Abstract The Notch signaling pathway is a highly conserved cell:cell communication pathway that plays critical roles in many aspects of metazoan development. Tight spatial and temporal regulation of this pathway is crucial in many developmental decisions, and understanding the molecular mechanisms contributing to this elegant control is an area of broad interest. The proposed research uses evolutionary changes in the segmentation clock to explore modes of Notch pathway regulation that rely on cell-autonomous functions of Notch ligands. The segmentation clock comprises a network of oscillatory gene expression that functions in the temporal regulation of somitogenesis. The mammalian clock requires expression of an atypical ligand called DLL3 that can not activate Notch signaling, but is able to modulate the pathway in a cell autonomous way. We have identified a chicken homolog of DLL3, and find that its predicted structure suggests that it too will be unable to activate Notch signaling. This finding highlights the segmentation clock as a developmental process that relies on cell autonomous ligand functions, providing a model to understand the novel mechanisms that regulate the Notch pathway in cis. The mechanisms that underlie cis interactions between Notch ligands and receptors, or between co-expressed ligands are poorly understood, and the segmentation clock provides an outstanding model to dissect these interactions. Two aims will provide support for the central model that cell autonomous ligand activities are critical for regulation of segmentation clock function. First, we will assess the expression and function of the chicken DLL3 protein. RNA in situ hybridization will assess cDll3 expression during chick embryogenesis and expression of tagged cDLL3 protein in cell culture will be used to identify the subcellular localization of the protein. Functional assays will asses the ability of cDLL3 to interact with other Notch pathway components, and how its expression affects Notch pathway activity. A second aim will directly examine whether cDLL3 expression is required for segmentation clock function in developing chick embryos. The work proposed here will provide critical data to support the central hypothesis that cell-autonomous ligand activity provides a critical mechanism to regulate Notch pathway activity in the segmentation clock. We anticipate that the results from this work will have broad implications for our understanding of how the Notch pathway is regulated, allowing a pathway that appears straightforward on the surface to contribute to complex developmental decisions across metazoans, and providing new insights into how pathway dysregulation can contribute to congenital anomalies in human development.