB-1 overlap in the C-subelement. To test if PHA-4 and PEB-1 act synergistically via the C-subelement to activate gene expression, we have been assembling the transcriptional regulatory network in yeast. We find that both PHA-4 and PEB-1 are (rather weak) transcriptional activators on their own. However, when both PHA-4 and PEB-1 are expressed together, we find no synergism, either positive or negative. To continue our investigation into the biochemical basis of PHA-4 action, we have produced all three forms of PHA-4 in baculovirus to define preferred binding sites.

Nassar, Layla et al. (2015) International Worm Meeting "Understanding how sex modulates the female nervous system to drive distinct reproductive behavior states."

Collins, Kevin, Nassar, Layla, Bode, Addys

[International Worm Meeting, 2015]

We are interested in understanding how mating and reproductive behaviors are coordinated in the female nervous system. Specifically, we are identifying the neural signaling systems that drive two mutually exclusive vulval motor behaviors: mating with males or the release of progeny during egg laying [1]. We hypothesize that: 1. female vulval muscle twitching contractions facilitate male spicule insertion during mating; 2. specific mechanical and chemical signals report successful copulation and insemination; and 3. mating initiates reproductive behaviors including oocyte production and egg release. To investigate these hypotheses, we are using calcium imaging techniques to record vulval signaling events during mating with males, after successful insemination, and during the resumption of normal egg laying behavior. We have found that hermaphrodites with sperm have reduced vulval muscle twitching behaviors resulting in inefficient mating. In contrast, hermaphrodites depleted of sperm have increased vulval muscle twitching that facilitates male spicule insertion and mating. After mating is complete, hermaphrodites have sustained vulval muscle twitching that can result in release of sperm from the uterus. This behavior may act as a mechanism for competition with self-sperm. We are now examining how the other cells in the egg-laying circuit, including the HSN and VC neurons and uv1 neuroendocrine cells, respond during steps of male mating. During egg laying behavior, we found that the uv1 cells are mechanically activated by passage of eggs through the vulva, driving release of tyramine that inhibits egg laying [2]. We predict that mechanical stimulation by male spicules may similarly activate the uv1 cells to inhibit egg release during mating. However, once mating is complete, we expect egg laying to resume or even increase. Together, these results will explain how internal and external signals modulate activity in the same neural circuit to drive distinct behavior states.1. Collins, KM, and Koelle, MR (2013). Postsynaptic ERG Potassium Channels Limit Muscle Excitability to Allow Distinct Egg-Laying Behavior States in Caenorhabditis elegans. J. Neurosci. 33, 761-775.2. Collins KM, Bode A, Fernandez R, Tanis JE, Creamer M, Koelle MR (2015). Unpublished results.

Stephen R Wicks et al. (2001) International C. elegans Meeting "A snip-SNP map of the C. elegans genome: Applications to positional cloning."

Robert H Waterston, Ronald H A Plasterk, Stephen R Wicks, Warren R Gish, Raymond T Yeh

[International C. elegans Meeting, 2001]

Single nucleotide polymorphisms (SNPs) are valuable genetic markers of human disease. They also comprise the highest potential density marker set available for mapping experimentally derived mutations in model organisms such as C. elegans . To facilitate the positional cloning of mutations we have identified polymorphisms in CB4856, an isolate from a Hawaiian isle that shows a uniformly high density of polymorphisms compared to the reference Bristol N2 strain. Based on 5.4 Mbp of aligned sequences, we predict 6222 polymorphisms. Furthermore, 3457 of these markers modify restriction enzyme recognition sites (snip-SNPs) and are therefore easily detected as RFLPs. Of these, we have experimentally confirmed 493 by restriction digest to produce a snip-SNP map of the worm genome (ref 1). A mapping strategy using snip-SNPs and bulked segregant analysis (BSA, ref 2) is outlined. CB4856 is crossed into a mutant strain, and exclusion of CB4856 alleles of a subset of snip-SNPs in mutant progeny is assessed with BSA. The proximity of a linked marker to the mutation is estimated by the relative proportion of each form of the biallelic marker in populations of wildtype and mutant genomes. This step bounds the mutation between flanking snip-SNPs. These flanking markers can be used to detect recombination in individual animals, and only recombinant strains need be phenotyped. By mapping the recombination points in individual animals, it is possible to rapidly zoom in on the site of a mutation. The advantages and limitations of this approach will be discussed. References: 1) Wicks, S.R., Yeh, R.T., Gish, W.R., Waterston, R.H. & Plasterk, R.H.A. (in press) Rapid gene mapping in C. elegans using a high density polymorphism map. Nature Genetics . 2) Michelmore, R.W., Paran, I. & Kesseli, R.V. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA 88, 9828-9832. (1991).