Viveiros, Ryan

During C. elegans embryogenesis, myoblasts initially form two rows along the left and right lateral midlines and at ~290 min of development migrate dorsally and ventrally to form the four muscle quadrants present upon hatching (Sulston et al, 1983). 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 (Schnabel et al, 1997). 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. In an attempt to identify genes mediating these migrations, I performed an RNAi based screen targeting 776 genes predicted to be members of the extracellular matrix (ECM), or one of its receptors. Using both feeding and injection based RNAi, I was able to identify three genes of interest. Knockdowns of F56B3.2 resulted in paralyzed animals with detached muscle, making it a good candidate for a new component of the muscle attachment complex. F33G12.4 knockdowns resulted in an embryonic arrest phenotype with an abnormal muscle lineage, possibly stemming from polarity defects. The only knockdown that resulted in muscle migration defects was that for lam-2, which encodes for the laminin gamma subunit. Analysis of the lam-2 knockdown, as well as knockdowns for the other laminin subunits, revealed dorsal/ventral migration defects 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 undescribed anterior muscle migration event and its dependency upon the extension of muscle processes from the leading cells.

Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for ∼900 predicted C. elegans promoters (∼5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners. [ABSTRACT FROM AUTHOR]

C. elegans body wall muscle is formed after a series of well-orchestrated steps. This thesis describes the plasma membrane dynamics of these migrating embryonic cells and the identification of two genes unc 54/MHB and mem-1 that appear to be involved negatively regulating post-embryonic muscle membrane extensions. During the characterization of embryonic muscle morphogenesis, whereby two rows of cells split and migrate dorsally or ventrally to form the final four muscle quadrants present upon hatching, I observed an anterior migration event, whereby the anterior-most pair of cells in each of the four muscle quadrants extends long processes to the anterior tip of the developing embryo. The anterior-most muscle cells then follow these extensions into their final positions in the developing embryo. Using RNAi and mutant analysis, I have identified laminin as being involved in mediating the dorsal-ventral muscle migrations and that the α-integrin INA-1, the ephrin VAB-2 and its receptor VAB-1 and the Robo receptor SAX-3 indirectly promote the proper extension of the ventral anterior muscle processes by organizing the embryonic neurons so as to provide a clear path for muscle membrane extension. Post embryonically, the loss of either unc-54 (a myosin heavy chain B), or mem-1 (a WD repeat domain protein) results in ectopic membrane extensions from the mature muscle cells. These extensions can be rescued via targeted depletion of actin remodeling or cell adhesion complex components. During the analysis of these mutations I identified a predisposition for generating these ectopic membrane extensions that is conferred by using ectopic expression of PAT-3/β-integrin that is bound to GFP and that using the PAT-3/β-integrin transmembrane domain to localize GFP to the plasma membrane is sufficient to generate this sensitivity.

C. elegans body wall muscle is formed after a series of well-orchestrated steps. With the onset of specification embryonic muscle cells accumulate under the hypodermal seam cells at the left and right sides of the embryo. Shortly thereafter they begin to migrate dorsally and ventrally resting beneath the dorsal and ventral hypodermis eventually forming the four muscle quadrants present upon hatching. In this study we describe the plasma membrane dynamics of these migrating cells and observe the extension of filopodia and lamellipodia during dorso-ventral migration but not during the earlier stages of accumulation. We also describe an anterior migration event during embryonic muscle morphogenesis, whereby the anterior-most pair of cells in each of the four muscle quadrants extends long processes to the anterior tip of the developing embryo. Anteriormost muscle cells then follow these extensions into their final positions in the developing embryo. Using RNAi and mutant analysis, we have identified laminin as being involved in mediating the dorsal-ventral muscle migrations. Finally we show that the α-integrin INA-1, the ephrin VAB-2 and its receptor VAB-1 and the Robo receptor SAX-3 indirectly promote the proper extension of the ventral anterior muscle processes by organizing the embryonic neurons so as to provide a clear path for muscle membrane extension.