Analysis of the functional divergence of the Notch ligands Delta1 and Delta4 in vitro and in vivo

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The evolutionary conserved Notch signaling pathway mediates the direct communication between adjacent cells, and is pivotal for the regulation of multiple developmental processes in diverse organisms such as nematodes, insects and vertebrates. DLL1 and DLL4 are two mammalian DSL proteins that are similar in their amino acid sequence (47% identical plus 14% similar amino acids) and domain structure (both contain the same number of EGF-like repeats, and a C-terminal PDZ binding domain) and both function as activating Notch ligands. These ligands are expressed both in unique and overlapping patterns during development and in adult tissues. In the intestinal epithelium DLL1 and DLL4 are coexpressed in crypt cells and act redundantly in maintaining the crypt progenitor cell pool. In the thymus DLL1 and DLL4 are both expressed in thymic epithelial cells, and both ligands expressed on stromal cells can induce T cell development of hematopoietic progenitors in vitro. However, in vivo DLL4 is the essential Notch ligand required for T-lymphopoiesis. DLL4 binds better to NOTCH receptors on thymocytes and a lower steady-state cell surface levels is required to induce T-cell development in vitro. Consistent with these observations biochemical studies with purified fragments of DLL1 and DLL4 and NOTCH1 have shown that DLL4 binds to NOTCH1 with approximately 10-fold higher affinity.Unexpectedly, our preliminary data using two mouse models show that -despite the stronger Notch activation potential of DLL4- during early embryonic development DLL4 cannot replace DLL1s function during somitogenesis and muscle differentiation, suggesting profound differences in the biochemical and functional properties of these closely related DSL proteins. In this study we propose to test the hypothesis that specific protein domains of DLL1 and DLL4 confer different biochemical properties to these ligands and thereby lead to non-redundant and redundant functions depending on the context. This will be achieved by the analysis of chimeric DLL1/4 ligand proteins in which domains of these proteins have been swapped. Chimeric ligands will be analyzed in vitro for their Notch activation potential and biochemical properties as well as during embryonic development in vivo using an established transgenic strategy. These experiments should elucidate the divergent biochemical properties of the Notch ligands DLL1 and DLL4 that are the basis for the different outcomes of NOTCH activation by two closely related DSL proteins and should provide novel insights how this critically important signaling pathway is activated and fine-tuned in vivo depending on the context.