Eric J Aamodt (2005) International Worm Meeting "PAG-3 may be a neuronal cell fate toggle switch"

Eric J Aamodt

[International Worm Meeting, 2005]

PAG-3 is a C2H2 zinc finger transcription factor that is involved in specifying neuronal cell fates and lineages in C. elegans. The pag-3 gene is expressed in numerous embryonic neuroblasts and in the larval ventral nerve cord P-neuroblast sublineages that give rise to the VA, VB and VC neurons. Except in the ventral nerve cord, the cells that express pag-3 also express unc-86, and UNC-86 is required for pag-3::gfp expression in these cells. In the P-neuroblast lineages, on the other hand, pag-3::gfp is expressed even in an unc-86 loss-of-function mutant background and PAG-3 represses unc-86::gfp expression. That is, unc-86::gfp is expressed in the VA, VB and VC neurons in pag-3 mutants. In pag-3 loss-of-function mutants, the BDU neurons adopt a touch receptor neuron fate rather than an interneuron fate. In the BDU neurons, pag-3 is expressed at low levels where it represses mec-3 expression, and in the touch receptor neurons, pag-3 is expressed at high levels where it activates mec-3 expression. The PAG-3 ortholog in Drosophila, Senseless, has a similar role. When Senseless is expressed at low levels, it represses a sensory organ precursor fate and when it is expressed at high levels it activates a sensory organ precursor fate. PAG-3, Senseless and perhaps their mammalian homologs, Gfi-1and Gfi-1B may represent a class of developmental toggle switches that control cell fate decisions by switching from repressors to activators in a concentration dependent manner. PAG-3 will self-associate in vitro at high concentrations so the switch from repressor to activator may occur when these proteins form heterodimers. There is a conserved C-terminal binding protein (CtBP) corepressor-binding site in the zinc finger regions of PAG-3 and Senseless. This region may be masked by self-association because the zinc finger region alone will self-associate. This model predicts that the PAG-3 self-association will occur over a narrow concentration range that is between the concentration of PAG-3 in the BDU nucleus and the concentration of PAG-3 in the ALM nucleus. And it predicts that PAG-3 requires CtBP for its repressor function.

Eric J Aamodt et al. (2003) International Worm Meeting "PAG-3 is in several complex transcriptional regulatory networks in neurons."

Eric J Aamodt, Nicole Toms

[International Worm Meeting, 2003]

The C. elegans pag-3 gene encodes a transcription factor involved in the control of neuronal specification and cell lineage. PAG-3 zinc fingers are 78% identical to the zinc fingers in the human proto-oncoprotein Gfi-1 and 88% identical to the zinc fingers in the Drosophila neurogenesis protein Senseless. PAG-3 and Gfi-1 bind to the same DNA sequences, and all three proteins are involved in controlling cell lineage and fate. Senseless, like PAG-3, specifies the fate of touch receptors and other neurons and both PAG-3 and Senseless are in similar regulatory networks. This project is aimed at identifying pag-3 transcriptional control networks and relating them to the Gfi-1 and Senseless control networks. We previously showed that PAG-3 represses its own expression and that it represses touch neuron gene expression in the BDU neurons. Here we show that PAG-3 prevented expression of touch neuron genes in the BDU neurons by repressing the expression of mec-3, a master regulator of touch neuron specific genes. We also found that pag-3, which is expressed in several neuronal types including both the BDU neurons and all of the touch receptor neurons, activated mec-3gfp 2-3 fold in the touch neurons. Furthermore, mec-3 was required for pag-3gfp expression in the touch neurons yet repressed pag-3gfp expression in a subset of head neurons including the FLP neurons. All of the neurons where pag-3 is expressed, except for the motor neurons that express pag-3, also express unc-86 and unc-86 was required for pag-3gfp expression in all of these cells. Interestingly, pag-3 repressed unc-86gfp expression in the motor neurons that express pag-3. lin-32 and vab-15 were required for pag-3gfp expression in a group of head neurons, FLP neurons and ALM neurons in early L1 larval development but not by the late L1/early L2 stage. unc-3 repressed pag-3gfp expression in the head, FLP neurons and the touch receptor neurons. The NeuroD homologue, cnd-1, activated pag-3gfp expression in the head neurons, FLP neurons and touch neurons. unc-37 repressed pag-3gfp in the FLP neurons and egl-44 repressed pag-3gfp in the head and FLP neurons. Overall, pag-3 appeared to be part of several complex transcriptional regulatory networks and the network in the head neurons was different from the network in the FLP neurons, which was different from the network in the touch neurons, which was different from the network in the motor neurons.

Dwyer, Donard et al. (2017) International Worm Meeting "Protophenotypes in C. elegans relevant for schizophrenia and major depression."

Dwyer, Donard, Dagenhardt, Julie, Aamodt, Eric

[International Worm Meeting, 2017]

Efforts to deconvolute the complexity of genetic contributions to psychiatric illnesses such as schizophrenia and major depressive disorder (MDD) have led to the concept of endophenotypes - heritable traits that reflect intermediary pathological processes of a complex disorder. We have coined the term 'protophenotypes' to describe endophenotypes that have been conserved across species during evolution. The goal of our studies is to characterize the genetics and pharmacology of protophenotypes in C. elegans related to pre-pulse inhibition and startle defects in schizophrenia, social withdrawal (a prominent feature of both schizophrenia and MDD) and avolition/anhedonia, which is characteristic of various affective disorders in man. To model pre-pulse inhibition we evaluated touch suppression of pharyngeal pumping in various mutant strains and with pharmacological agents. To gain insight into social withdrawal, we studied aggregation in social feeding strains in the absence and presence of drugs. Lastly, we developed a model of avolition/anhedonia or diminished motivation based on defects in insulin/IGF-1 signaling, exposure to DMSO and food deprivation. In this model, we observed "suicidal" worms that failed to seek food or escape, but remained in place until they died, despite preservation of responsiveness to external stimuli. Genetic and pharmacological studies revealed that dopamine affects touch suppression via a primitive counter-circuit that presages the arrangement of dopaminergic pathways in man controlling emotions and logical thought. We found that serotonin regulated social feeding, which was inhibited by 5-HT2 receptor antagonists. This finding was consistent with serotonergic defects in MDD and schizophrenia. Studies of the third protophenotype related to diminished motivation revealed that defects in insulin receptor signaling (DAF-2) and proteins that regulate insulin secretion (UNC-64, but not UNC-31) produced rapid immobility in animals subjected to food deprivation in the presence of 1% DMSO. The immobility state in daf-2(lf) mutants was maintained by elevated serotonergic and muscarinic cholinergic signaling. Immobility was reversed with antidepressants (e.g., amitriptyline, imipramine and amoxapine) and antipsychotics (e.g., clozapine and olanzapine, but not haloperidol) that decrease immobility in the forced swimming test - an established model of depression in man. These findings may help to explain the 2-3-fold increase in MDD in patients with diabetes. Finally, this work may establish mechanisms for how fundamental behaviors conserved through evolution contribute to the emergence of psychiatric illness in man.

Stroeher VL et al. (1991) International C. elegans Meeting "POSSIBLE LINEAGE-SPECIFIC ACTIVATORS OF THE C. elegans ges-1 GENE."

Stroeher VL, McGhee JD

[International C. elegans Meeting, 1991]

The ges-1 gene of Caenorhabditis elegans encodes a nonspecific carboxylesterase that is expressed exclusively in the intestinal lineage. Deletion and transformation analyses show that this restricted expression is due to lineage specific activators and repressors that bind to upstream promoter sequences (see abstract by E. J. Aamodt, M.A. Chung and J.D.M.). As a first step to clone these regulatory factors, we are identifying their binding sites using gel retardation assays (bandshifts) and DNasel footprinting experiments. At least seven regions of protein interaction have been found in the sequences 935 to 1309 basepairs (bp) upstream of the translation start site. One of these sites, which may bind a putative 'gut activator' molecule is being examined more closely. A doublestranded, 30 bp oligomer (30mer) representing this region has been synthesized. In gel retardation assays with crude nuclear extract from FUdR-blocked embryos, this 30mer gives a single major shifted species, which is effectively competed with unlabelled double-strand 30mer. DNA-protein binding reactions have been exposed to UV irradiation to crosslink the bound protein(s) to the labelled, doublestrand 30mer; a major 80 kd protein is detected. We are now investigating when this protein appears in development.

Irene Zelepuhin et al. (2005) International Worm Meeting "Genetics of sex Reversal of C. briggsae::C. remanei XO hybrids."

Scott Baird, Shannon Romer, Irene Zelepuhin

[International Worm Meeting, 2005]

Sterile F1 adult hybrids can be obtained from crosses of C. briggsae males to C. remanei females. Depending on the strains used, all hybrids are females. The absence of male hybrids results from sexual transformation, not male-specific lethality, as both XX and XO females are observed. Variant strains of C. briggsae and C. remanei suppress this hybrid sexual transformation, demonstrating genetic variation within both species the affects sex determination in F1 hybrids. This variation has been used to map hybrid sex reversal loci. In C. remanei, an ill-defined region of the X chromosome is implicated. In C. briggsae, three loci linked to hybrid sex reversal have been identified. The strongest linkage is to allelic variants in Cb-tra-2. A null mutation in Cb-tra-2 (thanks Eric) completely suppresses the transformation of XO hybrids. Other C. briggsae loci linked to sex reversal variants include Cb-daf-5 and Cb-mab-9.

Sawh, Ahilya et al. (2015) International Worm Meeting "Regulation of DCR-1-dependent RNAi pathways through DCR-1 phosphorylation."

Duchaine, Thomas, Wohlschlegel, James, Lewis, Alexandra, Sawh, Ahilya

[International Worm Meeting, 2015]

RNA interference (RNAi) pathways are directed by small (18-22nt) non-coding RNAs and orchestrate a variety of evolutionarily-conserved gene-silencing programs. The type III RNase protein Dicer is at the center of most of these pathways, including responses triggered by exogenous sources of long double-stranded RNA (exoRNAi). Other pathways, such as microRNA-mediated silencing and endoRNAi are triggered by endogenous loci, and control developmental and homeotic events. Dicer's role is crucial and three-fold: it cleaves long dsRNA precursors into smaller RNAs that direct silencing events, it loads the small RNAs into Argonaute proteins to form an active RNA-induced silencing complex (RISC), and it acts as a scaffold for several protein co-factors required for substrate recognition and processing. In C. elegans, Dicer (DCR-1) is essential in three small-RNA mediated silencing mechanisms including exoRNAi and ERI endoRNAi, two RNAi pathways that compete for downstream effectors. DCR-1 is part of distinct multi-protein complexes in each of these RNAi pathways: RDE complex in exoRNAi and ERIC in ERI endoRNAi.We discovered and validated several phosphorylated determinants of DCR-1 in the C. elegans. Particular attention was placed on a cluster of phosphorylated sites between the PAZ and RNase III domains, one of which could implicate AMPK signaling. Recent crystal structures of human Dicer have shown that this region could be involved in recognition of dsRNA substrates. We thus hypothesized that phosphorylation in this cluster could impair substrate recognition, or alter its specificity.To study the effect of DCR-1 phosphorylation on the three DCR-1-dependent RNAi pathways in C. elegans, transgenic strains were engineered expressing phosphorylation-deficient, and phosphorylation-mimetic mutants of DCR-1 in this cluster of sites. Functional analysis on these strains revealed that phosphorylation-deficient mutants display an impaired exoRNAi response, and severe developmental defects. Phosphorylation-deficient DCR-1 mutants also enhance interaction with ERI-endoRNAi DCR-1 cofactors. Taken together, these results support a model wherein DCR-1 phosphorylation may promote its shift from the ERIC to the RDE complex. Post-translational regulation of Dicer and RNAi pathways are largely unexplored and could potentially be incredibly impactful in light of their crucial roles in development and disease. The present study will shed light on how RNAi pathways integrate signals through phosphorylation signaling cascades.

Lee, Inhwan et al. (2013) International Worm Meeting "Epigentic regulation of L1 longevity."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2013]

Animals encounter food infrequently and starvation is a common physiological state in their natural circumstance [1]. Hatching in the absence of food, worms arrest their development at the L1 stage and survive starvation more than two weeks [2]. It was shown that adult longevity is regulated epigenetically by chromatin alterations and the genes that regulate epigenetics [3]. Here, we investigate whether L1 longevity is also regulated epigenetically. When we measured various histone modifications using Western blot, we found histone 3 lysine 4 tri-methylation, known to be a modification to activate gene transcription, is increased in L1 starvation. Moreover, mutants of set-2, which encodes a histone 3 lysine 4 tri-methyl transferase that functions in adult longevity [3], has reduced L1 longevity. There are several genes essential for normal L1 longevity, such as aak-2 and daf-16. Based on our data, we hypothesize that chromatin remodeling through histone 3 lysine 4 tri-methylation regulates transcription of genes involved in L1 longevity. Currently we are testing this hypothesis by measuring expression levels these genes by ChIP-qPCR during L1 starvation in set-2 mutant. References [1] Brian H. Lee, Plus Genetics, 2008 [2] Inhwan Lee, Plus One, 2012 [3] Eric L. Greer, Nature, 2010.

Hamill, Danielle R. et al. (2009) International Worm Meeting "Analysis of a spindle assembly mutant in C. elegans."

Hamill, Danielle R., Everett, Molly, Bowerman, Bruce, Price, Meredith, McNeeley, Marie, Mazhar, Sahar, Gearica, Amy

[International Worm Meeting, 2009]

Cell division is a highly conserved and complex process that must be carefully orchestrated. or452ts was isolated in a screen for temperature sensitive embryonic lethal cell division mutants. In or452ts mutant embryos, bipolar spindles often fail to form. In these mutants DNA localizes around microtubules emanating from a single, often misshapen, centrosome. Staining with centriole markers suggests that a centriole duplication defect may underlie the spindle assembly defect. Using live cell imaging and fluorescence transgenes (including GFP::gamma tubulin), we have noticed centrosome streaking and splitting that is not seen in wild-type embryos. We also have observed the formation of asymmetric spindles. Towards the goal of identifying the gene that is mutated in or452ts, we have done meiotic mapping using phenotypic markers and snip-SNPs. We have narrowed the genetic location down to about 0.5 cM around +2.5 on LGIII. This region contains less than 500 kbp of DNA, and there are about 40 predicted genes in this region. In collaboration with Doug Turnbull and Eric Johnson (University of Oregon), we are sequencing the DNA from this region to try to identify the mutation responsible for the or452ts mutant phenotype. We believe that characterization of this mutant, and identification of the gene responsible, will provide new insights into the process of mitotic spindle assembly and cell division.

Megan A Higginbotham et al. (2003) International Worm Meeting "A mod-5 Suppression Screen for Genes Involved in Serotonergic Neurotransmission"

Megan A Higginbotham, Bob Horvitz

[International Worm Meeting, 2003]

Wild-type animals that have been acutely food deprived slow their locomotory rate upon encountering bacteria more than do well-fed animals. This behavior, called the enhanced slowing response, is serotonin (5-HT) dependent. Animals mutant for the 5-HT reuptake transporter mod-5 slow even more than wild-type animals. Additionally, mod-5 animals are hypersensitive to exogenous 5-HT. To identify additional genes involved in 5-HT signaling and possibly the enhanced slowing response, we screened for suppressors of this 5-HT hypersensitivity. We screened 46,200 haploid genomes using Mos1 transposon mutagenesis (Bessereau et al. Nature, 413: 70-74, 2001). Four strong suppressors were identified. Two contain insertions in genes previously known to suppress mod-5 for both the exogenous 5-HT hypersensitivity and the hyperenhanced slowing response. One of these suppressors is an allele of mod-1, which encodes a 5-HT-gated chloride channel, and the other is an allele of goa-1, a predicted alpha subunit of a heterotrimeric G-protein. Four transposon insertions have been identified on chromosome I in the strain carrying the third suppressor, n4094. Four transposon insertions have also been identified in the strain carrying the fourth suppressor, n4095. Experiments are underway to determine which of these insertions, if any, causes suppression of the 5-HT hypersensitivity of mod-5. We are also working to define neural circuits through which both mod-5 and suppressors of mod-5 act to affect the hyperenhanced slowing response. Using antibodies raised against MOD-5 protein, we have identified several head neurons in which MOD-5 expression can be seen. Additionally, a translational mod-1::rfp reporter has been constructed (by Eric Miska), and we are currently identifying the cells in which it is expressed.

Kosalka, J. et al. (2015) International Worm Meeting "Mechanism and regulation of piRNA biogenesis in the C. elegans germline."

Weick, EM., Miska, E., Kosalka, J.

[International Worm Meeting, 2015]

Mechanism and regulation of piRNA biogenesis in the C. elegans germlineJoanna Kosalka, Eva-Maria Weick, Eric A MiskaWellcome Trust/CRUK Gurdon Institute/Department of Genetics, University of Cambridge, Cambridge, United KingdomPiwi-interacting RNAs (piRNAs) are the most recently identified and by far the largest class of endogenous small regulatory RNAs (sRNAs). In C. elegans, piRNAs are synthesised as single strand precursors from specific genomic loci known as piRNA clusters, processed into mature 21 nucleotides long, 5'U sRNAs that are recognised by the PIWI clade of Argonaute proteins. piRNAs are germline-expressed and are essential for fertility and genome stability. piRNAs can align to transposons and repetitive sequences in an antisense orientation, targeting them for silencing and the loss of the piRNA pathway results in reactivation of transposons. In addition, some endogenous protein-coding genes are under control of the piRNA pathway.Many aspects of the C. elegans piRNA pathway remain poorly understood and elucidating the biogenesis of 21U RNAs is of particular interest. Importantly, the fundamental questions of how the synthesis of piRNA precursors is initiated and directed to specific loci, and how the maturation of the precursor molecules is executed, remain unanswered. Furthermore, the developmental regulation of piRNA expression is unknown.In a forward genetic screen our lab has identified prde-1 (piRNA defective) as an essential germline-expressed factor for piRNA biogenesis (Weick et al. 2014). prde-1 is involved in the initial steps of piRNA biogenesis and is indispensable for piRNA precursor synthesis and/or stability. Interestingly, PRDE-1 protein localises specifically to parts of chromosome IV coinciding with the large piRNA cluster. Although the molecular mechanisms of prde-1 function are currently unknown, these findings provide an exciting starting point to elucidate the mechanisms of piRNA cluster transcription and regulation.Here we will present our most recent progress on understanding the molecular machinery required for the biogenesis of piRNAs. We used a range of biochemical approaches to identify and characterise PRDE-1-interacting proteins and their role in piRNA biogenesis.