Moerman, Donald G. et al. (2011) International Worm Meeting "The Million Mutation Project - A C. elegans Mutation Resource."

Edgley, Mark, Fernando, Lisa, Shendure, Jay, Turner, Emily H., Taylor, Jon, High, Amber, Waterston, Robert, Chaudhry, Iasha, Lee, Norris, Lau, Joanne, Strasbourger, Pnina, Flibotte, Stephane, Ewing, Brent, Miller, Angela, Moerman, Donald G., Raymant, Greta, Hillier, LaDeana, Thompson, Owen

[International Worm Meeting, 2011]

We are developing a library of 2,000 mutagenized strains and sequencing each to a depth of 15x as a community resource. From previous estimates this should yield a library of half to one million SNP's with 5-10 non-synonymous changes per average gene across the collection. After testing various mutagens we are using EMS, ENU and a cocktail of EMS plus ENU. In the F1 we select for unc-22 mutations to ensure that the genome has been mutagenized. In the F2 we select for non-unc-22 animals and then these animals are self-crossed for 10 generations to ensure that the final isolate is homozygous across all regions of the genome. We will supplement these strains with natural isolates to recover additional mutations. For sequencing we load size-selected, barcoded samples on either a GAII, or Hi-Seq sequencing machine (Illumina) and do paired-end reads. Data analysis employs BWA, SamTools and custom filters. Sequence data will be deposited in WormBase and the individual strains will be available from the Caenorhabditis Genetics Center for detailed study. Eventually we hope to distribute the 2,000 strains as a single kit, allowing parallel experimentation on a wide spectrum of mutant genes. To date we have constructed and tested libraries for more than 1,000 strains and completed analysis of 451 strains. The analysis has yielded 142,566 SNP's including 32,835 non-synonymous changes in 13,685 genes. These comprise 1,404 nonsense mutations in 1,310 genes, 804 splicing mutation in 777 genes, 30,607 missense mutations in 13,269 genes and 20 readthrough mutations in 20 genes. Of 2,200 genes with either a nonsense, or a splicing alteration, over 1,000 have no previously reported mutations. Based on read numbers, the rDNA repeat copy number is surprisingly variable, with some strains having fewer than 60 copies and a few having more than 200. By the meeting in Los Angeles we will report on the analysis of the first 1,000 strains.

Moerman, Donald G. et al. (2012) Worm Breeder's Gazette "The million mutation project a genetic resource for C. elegans."

Chaudhry,Iasha, Flibotte, Stephane, Raymant, Greta, Ewing, Brent, Lee, Norris, High, Amber, Adair, Ryan, Au, Vinci, Strasbourger, Pnina, Hillier, LaDeana, Fernando, Lisa, Edgley, Mark, Moerman, Donald G., Hutter, Harald, Turner, Emily H., Taylor, Jon, Miller, Angela, Waterston, Robert, Thompson, Owen, Shendure, Jay, Lau, Joanne

[Worm Breeder's Gazette, 2012]

Donald G. Moerman (2006) WormBook "Sarcomere assembly in C. elegans muscle."

Donald G. Moerman

[WormBook, 2006]

Sarcomeres within body wall muscle in C. elegans include attachments to the sarcolemma that are remarkably similar in structure to vertebrate adhesion complexes. Crucial early steps in muscle sarcomere assembly, a highly orchestrated affair involving many proteins, involve the assembly of these sarcomere attachments. The steps involved in initiating the correct placement of these attachments and other sarcomere substructures are poorly understood. Using mutants in C. elegans we are attempting to dissect the various steps in this process. We review what has been discovered to date and present a model of sarcomere assembly that initiates at the plasma membrane and involves proteins within muscle, the hypodermis and within the extracellular matrix.

Donald Moerman et al. (2005) International Worm Meeting "SAGE on specific cells and tissues during C. elegans embryogenesis."

Susanna Chan, Jaswinder Khattra, Adam Lorch, David Charest, Sheldon McKay, Jennifer Asano, Kim Wong, Donald Moerman, Peter Huang, Barbara Goszczynski

[International Worm Meeting, 2005]

We are determining gene expression profiles in specific cells and tissues of the developing embryo using serial analysis of gene expression (SAGE; using a longSAGE 21 base pair tag). Embryonic tissues and specific cell populations are isolated using a fluorescence activated cell sorter to sort GFP-marked cell populations (Christensen et al, Neuron 14:503). To date we have libraries for all the major germ layers, including ectoderm (pan-neural and hypodermis) mesoderm (body wall muscle and the pharyngeal muscle) and endoderm (gut). We also have a library to the maternal genome (oocytes). More recently we have subdivided the developing nervous system; constructing libraries to first, the ciliated neurons (32 cells), and second, to the AFD neurons (2 cells). Other libraries specific to subpopulations of the nervous system and muscle are in progress. Our sampling depth has permitted the detection of over 400 different transcription factors within the developing embryo, although many of these are at very low tag frequencies. We find that several tissue specific TFs, including Pha-4, Elt-,2 Ttx-1, Daf-19 and Hlh-1, are in fact expressed in many tissues. However, in each case they are indeed enriched (3 to 10 fold) in the tissue where they have been determined to be critical, including developing pharynx (Pha-4), gut (Elt-2), AFD neurons (Ttx-1), ciliated neurons (Daf-19) and muscle (Hlh-1). Our database of over 3 million SAGE tags is the largest of its kind for C. elegans. With this data we have confirmed the expression of a minimum of 7,000 genes during embryogenesis. Over 3,000 genes are common to all tissues, and an additional 1,000 to 2,000 genes are specific, or at least enriched in the various tissues. Many of the SAGE tags map to unannotated regions of the genome, and thus may identify new genes. The SAGE data are available at http://elegans.bcgsc.bc.ca and WormBase.

Donald G Moerman et al. (2002) Japanese Worm Meeting "Sarcomere assembly in C. elegans muscle"

Teresa Rogalksi, Hiroshi Qadota, Donald G Moerman, Shaun Cordes, Kenneth Norman

[Japanese Worm Meeting, 2002]

The spacing of dense bodies and M-lines within C. elegans body wall muscle is precise. What we wish to understand is how this subcellular pattern is generated because it is this pattern that ultimately leads to the organization of sarcomeres. The position, length and interdigitation of the thick and thin filaments of a sarcomere are dependent on the position, spacing and depth of the dense bodies and the M-lines. The work of several laboratories has contributed to our understanding of the structure of these key anchoring complexes. We now know that dense bodies and M-lines are both analogs and homologs of vertebrate adhesion complexes. The major structural proteins, integrin, talin and vinculin are present in the anchoring complexes, and recently we, and our collaborators, have identified UNC-97/PINCH, UNC-112, and PAT-4/ILK as components of these structures. Analysis of mutants for each of these genes reveals the order of addition of these components to an adhesion junction. The mutants also yield a hint at the possible functional role of each protein, whether it be in nucleating a complex, spacing of the complex, or further downstream assembly. Our current model has UNC-52/Perlecan and PAT-2/alpha-integrin, PAT-3/beta-integrin as the early nucleating steps with talin and other structural components being added even before correct spacing is organized. The UNC-97/UNC-112/PAT-4 complex appears critical for correct spacing of adhesion complexes but not their initiation, since these mutants have no effect on polarization. UNC-112 and PAT-4 require each other for correct localization and function within the dense body . UNC-97/PINCH which binds to PAT-4/ILK is dependent on this protein for its localization to the dense body. While this smaller complex of UNC-97, PAT-4 and UNC-112 does not impede the addition of talin or vinculin to the dense body, spacing of integrin complexes is altered and no sarcomere with interdigitating thick and thin filaments capable of contraction is generated when any of these three molecules is missing. In all cases the absence of the protein leads to a Pat phenotype similar to what we observe for unc-52 or pat-3 null mutations.

Donald G Moerman et al. (2004) West Coast Worm Meeting "Gene expression profiling of cells, tissues and developmental stages of C. elegans."

R Viveiros, D Anastis, R Zhapf, Donald G Moerman, H Tian, D Li, J McGhee, K Wong, R Johnsen, M Marra, DL Baillie, E Tang, D Tu, L Chen, C Mills, A Pouzyrev, A Lorch, J Asano, S Chan, DL Riddle, P Huang, S J McKay, J Khattra, A Warner, J Perkins, SJM Jones, Z Zhao, A Mah, R Schnabel, B Goszczynski, R Newbury

[West Coast Worm Meeting, 2004]

Perkins, Jaryn et al. (2009) International Worm Meeting "Filling in the gaps of a C. elegans fosmid library."

Moerman, Donald G., Perkins, Jaryn

[International Worm Meeting, 2009]

The fosmid genomic library produced and sequenced by the Moerman laboratory and the Genome Sciences Center, in Vancouver, was created to provide a resource for genome wide studies (IWM 2005 Abstract 1299B). It is meant to supplement, and in some cases replace, older cosmid libraries. With 15,744 clones characterized, the inserts should theoretically contain 5x coverage of the Caenorhabditis elegans genome. However, the mapping of paired end reads display gaps in the sequence that correspond to areas of highly repetitive sequence, possibly suggesting mismapping in these regions. The 12,481 clones, which aligned contiguously, cover approximately 80% of the genomic sequence, and account for 90% of genes within one or more fosmids. To increase the usefulness of the resource to the community we have begun PCR screening the 3,263 ambiguously mapped clones for the presence, using internal primer sets, of the missing genes. Initially we are screening for transcription factors, as 197 of the 936 annotated genes are not represented among the mapped fosmids. Completion of this large and important functional gene family will provide a benchmark for completion of gapped sequences. Initial positive hits for TF genes in the screen provides evidence for the hypothesis that the gaps are caused by ambiguous mapping of some clones, and suggest that a large portion of genes currently missing from contiguous coverage can be found in the remaining ambiguously mapped clones. From the initial 56 transcription factors screened, we have observed amplicons of correct size for 43. These results demonstrate that screening rather than de novo library production may be a cost effective way to complete gapped sequences, as any new clones may be susceptible to ambiguity in the same regions. Using this approach, we hope to close many of the 400 gaps in the current fosmid coverage of the genome.

Viveiros, Ryan et al. (2009) International Worm Meeting "Investigation of myoblast migration during embryogenesis in Caenorhabditis elegans."

Viveiros, Ryan, Moerman, Donald, Hutter, Harald

[International Worm Meeting, 2009]

C. elegans body wall muscle is formed after a series of well-orchestrated steps. Initially, embryonic muscle cells accumulate under the hypodermal seam cells, at the left and right lateral midline, and shortly thereafter, begin to migrate dorsally and ventrally, to form the final four muscle quadrants present upon hatching. As the myoblasts migrate they are still dividing, as are many other cells in their immediate environment. This means the cell-cell contact of cells during migration is dynamic and can vary from animal to animal. This situation creates an environment where the extracellular matrix (ECM) and cell surface contacts are in constant flux, which begs the questions as to how these cells navigate unerringly to their final destination. Though a number of ECM and cell surface molecules have previously been shown the be involved in cell migration, positioning and attachment, including perlecan, type IV collagens, the NCAM, cadherin and integrin families, and the UNC-6/netrins, previous studies have not shown any of these molecules to affect the early muscle migration. Using RNAi to target ECM components, we found that loss of laminin results in a number of muscle migration defects. Analysis of the laminin knockdown phenotype reveals defects in the aforementioned dorsal and ventral muscle cell migrations, as well as a posterior displacement of the anterior-most ventral muscle cells. Investigation of this posterior displacement has led to the identification of a previously un-described anterior migration event of the embryonic muscle cells, MSapappp, MSapaaap, MSapapap and MSsappapp. This anterior migration is dependent upon the extension of processes from the anterior-most muscle cells, as these processes appear to be absent in the ventral anterior-most cells of the laminin knockdown embryos. Using spinning disk confocal microscopy we observe that the extension of the muscle processes to the anterior coincides with the onset of elongation and is concurrent with the dorsal and ventral migrations. Wildtype analysis has also revealed the persistence of cell-cell contacts between the dorsally and ventrally migrating muscle cells with the degree of contact increasing towards the posterior. These contacts are eventually lost as the embryo elongates. While laminin is required for a subset of these processes, their extension does not require pat-3/beta-integrin, suggesting either this is an integrin independent migratory event, or that there is a yet to be identified beta-integrin. Investigations into what other genes are involved in mediating these anterior migrations are currently underway.

Viveiros, Ryan et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Investigation of muscle migration during embryogenesis in Caenorhabditis elegans"

Moerman, Donald, Viveiros, Ryan, Hutter, Harald

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

C. elegans body wall muscle is formed after a series of ordered steps. Initially, embryonic muscle cells accumulate under the hypodermal seam cells and shortly thereafter, begin to migrate dorsally and ventrally, to form the final four muscle quadrants present upon hatching. As the myoblasts migrate they are still dividing, as are many other cells in their immediate environment. This means the cell-cell contact of cells during migration is dynamic and can vary from animal to animal. This situation creates an environment where the extracellular matrix (ECM) and cell surface contacts are in constant flux, which begs the questions as to how these cells navigate unerringly to their final destination. To better characterize these muscle cells we used a membrane-bound GFP to visualize their membrane dynamics. This allowed us to identify filapodia and lamelapodia in the embryonic muscle cells and led to the identification of processes extending from the anterior-most muscle cells. Using spinning disk confocal microscopy, we observe that these extensions to the anterior coincide with the onset of elongation and are concurrent with the dorsal and ventral migrations. Using RNAi and mutant analysis to identify genes involved in mediating these migrations, we found that laminin, the a-integrin INA-1, the ephrin VAB-2, and its receptor VAB-1 and the ROBO receptor SAX-3 are required for proper muscle migration. Disrupting any of these genes results in defects in the extension of the ventral anterior processes, which leads to the posterior displacement of the ventral muscle quadrants. As most of these genes have a known role in hypodermal morphogenesis, we are currently investigating whether the improper muscle migration is due to hypodermal defects.

Edgley, Mark L. et al. (2021) MicroPubl Biol

Flibotte, Stephane, Moerman, Donald G, Edgley, Mark L.

[MicroPubl Biol, 2021]

We used whole-genome sequencing (WGS) data from a number of balanced lethal strains in Caenorhabditis elegans to show that the crossover suppressor qC1 is an inversion. The rearrangement is complex, with a large primary inversion that contains several other smaller inverted regions. The graphical representation below depicts these various qC1 rearrangements for ease of conceptualization. It is the simplest chromosomal structure compatible with the data currently available, but even then it is worth noting that the complexity of the qC1 chromosome can make the graphical reconstruction difficult to understand, and it may seem a bit like relativity theory or artwork from M.C. Escher (https://moa.byu.edu/m-c-eschers-relativity/).