Deehan, Kevin et al. (2015) International Worm Meeting "Spinal Muscular Atrophy (SMA) Genes Regulate Insulin Signaling in Response to High-Glucose Diet."

Sikes, Emma, Hanover, John, Mastroianni, Michael, Krause, Michael, Mondoux, Michelle, Deehan, Kevin

[International Worm Meeting, 2015]

Spinal muscular atrophy (SMA) is a neurodegenerative disease that results in the loss of motor neurons, muscle atrophy, paralysis, and in the majority of cases, infant mortality. The causative gene of SMA is SMN1, which encodes a survival motor neuron protein and is implicated in the regulation of splicing. Our project seeks to establish and characterize a new, promising link between SMA and glucose intake in C. elegans. Studies have shown that in humans, glucose consumption has increased in recent years along with the prevalence of type 2 diabetes. Caenorhabditis elegans is an excellent model to study SMA, as the smn-1 gene is nearly 80% identical in worms and humans, and it is also a good model to study the response to high-glucose diet due to its conserved insulin-signaling pathway.Previous studies have shown that a high-glucose diet decreases dauer formation in daf-2 insulin receptor mutants due to the up-regulation of insulin signaling. An RNAi screen identified approximately 160 candidate genes that may be required for this insulin signaling response to high glucose. Secondary screens for insulin- and glucose-specificity reduced the gene list to 44 candidates. Interestingly, two of the most promising candidates are smn-1 and nekl-3, the latter of which has been identified as a modifier of smn-1. This identifies a potential link between SMA, high-glucose diet, and insulin signaling.We have completed dauer assays to verify and quantify the insulin specificity of these 44 candidate genes, and have partially verified and quantified the glucose specificity. We are currently investigating the role of ~30 other genes that have been identified as smn-1 genetic interactors, and how insulin signaling and a high glucose diet effect other smn-1 phenotypes such as lifespan, fertility, and stress resistance in a daf-2 background.

Alcorn, Melissa et al. (2015) International Worm Meeting "The propensity for stochastic PCD during male tail development varies across C. elegans isotypes."

Alcorn, Melissa, Rothman, Joel, Birsoy, Bilge, Cieslak, Matthew, Lopez Santos, Agustin, Callander, Davon

[International Worm Meeting, 2015]

While somatic programmed cell death (PCD) occurs in a largely invariant pattern in C. elegans, we have found that it arises sporadically and inappropriately during the formation of the 18 bilaterally symmetric sensory structures (rays) of the adult male tail. The rays, each consisting of the dendritic ends from two neurons and a structural support cell, arise during post-embryonic development. Wild-type N2 males frequently lack one or more rays. Males defective for components required for PCD contain few or no missing rays, suggesting that ray loss is the result of stochastic, inappropriate PCD. We found that the propensity for ray loss varies widely between 97 unique isotypes: while some isotypes virtually never show missing rays, in others, up to ~35% of males lack rays. As with N2, the loss of rays in these other strains appears to be the result of PCD, as they are restored by egl-1(RNAi). EMMA analysis applied to the full dataset of the 97 strains revealed that the variation is associated with a region on Chromosome I. We have also developed a complementary computational approach to analyzing this type of data by using a predictive regression model based on machine learning. This method applied to this and other datasets has identified regions of significance on Chromosomes II, III, V and X that were not predicted from standard EMMA analysis. Both approaches indicate that the sporadic PCD in the male is a polygenic trait. Ongoing analysis of RILs between two strains that represent phenotypic extremes is being used to identify the specific causal genes that control stochastic PCD during formation of the male rays. .

Rachel McMullan et al. (2006) European Worm Meeting "Rho is a Pre-Synaptic Activator of Neurotransmitter Release at Pre-Existing Synapses in C.elegans"

Rachel McMullan, Emma Hiley, Paul Morrison, Stephen J. Nurrish

[European Worm Meeting, 2006]

Rachel McMullan, Emma Hiley, Paul Morrison and Stephen J. Nurrish. Rho GTPases have important roles in neuronal development but their function in adult neurons is less well understood. We demonstrate that pre-synaptic changes in Rho activity at C.elegans neuromuscular junctions can radically change animal behaviour via modulation of two separate pathways. In one, pre-synaptic Rho increases acetylcholine (ACh) release by stimulating the accumulation of diacylglycerol (DAG) and the DAG-binding protein UNC-13 at sites of neurotransmitter release; this pathway requires binding of Rho to the DAG kinase DGK-1. A second DGK-1-independent mechanism is revealed by the ability of a Rho inhibitor (C3 transferase) to decrease levels of release even in the absence of DGK-1; this pathway is independent of UNC-13 accumulation at release sites. We do not detect any Rho induced changes in neuronal morphology or synapse number, thus Rho facilitates synaptic transmission by a novel mechanism. Surprisingly, many commonly available human RhoA constructs contain an uncharacterised mutation that severely reduces binding of RhoA to DAG kinase. Thus a role for RhoA in controlling DAG levels has not been previously appreciated.

Michael Briese et al. (2006) European Worm Meeting "A Worm Model for Spinal Muscular Atrophy"

David B. Sattelle, Laurence A. Brown, Michael Briese, Emma Burt, Paula Towers, Behrooz Esmaeili

[European Worm Meeting, 2006]

Michael Briese1, Behrooz Esmaeili2, Laurence A. Brown1, Emma Burt1, Paula Towers1 and David B. Sattelle1. Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder in which lower motor neurons in the spinal cord degenerate causing progressive muscle wasting. The predominant form of SMA shows childhood-onset and is associated with deletions or mutations of the Survival Motor Neuron 1 (SMN1) gene. Its C. elegans orthologue smn-1 is expressed in neurons, muscles and other cells and knockdown of smn-1 by RNA interference (RNAi) results in embryonic lethality and sterility. We have investigated the smn-1(ok355) deletion mutant which is sterile and arrests at larval stage L2-L3. Mutant worms show defects in pharyngeal pumping, movement and defecation. The movement defect becomes apparent at the late L1 stage in the form of an abnormal backward motion. As development proceeds, mutant animals become uncoordinated for both forward and backward movement. Using GFP reporters expressed pan-neuronally (F25B3.3::GFP) or in cholinergic neurons (unc-17::GFP), we could not detect any gross morphological abnormalities of the nervous system architecture. We are currently in the process of scoring smn-1(ok355) worms for synaptic defects using synaptic markers, including unc-25::SNB-1::GFP. We are also investigating the neuromuscular functions of SMN-1 in C. elegans by attempting to knock down smn-1 through promoter-driven hairpin RNAi. To this end we are using the plasmid pWormgatePro which we have previously tested on genes expressed in muscle cells and neurons. Additionally, C. elegans embryonic cell cultures are being deployed to investigate cell-autonomous functions of smn-1 in neurons and muscles.

Alcorn, Melissa et al. (2017) International Worm Meeting "Evidence that machine learning tools provide a more sensitive strategy than conventional mapping methods for identifying loci underlying genetically complex traits."

Cieslak, Matthew, Torres Cleuren, Yamila, Alcorn, Melissa, Rothman, Joel

[International Worm Meeting, 2017]

Genome-wide association studies (GWAS) and quantitative trait loci (QTL) mapping have been successful in identifying genetic variants in humans and model organisms. However, for complex traits, in which multiple loci contribute, genomic variants with small individual effects may be missed by these methods owing to limited statistical power from testing thousands of single-nucleotide polymorphisms (SNPs). Here we present a case-study of the application of machine learning algorithms, specifically ElasticNet regression, as a complementary approach to conventional GWAS and QTL mapping techniques, with the goal of identifying major loci and others that may contribute minor effects on their own, but collectively drive substantial phenotypic diversity. As a test of the approach, we applied ElasticNet regression to identify loci that control the plasticity in the gene regulatory network for C. elegans endoderm development. Our phenotype analysis has revealed large variation in the requirement for SKN-1 in endoderm development: while the laboratory N2 strain shows a partially penetrant phenotype loss of gut (30% of embryos produce gut) in skn-1(-), we found that this varies widely across 94 wild isotypes, ranging from 0% to ~60%. GWAS using efficient mixed-model analysis (EMMA) identified a single highly significant peak on chromosome IV that accounts for at least some of the variation across these strains. QTL mapping using RILs obtained from crosses between N2 and the MY16 isotype (in which only ~2% of embryos make gut in skn-1(-)) identified a significant QTL on chromosome IV, likely corresponding to the locus identified by GWAS, as well as three additional QTL on chromosomes I, II, and X. We found that the ElasticNet machine-learning tool applied to the same dataset not only effectively identified all four regions found by conventional QTL mapping, but also uncovered additional loci on chromosomes III and V (R2 = 0.50, p = 1.95x10 -7), revealing that this method may provide a more sensitive strategy for identifying genomic variations responsible for complex genetic traits. We are currently testing the novel regions identified by the machine learning approach in near-isogenic lines of different genetic backgrounds to assess their impact on the observed variation in the endoderm gene regulatory network.

Torres Cleuren, Yamila et al. (2015) International Worm Meeting "Extreme variation in the requirement for SKN-1 in endoderm development reflects both genomic variation and stably heritable epigenetic states that differ between isotypes."

Chipman, Kyle, Alami, Coco, Rothman, Joel, Snell, Russell, Torres Cleuren, Yamila

[International Worm Meeting, 2015]

We have analyzed the well-described gene regulatory network (GRN) for endoderm specification in C. elegans to investigate the basis for evolutionary plasticity in GRN architecture. Deployment of the endoderm GRN is initiated by the combined action of the SKN-1 transcription factor and an inductive signal mediated in part by MOM-2/Wnt. While removal of either maternal SKN-1 or MOM-2 in the N2 strain results in a partially penetrant loss of gut, simultaneous removal of both effectively eliminates endoderm. We found that the requirement for SKN-1 and MOM-2/Wnt in endoderm development varies dramatically across the 97 haplotypes: while RNAi knockdown of skn-1 or mom-2 results in fully penetrant lethality, we observed broad variation in penetrance across the spectrum of strains, ranging from <1% to 100% of arrested embryos with differentiated gut. The extreme variation in requirement for SKN-1 reflects bona fide differences in GRN function, as introgression of a skn-1(-) chromosomal mutation in selected isolates yield quantitatively the same effect as for RNAi. The penetrance of the skn-1(-) vs. mom-2(-) phenotypes did not correlate across the isolates, indicating that this variation does not arise from complementarity in the relative strengths of the SKN-1 and Wnt inputs into the GRN. Genome-wide association studies (GWAS) and Efficient Mixed Model Analysis (EMMA) revealed that much of the variation across the strains is attributable to genomic regions on LG IV and V. Further, analysis of recombinant inbred lines (RILs) with large differences in SKN-1 requirement by bulk segregant analysis (BSA) and whole-population genotyping (WPG) revealed two major contributing regions, including one identified by GWAS, as was also confirmed in introgressed lines. While these studies revealed that a multiplicity of genes and genetic interactions are responsible for the quantitative differences in GRN regulatory inputs, there also appear to be very substantial epigenetic contributions to the variation that follows the maternal line, as shown by reciprocal crosses of four different pairs of isotypes. This striking parent-of-origin effect persisted through at least five generations, suggesting a parental imprinting phenomenon. These findings demonstrate that the GRN for embryonic endoderm specification has undergone unexpectedly rapid evolutionary modification during radiation of the C. elegans species and reveal that some divergence between isotypes has a non-genetic origin.

Emma Hiley et al. (2006) European Worm Meeting "Regulation of RHO-1 by G12 Alpha Controlling Neurotransmitter Release"

Emma Hiley, Rachel McMullan, Stephen Nurrish

[European Worm Meeting, 2006]

Emma Hiley, Rachel McMullan and Stephen Nurrish . Gq alpha increases and Go alpha decreases levels of acetylcholine (ACh) release by respectively increasing or decreasing levels of diacylglycerol (DAG) at release sites. The accumulation of DAG at release sites recruits the DAG binding neuromodulator UNC-13, where it is essential for all neurotransmitter release. Data from our lab and others suggests that accumulation of DAG and UNC-13 at release sites is necessary for Gq alpha mediated increase in ACh release. A critical inhibitor of release is a diacylglycerol kinase (DGK-1), which lowers DAG levels by phosphorylation to phosphatidic acid. We have found that DGK-1 is bound by and it's activity inhibited by the single C.elegans homolog of the small GTPase RhoA, RHO-1. Thus increases in RHO-1 activity inhibit DGK-1 and cause an increase in ACh release.. Inhibition of endogenous RHO-1 using the C. botulinum C3 transferase causes a decrease in locomotion and ACh release suggesting that RHO-1 signals to control locomotion under normal laboratory conditions. What are the upstream regulators of RHO-1? Evidence from mammalian studies suggests that RhoA activity is mainly regulated via RhoGEFs that convert the inactive RhoA-GDP to the active RhoA-GTP. We have therefore been testing for the role of RhoGEFs in control of ACh release. Our studies indicate that the trimeric G-protein G12 alpha (GPA-12) activates RHO-1 via the single RGS RhoGEF (RHGF-1) in C. elegans. Over-expressors of constitutively active G12 alpha, GPA-12 (Q205L) have increased ACh release and both mutation of rhgf-1 and inhibition of RHO-1 by C3 transferase suppresses this effect of GPA-12 (Q205L). We have defined the site of action of RHGF-1 to the cholinergic motor neurons for it's effect on ACh release. Co-incident with it's effect on ACh release GPA-12 (Q205L) causes an increase in UNC-13S puncta located at release sites that is also suppressed by the rhgf-1 mutation. Both recruitment of UNC-13S to release sites and increases in ACh release by GPA-12 (Q205L) expression are blocked by a mutant of UNC-13 which is unable to bind DAG. Interestingly our previous results show that RHO-1 increases ACh release by both UNC-13 dependent and independent pathways whereas GPA-12 acts only via the UNC-13 dependent pathway suggesting that GPA-12 activates only a subset of neuronal RHO-1 signalling pathways.. These results show there is a third G protein coupled pathway controlling DAG mediated ACh release in the motor neurons. G12 alpha activates RHGF-1 converting RHO-1 to its active GTP bound form enabling it to inhibit DGK-1 thus increasing DAG levels. The increase in DAG causes UNC-13 recruitment to the membrane, increasing synaptic vesicle fusion and neurotransmitter release.