Lloret-Fernandez, Carla et al. (2017) International Worm Meeting "Conserved transcriptional regulatory rules define the serotonergic-neuron identity."

Maicas, Miren, Chirivella, Laura, Flames, Nuria, Lloret-Fernandez, Carla, Jimeno, Angela, Weinberg , Peter, Artacho, Alejandro, Mora, Carlos

[International Worm Meeting, 2017]

Cell differentiation is controlled by individual transcription factors (TFs) that together activate a selection of enhancers in specific cell types. How these combinations of TFs identify and activate their target sequences remains unknown. Here, we identify the cis-regulatory transcriptional code that controls the differentiation of serotonergic (5HT) HSN neurons in C. elegans. Loss of function mutant and cis-regulatory analyses reveal that direct activation of the HSN transcriptome is orchestrated by a collective of six TFs. This TF code is composed by AST-1 (ETS TF family), UNC-86 (POU TF family), SEM-4 (SPALT TF family), HLH-3 (bHLH TF family), EGL-46 (INSM TF family) and EGL-18 (GATA TF family). Bioinformatically identified binding site clusters for these six TFs are enriched in known HSN expressed genes compared to a random set of genes. Through in vivo reporter analysis, we demonstrate that the clustering of TF collective binding sites constitutes a regulatory signature that is sufficient for de novo identification of HSN neuron functional enhancers. This regulatory signature contains certain syntactic constrains that further improve the prediction of enhancer expression in the cell. Mouse orthologs of most members of this TF collective are known regulators of mammalian 5HT differentiation programs and can functionally substitute for their worm counterparts. Finally, Principal Coordinates Analysis suggests that, among C. elegans neurons, the HSN transcriptome most closely resembles that of mouse 5HT neurons, which reveals deep homology. Our results?identify rules governing the transcriptional regulatory code of a critically important neuronal type in two species separated by over 700 million years.

Rook, Vicky et al. (2021) International Worm Meeting "Sexually dimorphic glia-neuron reprogramming in Caernorhabditis elegans."

Marti I Sabari , Albert, Rook, Vicky, Sammut , Michele, Bonnington, Rachel, Poole, Richard, Lloret-Fernandez , Carla

[International Worm Meeting, 2021]

How fully differentiated cells can retain their neurogenic potential remains a central question in developmental biology. Previously, we described a glia-to-neuron cell fate switch during sexual maturation in the nervous system of male Caenorhabditis elegans; whereby stably differentiated amphid socket (AMso) glial cells in the head undergo asymmetric cell division to self-renew and give rise to the MCM (Mystery Cells of the Male) neurons1. Using forward genetic screening and candidate gene approaches we have identified a particularly exciting class of mutants (Class III) in which the AMso divides but fails to adopt MCM neuronal identity, instead retaining glial marker expression. We also find that mutations in the zinc finger transcription factor lin-48, an ortholog of vertebrate OVO1 and in the proneural bHLH transcription factor hlh-14, a homolog of vertebrate ASCL1 generate a Class III phenotype. In addition, we have isolated two novel Class III mutants; nom-7 and nom-9 and we are currently performing mapping-by-sequencing to identify the causal locus and will present our most recent findings here. Our analysis of Class III genes will help us to elucidate the molecular mechanisms regulating the plasticity underlying this sexually dimorphic AMso-to-MCM transdifferentiation event. 1Sammut, M. et al. Glia-derived neurons are required for sex-specific learning in C. elegans. Nature 526, 385-390, doi:10.1038/nature15700 (2015).

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.

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. 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 vivo imaging experiments. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022),¹ Fernandez-Abascal et al. (2022),² and Johnson et 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.

John Kalb et al. (2001) International C. elegans Meeting "Analysis of PHA-4 activity on the myo-2 promoter and identification of a new PHA-4 DNA binding site"

Pete Okkema, Jim McGhee, John Kalb

[International C. elegans Meeting, 2001]

PHA-4 is a forkhead/winged helix transcription factor that is essential for pharyngeal organogenesis. PHA-4 is expressed in all five classes of pharyngeal cells. Ectopic expression of PHA-4 leads to ectopic expression of pharyngeal markers. We wish to identify other factors that cooperate with PHA-4 to confer cell-type specificity. In pharyngeal muscles, PHA-4 binds to a site in the myo-2 promoter to activate myo-2 transcription. A potential transcriptional cofactor of PHA-4 is PEB-1, a novel DNA binding protein that is expressed in the pharynx and binds to the myo-2 promoter at a site that overlaps with PHA-4 ( 1 ). To test for PHA-4 and PEB-1 interactions we have expressed both factors in yeast to investigate their ability to activate transcription of a reporter gene regulated by the binding sites found in the myo-2 promoter. Both PHA-4 and PEB-1 (weakly) activate transcription when expressed alone. However, we detect neither synergy nor interference between PHA-4 and PEB-1 when they are coexpressed. Other experiments also suggest no evidence for cooperative binding but rather that PEB-1 may interfere with PHA-4 binding ( 1 ). To identify PHA-4 target genes, we are determining the preferred PHA-4 binding site. The pha-4 gene produces three PHA-4 protein isoforms called PHA-4A, B and C that differ only at their N-termini. After six rounds of DNA site selection from a pool of random oligonucleotides 20 bp long, baculovirus-produced PHA-4B selects the sequence TGTGG(C/T); this differs somewhat from the known TGTTTG PHA-4 binding site in the myo-2 promoter. We are currently putting this selected sequence in the promoter of a GFP reporter gene to determine if it functions as an enhancer in vivo and continuing site selection with PHA-4A and C. ( 1 ) See the abstracts by Laura Beaster-Jones and Anthony Fernandez.

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.

Laura Beaster-Jones et al. (2001) International C. elegans Meeting "Comparative and functional analyses of the novel pharyngeal factor PEB-1"

Peter Okkema, Anthony Fernandez, Laura Beaster-Jones

[International C. elegans Meeting, 2001]

The C.elegans pharynx is a complex neuromuscular organ consisting of five distinct cell types: muscles, neurons, glands, epithelial, and marginal cells. Pharyngeal cell-type specific gene expression is regulated in part by organ-specific signals. In the pharyngeal muscle myosin gene, myo-2 , these signals target an enhancer sequence called the C subelement. The C subelement contains overlapping binding sites for PHA-4, a winged helix transcription factor, and PEB-1, a novel factor that may define a new class of DNA binding proteins. PEB-1 and PHA-4 are co-expressed in many pharyngeal cell types and we hypothesize they participate in organ-specific regulation of gene expression. PEB-1 is a novel 427 amino acid protein with a unique DNA binding domain. To help recognize functionally important regions in PEB-1, we have identified a peb-1 homologue from the related nematode C. briggsae (1). peb-1 intron/exon structure, splice acceptor and splice donor sites are highly conserved between the two species. Overall, the predicted C. briggsae PEB-1 protein is 77% identical to C. elegans PEB-1. The PEB-1 DNA binding domain exhibits 84% identity over 158 amino acids. While this level is higher than the overall sequence identity, it is lower than that found in other DNA binding domains that have been characterized in both species. A match to a consensus sequence found in the Drosophila Mod(mdg4) proteins is found in both C. elegans and C. briggsae PEB-1. Among the most highly conserved regions are a putative PEST sequence located near the N-terminus and a Cys-rich region at the C-terminus. We hypothesize the DNA binding domain and these conserved regions are important for PEB-1 function. To better understand PEB-1’s role in regulating pharyngeal gene expression, we are characterizing how PEB-1 interacts with PHA-4 and with DNA. We have observed no evidence for cooperative binding between PEB-1 and any of the three PHA-4 isoforms in vitro (2). Rather PEB-1 competes effectively for PHA-4 binding to the C subelement, suggesting these proteins may not bind the C subelement together. Indeed when PEB-1 and PHA-4 are coexpressed in vivo using a heat shock promoter, PEB-1 interferes with PHA-4’s ability to activate the C subelement enhancer (3). We are currently identifying a consensus PEB-1 binding site by PCR-assisted binding site selection to facilitate identification of other genes that may be targeted by PEB-1. Genome Sequencing Center, Washington University, personal communication. We thank J. McGhee and J. Kalb for PHA-4 protein. See abstract of A. Fernandez and P. Okkema.

Powell-Coffman JA et al. (1997) Worm Breeder's Gazette "A C. elegans Dioxin Receptor"

Wood WB, Powell-Coffman JA

[Worm Breeder's Gazette, 1997]

The vertebrate aryl hydrocarbon receptor (AHR; a.k.a. the dioxin receptor) mediates the teratogenic and carcinogenic effects of certain environmental contaminants. AHR ligands include benzo(a)pyrene, the primary carcinogen in cigarette smoke, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous man-made pollutant. Unliganded AHR resides in the cytoplasm in a complex with the 90-kDa heat shock protein (HSP90). Upon binding ligand, AHR translocates to the nucleus, dissociates from HSP90, and dimerizes with the AHR nuclear translocator protein (ARNT). Both AHR and ARNT belong to a class of regulatory proteins that contain a basic-helix-loop-helix DNA binding motif and a PAS domain (also found in Drosophila period and single-minded). The AHR-ARNT heterodimer binds specific promoter sequences to regulate transcription. (1) In mice AHR function is required for the proper development of the immune system and liver, but the presumptive endogenous ligand(s) is unknown. (2) Until recently, it had not been possible to study AHR signaling in a model system amenable to genetic analysis, as no invertebrate orthologues of AHR or ARNT had been reported. We have cloned the C. elegans homologues of AHR and ARNT. CeAHR may be the egl-33 gene; it is included in the 15kb genomic fragment that rescues egl-33 (ct315). (3) CeARNT maps to the right of egl-33 on LGI. We have expressed these gene products in rabbit reticulocyte lysates. Initial gel electrophoretic shift experiments demonstrate that CeAHR and CeARNT interact to bind the mammalian AHR-ARNT enhancer site. Competition experiments with wild-type and a mutant form of the enhancer site confirm that this binding is specific. These data suggest that we have cloned true C. elegans orthologues of AHR and ARNT. We are also collaborating with C. Bradfield and colleagues at the University of Wisconsin to determine the binding affinity of CeAHR for TCDD. To identify the cellular decisions regulated by the AHR signaling complex in C. elegans, we are screening for Tc1-induced deletions in CeAHR and CeARNT, and we are examining the expression patterns of these two genes. RNA blots indicate that CeAHR and CeARNT are coordinately expressed and are most abundant during embryogenesis and the first larval stage. We have constructed lacZ and GFP reporter genes and have detected expression of CeAHR::lacZ as early as the 30-cell stage. At the 300-cell stage, it is expressed in approximately 20% of the cells. As morphogenesis begins, most cells cease to express CeAHR::lacZ, and at hatching, it is detectable in a subset of neuronal and hypodermal cells. These experiments will establish the foundation for further genetic analysis of the AHR signaling pathway. 1. Hankinson, O. (1995) Annu. Rev. Pharmacol. Toxicol. 35: 307-34; Whitlock, J. P. (1993) Chem. Res. Toxicol. 6: 754-763; Bock, K. W. (1993) Rev. Phys. Biochem. Pharmacol. 125: 1-42 2. Fernandez-Salguero, P. et al. (1995) Science 268: 722-726 3. Powell-Coffman, J. A. and B. Wood. 1996 West Coast Worm Mtg.