Van Wynsberghe, Priscilla et al. (2017) International Worm Meeting "The period protein homolog LIN-42 regulates early germline development in C. elegans."

Van Wynsberghe, Priscilla, Stine, Laurel, McFall, Alanna, Graham, Hillary, Toledo-Hernandez, Amanda

[International Worm Meeting, 2017]

Germline stem cells are maintained in the distal region of the gonad. These cells undergo mitotic divisions and are maintained in this state through GLP-1 Notch signaling. The somatic distal tip cell (DTC) caps the end of the distal gonad and is essential for maintenance of the germline mitotic zone. As germ cells move away from the DTC they pass through the transition zone and enter early meiotic prophase. Here we present our work that identifies the Period protein homolog LIN-42 as a new regulator of germline development in C. elegans. Previous work in our lab and others has shown that LIN-42 is a transcription factor that regulates expression of both microRNAs and some mRNAs. LIN-42 is important for multiple developmental events including molting and hypodermal cell development. In addition, in the absence of LIN-42, late-stage specific events occur precociously in vulval precursor cells, sex myoblasts and the DTC. LIN-42 is expressed in all of these cells as well as multiple other cells in the muscles, neurons, intestines and hypodermis. Because of this we sought to understand the role of LIN-42, if any, in germline development. In lin-42(n1089) mutants we find that the mitotic zone size, determined by both the number of rows of cells and the total number of cells in the mitotic zone, is significantly reduced by 25% compared to wild type in young adult worms. We further find that mRNA levels of the GLP-1 Notch ligand, lag-2, are significantly decreased in lin-42(n1089) mutant worms relative to wild type. Thus, LIN-42 likely impacts germline development by regulating LAG-2 expression and subsequent GLP-1 Notch signaling. Current work aims to determine if LIN-42 acts directly or indirectly to control lag-2 levels. In Drosophila, the ATAXIN-2 protein is an important regulator of Period expression. In C. elegans, the ATAXIN-2 homolog atx-2 promotes germline proliferation. We find that lin-42 levels are significantly decreased in atx-2 mutant worms relative to wild type, suggesting that ATX-2 impacts germline development in C. elegans at least in part through its affects on LIN-42. Altogether our results establish a new role for the conserved, important Period protein homolog LIN-42 in regulating early germline development.

Wang, Xin et al. (2013) International Worm Meeting "Mutations in C9ORF72/F18A1.6 produce developmental and behavioral defects."

Buttner, Edgar, Wang, Xin

[International Worm Meeting, 2013]

Expanded hexanucleotide repeats in the human C9ORF72 gene cause frontotemporal dementia and amyotrophic lateral sclerosis (Boeve et al., 2012; Renton et al., 2011). The normal and abnormal mechanisms of action of this human disease gene are not fully understood. We are characterizing phenotypes associated with mutations in F18A1.6, the C. elegans homolog of C9ORF72. We found that feeding RNAi of F18A1.6 caused developmental delay, while the insertion/deletion allele ok3062 did not. F18A1.6(RNAi) also reduced locomotor behavior in a thrashing assay, compared to empty-vector control. Surprisingly, ok3062 animals displayed increased thrashing, compared to N2. To better understand the cellular basis of the locomotion phenotypes, we are now conducting pharmacological and neuronal GFP reporter assays. We are also generating transgenic animals to test the ability of C9ORF72 to rescue F18A1.6-associated phenotypes and to test the effects of expressing expanded hexanucleotide repeats in C. elegans. References Boeve, K. B. Boylan, N. R. Graff-Radford, M. DeJesus-Hernandez, D. S. Knopman, O. Pedraza, and P. Vemuri (2012). Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72. Brain 135, 765-783. Renton, E. Majounie, A. Waite, J. Simon-Sanchez, S. Rollinson, J. R. Gibbs, J. C. Schymick, and H. Laaksovirta (2011). A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257-268.

H Van Luenen et al. (1999) International C. elegans Meeting "World-wide worm SNPs: Dissecting the molecular evolution of the species using a high density SNP map"

R Plasterk, R Koch, H Van Luenen

[International C. elegans Meeting, 1999]

We would like to reconstruct the history of the C. elegans species at the genome level, therefore we sampled the genomes of four natural isolates (strain CB4857 isolated in Claremont, California, RC301 from Freiburg, Germany, TR403 from Madison, Wisconsin and AB1 from Adelaide, Australia) for single nucleotide polymorphisms (SNPs). Random genomic DNA fragments from the 4 strains were shotgun cloned and sequenced. There was no selection for transcribed or non-transcribed regions of the genome. In total we sequenced 1572 clones resulting in over 1 Mb of sequence information. The sequences are compared to the canonical Bristol N2 sequence to ask the question whether the clone maps to a unique sequence, and -if so- whether it contains polymorphisms. Once a SNP is identified we check other strains for the presence of the same polymorphism by PCR amplification and sequence analysis. In an initial experiment we found approximately one SNP per 3000 bp sequenced. The SNPs are randomly spread over the genome. Based on these observations we expect to find approximately 500 SNPs, one in every 200 kb. In the initial experiment we found, as expected, that several SNPs initially detected in one strain were also present in some but not all other strains. For example: a T in the Australian AB1, is a G at the same position in Bristol N2 in cosmid K10D2 at position 27946, and we found it to be like AB1 in the Californian CB4857 strain and the German RC301 strain, while the TR403 strain from Wisconsin resembles the Bristol N2 strain. Thus different patches of the genome have different ancestors. With our high density SNP map we will generate a genome map for each isolate which will show how each genome is patched together from a limited set of parental strains. The SNP's will be added to ACeDB, and can also be used as markers on the genetic map. They can be recognised by PCR followed by sequencing, but we also found that the SNPs we looked at could be visualised by SSCP analysis. We thank Jane Rogers and Amanda McMurray for their assistance in sequencing the clones.

Benian GM et al. (2000) East Coast Worm Meeting "UNC-89 is a putative muscle specific activator of rhoA"

Kindt RM, Benian GM, Blangy A, Tinley TL

[East Coast Worm Meeting, 2000]

unc-89 has a myofilament lattice that is thinner than normal, with disorganization of thick filaments, and for most alleles, no M-lines. UNC-89 is located throughout the depth of the myofilament lattice, in the middle of A-bands. UNC-89 is a 732 kD polypeptide composed of Ig domains, as well as SH3, DH (guanine nucleotide exchange factor) and PH domains (Benian et al., JCB 132: 835, 1996). We hypothesize that unc-89 receives a signal from outside the muscle cell and recruits molecules to organize the myofilament lattice. Genetic data indicates interaction between unc-89 and two mutationally defined Rho-GTPases, mig-2 and ced-10. mig-2 encodes a Rho family member and when mutant results in the failure of many migrating cells to complete their journeys. mig-2::GFP is expressed in many cells, including muscle, and is localized at or near the cell membrane (Zipkin et al., Cell 90: 883, 1997). When a null allele of mig-2, mu28, is combined with two hypomorphic alleles of unc-89, the Unc-89 phenotype is enhanced. When mu28 is combined with an unc-89 null allele, the Unc-89 phenotype remains the same. Similar experiments with an activated allele of mig-2 gave the same results. ced-10 encodes a Rac1 homolog that when mutant results in defects both in the engulfment of apoptotic cells and in the migrations of distal tip cells (Reddien & Horvitz, Nat. Cell Biol. 2: 131, 2000). When loss of function alleles of ced-10 are combined with hypomorphic alleles of unc-89, the Unc-89 phenotype is enhanced. ced-10, mig-2, or ced-10;mig-2 double mutants all have normal appearing muscle. By use of a novel method (yeast exchange assay; De Toledo et al., submitted), we can demonstrate guanine nucleotide exchange activity by the UNC-89 DH domain for mammalian RhoA, but not for nematode MIG-2 or CED-10, nor a number of other mammalian Rho-GTPases. There are no known mutations in C. elegans RhoA. RNAi for RhoA results in embryonic lethality (Malone, Orital & Han, pers. comm.; our unpub. data). We plan to use the yeast exchange assay on C. elegans RhoA (88% identical to human RhoA), localize RhoA in unc-89 mutants, and determine the phenotype of an unc-89 mutant in which antisense RhoA is driven by a muscle promoter.