Sebastien Holbert et al. (2001) International C. elegans Meeting "The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis"

Robert J Ferrante, Adam Rosenblatt, Sebastien Holbert, Michael R Hayden, Tamara Kiechle, Russell L Margolis, Christopher A Ross, Cheryll Wellington, Jean Dausset, Isabelle Denghien, Christian Neri

[International C. elegans Meeting, 2001]

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). Pathogenesis in HD appears to involve the formation of ubiquitinated neuronal intranuclear inclusions containing N-terminal mutated htt, abnormal protein interactions, and the aggregate sequestration of a variety of proteins (noticeably transcription factors). To identify novel htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library (R. Barstead, ORMF, Oklahoma City, OK). We found a predicted WW domain protein (ZK1127.9) that interacts with N-terminal fragments of htt in two-hybrid tests. A human homologue of ZK1127.9 is CA150, a transcriptional coactivator with a N-terminal insertion that contains an unperfect (Gln-Ala)38 tract encoded by a polymorphic repeat DNA. CA150 interacted in vitro with full length htt from lymphoblastoid cells. The immunohistochemical expression of CA150 was markedly increased in human HD brain tissue, in comparison to normal age-matched human brain tissue, and showed aggregate formation with colocalization to ubiquitin-positive aggregates. In 432 HD patients, the CA150 repeat length explains a small but statistically significant amount of the variability in HD onset age. Our data suggest that abnormal expression of CA150, as mediated by interaction with polyQ-expanded htt, may alter transcription, and have a role in HD pathogenesis. Our data illustrate the value of using C. elegans for the direct identifcation of novel biochemical and genetic modifiers of human neurodegenerative disease pathogenesis [Holbert et al. (2001) Proc. Natl. Acad. Sci. USA 98 1811-1816].

Christopher J Cronin et al. (2005) International Worm Meeting "The Combined UC San Diego/Caltech Quantitative Behavior Analysis System"

Kuang-man Huang, Paul W Sternberg, William R Schafer, Zhaoyang Feng, Christopher J Cronin

[International Worm Meeting, 2005]

California Institute of Technology and University of California, San Diego have each developed practical, automated movement analysis systems to quantify aspects of worm behavior and morphology (see An imaging system for standardized quantitative analysis of C. elegans behavior, Zhaoyang Feng, et al, BMC Bioinformatics 2004, 5:115, An automated system for measuring parameters of nematode sinusoidal movement, Christopher J Cronin, et al, BMC Genetics 2005, 6:5, and previous meeting abstracts). We have consolidated our development efforts and merged our independent system designs to create a single, unified behavior analysis system. The combined system provides researchers with all of the quantification tools and database functionality of the individual UCSD and Caltech systems, but also establishes a common, open software and hardware foundation for continued development by Caltech, UCSD and the community. Current efforts include expanding system capabilities for the automated analysis of mating behavior with the challenge of tracking multiple moving individuals, both in contact with and separated from each other.

Winnier AR et al. (1998) Midwest Worm Meeting "THE UNC-4 ENGRAILED-LIKE REPRESSOR DOMAIN INTERACTS WITH UNC-37 TO REPRESS MOTOR NEURON SPECIFIC GENES."

Ross JM, Miller DM, Winnier AR, Meir JYJ

[Midwest Worm Meeting, 1998]

The gene regulatory function of specific homeobox sequences may be augmented by adjacent domains that interact with cofactor proteins. The combinatorial action of the homeoprotein and these accessory proteins then leads to either activation or repression of target genes. unc-4 encodes a homeoprotein that specifies synaptic input to VA motor neurons. In unc-4 mutants, VAs are miswired with inputs from interneurons normally reserved for their lineal sister cells, the VB motorneurons. UNC-4 function depends on the simultaneous presence of UNC-37, a ubiquitously expressed Groucho-like protein. In flies, Groucho interacts with specific DNA-binding proteins to repress gene transcription. Thus, we have proposed that UNC-4 and UNC-37 function together to repress target genes that would otherwise lead to the expression of VB-specific traits in VA motor neurons (1). Here we show that UNC-4 contains a C-terminal Engrailed-like repressor domain that physically interacts with UNC-37 to negatively regulate VB-specific genes. The UNC-4 C-terminal region includes an amino acid sequence that is similar to a conserved repressor domain first defined for the Engrailed family of homeoproteins (2). Five different randomly generated unc-4 mutants contain ELD missense mutations. Three of the five UNC-4 ELD mutations, as well as a missense mutation in the UNC-4 homeodomain, are suppressed by a specific amino acid substitution in UNC-37 (3). The UNC-4 ELD interacts with UNC-37 in the yeast two hybrid assay and specific ELD missense mutations disrupt this interaction (J. Meir et al., this meeting). UNC-4 and UNC-37 are required for repression of at least two VB motor neuron specific genes, acr-5 and del-1, in the VA motor neurons (J. Ross et al., this meeting) (4). ACR-5, an alpha subunit of a nicotinic acetylcholine receptor, and DEL-1, a degenerin-like sodium channel, are putative cell surface proteins that may function to distinguish VB from VA motor neurons. Missense mutations in the UNC-4 ELD result in ectopic expression of acr-5 and del-1 in VA neurons suggesting that the ELD is indeed functioning as a repressor domain in vivo. Our results are consistent with a recent report that the canonical Engrailed repressor domain (eh-1) physically interacts with fly Groucho and that the repressor function of eh-1 in vivo depends on Groucho activity (5). 1. Pflugrad, A., et al. (1997) Development 124, 1699-1709. 2. Smith and Jaynes (1996) Development 122: 3141-3150. 3. Winnier, A. et al., 1997 International C. elegans Meeting. 4. Ross, J. et al., 1997 International C. elegans Meeting. 5. Jimenez et al. (1997) Genes & Development 11:3072-3082.

Hall DH (1997) International C. elegans Meeting "EM-IMMUNOCYTOCHEMISTRY OF MH27 AB"

Hall DH

[International C. elegans Meeting, 1997]

This method has been used to study the expression pattern of a protein at high resolution in identified cells. A post-embedding procedure was performed on LR Gold thin sections of lightly fixed animals, marking MH27 binding with a gold-linked anti-mouse IgG secondary antibody. Methods for fixation, embedding, sectioning and antibody procedures were modified from those of Selkirk et al. (1). We will discuss improvements to the procedure, particularly to label fragile tissues and embryos. Adherens junctions (AJs) were intensely labelled in intestine, pharynx, seam cells and hypodermis. In the intestine, a continuous narrow band of apical AJs link adjacent pairs of intestinal cells. Gap junctions are known to lie very near to the apical AJs in both intestine and hypodermis, but were not labelled by MH27. Smooth septate junctions were heavily labelled between epithelial cells in the spermatheca. Pleated septate junctions immediately adjacent in the same membranes showed no labelling. Negative staining with lanthanum was used to further characterize the septate junctions. The high antigenicity and ubiquitous nature of AJs in intestine and hypodermis along the length of the nematode make the MH27 antibody useful when testing immunochemical procedures in C. elegans. MH27 antibody was generously provided by Jim Waddle and Ross Francis (2). 1. M.E. Selkirk et al. (1990) Mol. & Biochem. Parasitol. 42: 31. 2. G.R.Francis & R.H.Waterston (1985) J. Cell Biol. 101: 1532.

Mouanoutoua, Hanson et al. (2015) International Worm Meeting "Investigation of genetic mechanisms of hybrid dysfunction reveals evidence of male mitochondrial transmission."

Mouanoutoua, Hanson, Ross, Joseph, Chang, Chih-Chiun, Ortega, Brandon, Velazco-Cruz, Leonardo

[International Worm Meeting, 2015]

The Dobzhansky-Bateson-Muller model suggests that neutral genetic differences unique to one of two populations can lead to a reduction in hybrid fitness (hybrid dysfunction) when individuals from those two populations hybridize, combining those alleles. Additional mutations also contribute to hybrid dysfunction, pushing the two populations along the continuum to becoming distinct species. Thus, although it is easy to identify different species, it is difficult to determine the identity and order of genetic events that establish hybrid dysfunction and species formation. One way to circumvent this issue is to study cases in which hybrids are only slightly less fit than their parents, a situation that might represent the initiation of speciation. Such a situation exists in inter-population hybrids of Caenorhabditis briggsae. Two populations, AF16 and HK104, can be hybridized, but two distinct dysfunction phenotypes have been identified in their hybrids. In the first (Ross et al. 2011 PLoS Genetics; Baird and Stonesifer 2012 Worm), a small percent of F2 hybrids exhibit developmental delay, taking longer to reach adulthood than their wild-type siblings. In the second, genetic evidence from AF16-HK104 advanced-intercross recombinant inbred lines (AI-RIL) also suggested the presence of negative mitochondrial-nuclear epistatic interactions that might reduce hybrid fitness (Ross et al. 2011 PLoS Genetics). We present evidence from F10 mitochondrial-nuclear hybrids that a range of dysfunction phenotypes (including mitochondrial physiology and life history traits) are produced by exchanging the mitochondrial genome of one strain for the other, confirming the existence of mito-nuclear incompatibility. We also unexpectedly uncovered empirical evidence of occasional male mitochondrial transmission, challenging the common knowledge that mitochondria are maternally inherited and raising the possibility of using C. briggsae to study the cellular mechanisms that normally act to prevent such inheritance. Finally, three independent mapping approaches, involving F2 bulk segregant mapping, AI-RIL mapping, and near-isogenic line (NIL) mapping have begun to narrow down the region of C. briggsae chromosome III that contains one of the epistatic loci contributing to hybrid developmental delay. In sum, we present multiple lines of evidence that suggest the genetic divergence between AF16 and HK104 has placed these populations on the speciation continuum. Ongoing efforts seek to identify the molecular causes for these hybrid dysfunction phenotypes to pinpoint the genetic basis of species formation.

Lickteig KM et al. (1997) International C. elegans Meeting "IDENTIFYING UNC-4 HOMEODOMAIN BINDING SITES"

Lickteig KM, Miller DM

[International C. elegans Meeting, 1997]

unc-4 encodes a paired-like homeodomain (HD) protein expressed in the VA motor neurons but not in their VB sisters cells. unc-4 functions to specify synaptic inputs to the VAs; in unc-4 mutants, the VA motor neurons receive synaptic input from an interneuron that is normally reserved for VB sister cells. Therefore, we have proposed that unc-4 regulates genes in the VAs that distinguish them from VBs. In an effort to identify these UNC-4 target genes, we are determining the sequence of the UNC-4 DNA binding site. As a member of the paired-like class of HD proteins, UNC-4 is predicted to bind to a palindromic sequence (TAATYNRATTA) as a homodimer or as a heterodimer with other paired-like HDs. In gel shift assays, His-tagged UNC-4 HD binds cooperatively as a homodimer to the predicted palindromic sequence. UNC-4 HD also binds as a heterodimer with ALX4, a mammalian paired-like HD of the aristaless family. Heterodimeric binding presents the interesting possiblity that UNC-4 could regulate transcription in cooperation with other HD proteins. We are currently using the PCR based site selection assay to define an optimal UNC-4 HD DNA binding site. This lab has identified two genes, acr-4 and del-1, that are negatively regulated by unc-4 in the VAs (see Ross et al.). Therefore, acr-4 and del-1 are candidate UNC-4 target genes. Transgenic gfp constructs will be used to identify sites in the regulatory regions of the del-1 and acr-4 genes that respond to UNC-4 regulation. In addition, these regions will be scanned for UNC-4 DNA binding sites and directly tested for UNC-4 HD binding in gel shift assays.

Stegeman, Gregory W. et al. (2013) International Worm Meeting "Quantitative trait loci mapping of temperature-dependent behaviour in Caenorhabditis briggsae."

Stegeman, Gregory W., Ryu, William S., Cutter, Asher D.

[International Worm Meeting, 2013]

In order to understand how evolution acts on complex traits like behaviour, we need to define the causative genetic variants underlying these traits of interest. To further this aim we are working to determine the genetic basis of natural variation in temperature-dependent behaviour among Caenorhabditis briggsae isolates from temperate and tropical groups. Compared to C. elegans, C. briggsae has a strong phylogeographic pattern which indicates latitudinal differentiation. Most C. briggsae wild isolate strains fit into one of two genetically similar groups which have been named the 'tropical' clade or temperate 'clade' according to the latitudes at which strains from these groups have been isolated. We found that worms of strain AF16 from the 'tropical' clade tolerate a higher temperature and are able to continue locomotion at higher temperatures than worms of strain HK104 from the 'temperate' clade. We developed a droplet-based behavioral assay where the swimming behaviour of individual worms is monitored while the temperature of the system is programmatically controlled. Because we suspect that natural variation in behaviour is multigenic we took a quantitative genetic approach and assayed the C. briggsae AF16-HK104 recombinant inbred lines (RIL) (Ross, J.A., et al., 2011. PLoS Genetics, 7(7), e1002174) using our droplet assay. We found a gradient of behavioural variation along with some transgressive segregation among the RIL phenotypes. According to our quantitative trait loci (QTL) mapping analysis, two loci may explain the natural thermal behaviour variation observed between AF16 and HK104. The most striking aspects of behaviour which differ most between the "temperate" and "tropical" parents, map to the center of Chromosome 2 and a larger region covering the left and centre region of Chromosome 5. This analysis suggests this temperature-dependent behaviour is at least digenic and possibly involves more loci and further work will attempt to resolve these loci and ultimately locate the causative nucleotide differences.

Pena-Gonzalez, I. et al. (2013) International Worm Meeting "Suppressors of TDP-1 toxicity in Caenorhadbitis elegans."

Link, CD., Pena-Gonzalez, I.

[International Worm Meeting, 2013]

Suppressors of TDP-1 toxicity in Caenorhadbitis elegans Ilana Pena-Gonzalez1 and Christopher D Link1, 2 1 Integrative Physiology, University of Colorado, Boulder, CO 80309, USA 2 Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309, USA RNA binding protein TDP-43 forms damaging aggregates in multiple neurodegenerative diseases. We have found that the deletion of tdp-1, the C. elegans ortholog of TDP-43, leads to increased accumulation of double stranded RNA. TDP-1 is not believed to bind to dsRNA itself; however the marked increase in dsRNA accumulation in worms deleted for tdp-1 implies TDP-1 normally participates in limiting the stability and structure of dsRNA. Although worms deleted for TDP-1 do not have a severe phenotype, overexpressed nuclear TDP-1 is toxic in worms. One possible explanation for this is that too much TDP-1 leads to excessive disruption of structured RNAs needed for normal RNA metabolism. Making use of this overexpression model we have established a heat shock induction system to help identify components associated with TDP-1's prevention of dsRNA accumulation. Identifying mutations that suppress the neurotoxic effects of overexpressed TDP-1 could help identify others factors contributing to the complex mechanism of disassembling dsRNA. A mutagenesis screen has identified four mutant strains that partially suppress the toxicity of overexpressed TDP-1. We have also tested suppressors of other toxic proteins. Further identifying the pieces of the mechanism regulating dsRNA breakdown could help better explain the pathogenic pathway of TDP-1 and consequently TDP-43 in disease.

Shi, Yeu Ching et al. (2011) International Worm Meeting "Monascus-fermented dioscorea enhances oxidative stress resistance via insulin signaling pathway."

Pan, Tzu-Ming, Shi, Yeu Ching, Liao, Vivian

[International Worm Meeting, 2011]

Monascus-fermented products offer valuable therapeutic benefits and have been extensively used in Eastern Asia for several centuries. Monascus spp. produces many types of polyketide secondary metabolites and some of them have been reported to have antioxidant, antiinflammatory, and antitumor-initiating effects. In this study, we investigated Monascus-fermented products-modulated antioxidant effect in C. elegans. We examined oxidative stress resistance of ethanol extract of red mold dioscorea (RMDE) in C. elegans, and found that it responded to 10 mg/mL RMDE with an increased survival rate and reduced intracellular reactive oxygen species (ROSs). The subcellular distribution of the FOXO transcription factor DAF-16 was affected by RMDE. We analyzed genetic determinant that might influence oxidative stress resistance by RMDE. We showed that the antioxidant phenotype was absent from the daf-2, age-1, akt-2, and daf-16 mutants. These finding suggested that RMDE may act as an antioxidant via insulin signaling pathway and DAF-16/FOXO transcription factor.

Joseph Watson et al. (2002) West Coast Worm Meeting "GFP screens for regulators of motor neuron differentiation."

Joseph Watson, David M Miller III

[West Coast Worm Meeting, 2002]

A-type (DA,VA) and B-type (DB,VB) C. elegans motor neurons are morphologically distinct and receive inputs from separate sets of command interneurons. The UNC-4 homeodomain is expressed in A-type motor neurons where it functions with its Groucho-like cofactor protein, UNC-37, to block expression of B-type genes such as acr-5 (nicotinic acetylcholine receptor). This UNC-4 repressor function is essential to the fidelity of the motor neuron network; mutations that disrupt unc-4 or unc-37 result in the miswiring of VA motor neurons with VB-type inputs. Conversely, ectopic expression of UNC-4 in VB motor neurons inhibits expression of acr-5 and may miswire VB motor neurons with VA-type inputs (J. Ross & D. M.M. unpublished). Thus, proper regulation of unc-4 expression is clearly important to the creation of a functional motor neuron circuit. We have utilized a reporter gene and unc-4 target, acr-5::YFP, to conduct a visual screen for upstream regulators of unc-4. acr-5::YFP is normally expressed in B-type motor neurons but is also de-repressed in the A-type motor neurons in unc-4 and unc-37 mutants. The net two-fold increase in the number of YFP-expressing neurons in the ventral nerve cord (VNC) can be easily observed in a fluorescence stereodissecting microscope. (acr-5::YFP is also expressed in neurons in head and tail ganglia where it is not regulated by unc-4.) A clonal screen of 2200 F1 mutant worms revealed two new unc-4 alleles, but did not detect mutations in other genes that also result in ectopic acr-5::YFP expression in the VBs. Our screen did uncover, however, seven mutants with decreased acr-5::YFP expression in the VNC. One particular isolate, wd54, lacks acr-5::YFP expression in the ventral cord but shows normal expression in head and tail neurons. wd54 animals also display a forward movement defect. Although wd54 does not affect unc-4 expression, it appears to be required for proper differentiation of B-class motor neurons.