Meissner, Barbara et al. (2009) International Worm Meeting "Body wall muscle in C. elegans - from expression profiling to profiling expression."

Meissner, Barbara, Lorch, Adam, Moerman, Don, Rogalski, Teresa, Granger, Laure, Warner, Adam, Segalat, Laurent, Viveiros, Ryan

[International Worm Meeting, 2009]

The large size of body wall muscle cells and the beautifully organized muscle sarcomeres within C. elegans muscle offer an opportunity to identify the precise position of proteins within cell substructures and the possibility of determining how such structures are assembled. The close relationship between subcellular localization and function is such that determining the preferential localization of a protein is often an essential step towards determining its function. GFP tagging has been shown to be a powerful tool in large-scale protein localization studies1-3. Our goal is to generate a comprehensive ''localizome'' for the C. elegans body wall muscle by systematic GFP-tagging and localization of proteins expressed in muscle. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. To date, four SAGE libraries are available generated from FACS sorted embryonic muscle cells (http://elegans.bcgsc.bc.ca). For this subcellular localization project, we are focusing on genes which a) exhibit expressed tags in the muscle SAGE libraries b) display a muscle phenotype in the RNAi screen performed previously (#981A, 16th IWM 2007) and c) are orthologs or at least homologs of human genes. We are using Gateway cloning technology to express protein::GFP fusions of candidate genes exclusively in muscle cells. To date we have analyzed the expression of about 300 genes, 240 of which display localized expression in the C. elegans body wall muscle (e.g. dense bodies, M-lines, golgi, mitochondria, cell membrane, nucleus or nucleolus). For most proteins localized in this study no prior data on localization was available. In addition to discrete subcellular localization we observe overlapping patterns of localization including the presence of protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 15 subcellular localization patterns within nematode body wall muscle. The localization of all proteins within a muscle cell will be an invaluable resource in our attempt to understand how proteins interact within muscle to form properly organized and regulated sarcomeres. 1Ding et al, Genes Cells 2000 2 Huh et al, Nature 2003 3Heazlewood et al, Nucleic Acids Res 2007.

Andrew Chisholm et al. (2006) Development & Evolution Meeting "The role of the substrate in epidermal enclosure"

Andrew CHISHOLM, Martin Hudson, Leilani Miller

[Development & Evolution Meeting, 2006]

Epidermal enclosure is an example of an epiboly process that involves spreading of the epidermal epithelium over substrate cells. The substrate for epidermal enclosure is formed predominantly by ventral neurons and support cells. Disruption of several signaling pathways (bidirectional Eph signaling, PTP-3, SAX-3, KAL-1/HSPGs) leads to aberrant development of the substrate and consequent defects in epidermal enclosure. The primary defect in these mutants arises in movement of ventral neuroblasts (VNBs) to the ventral midline, a process that normally closes a ventral cleft formed in gastrulation. The cellular forces driving VNB movement are not known. To address how VNBs move, we are using a combination of imaging, laser ablation and pharmacology. We will describe progress in our analysis of the basis of neuroblast migrations. Elimination of any one of the VNB pathways results in incompletely penetrant defects in development whereas loss of two or more pathways confers synergistic defects, indicating a high level of genetic redundancy in the control of VNB movement. We have exploited this synergism by using RNA interference screens for enhancers of weak morphogenetic phenotypes. Such enhancers may define additional pathways involved in VNB movement, or additional components of the known pathways. We are especially interested in genes that might function in the ephrin reverse signaling pathway that is required in VNB migration, as little is known about how GPI-linked ephrins promote reverse signals. From an RNAi screen of 1/3 of the genome in an efn-4(wk) rrf-3 background we identified one strong unexpected enhancer and a large number of weaker enhancers. We will describe our characterization of these enhancers. Studies of epiboly processes in other organisms suggest that substrate contractility plays an active role in promoting the movement of the overlying epithelium. To test if such coordinated movements may be important in C. elegans we have begun dual imaging of epidermis and substrate during enclosure.