DESCRIPTION (applicant's description): The long-term goal of this program is to obtain a molecular understanding of the developmental processes that involve extracellular 0-linked sugars and glycoproteins, synthesized by the epidermis. During development, epithelial, neuronal and muscle cells undergo a concerted process of programmed changes in cell shape, attachment, positioning, which depend on cell-cell and cell-matrix interactions. Epithelial cells synthesize an extracellular matrix, which is rich in mucin-type glycans. Mucin-type 0-glycosylation is regulated by a family of polypeptide Ga1NAc transferases (ppGaNTases), which areconserved in man and the nematode Caenorhabditis elegans. Mutationsin some nematode mucin glycoproteins produce defects in cell migration, cell fusion, embryo elongation, epidermal and muscle attachment to the extracellular matrix. Because the extracellular environment of a developing organism is rich in a vast array of mucin-type 0-linked glycans, we hypothesize that 0-linked sugars are critical for fundamental developmental processes involving epidermal tissues during embryogenesis. To test this hypothesis, we propose to modulate mucintype0-glycosylation in epidermal tissues during C. elegansdevelopment and identify their target substrates. To understand how the 0-glycosylation machinery changes in temporal and spatial patterns, we propose to focus thisstudy by mapping, at single-cell resolution, the expression pattern of only those transferases that are expressed in the nematode epidermis. To identify ppGaNTases that areessential for hypodermal cells, we will initially screen the complete family of ppGaNTases, using double stranded RNA interference andpromoter-driven antisense RNA, for hypodermal cell defects, and later select only the most critical ppGaNTase(s) for producing knockout worms. Thisfunctional screening approach will be coupled with the expression pattern data to select ppGaNTases isoforms for an in-depth substrate analysis. The critical ppGaNTase(s) will be used as tools for identifying the target proteins that are 0-glycosylated in the hypodermis. To address if the isoform specificity and the timing of ppGaNTase gene expression is critical for the 0-glycosylation machinery, we will over-express different isoforms in hypodermal cells and attempt to rescue any ppGaNTase loss-of-function phenotype with different family members.