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Notch signaling is a highly conserved pathway that mediates communication between adjacent cells during development. Notch signaling plays a major role in the segmentation clock. The segmentation clock regulates the oscillatory gene expressionthat times the periodic patterning of undifferentiated posterior mesoderm into skeletal tissue precursors. These precursors then develop into the spinal elements of the axial skeleton. Across evolution, the cycling of Notch activation is timed by the interaction between the Notch receptor and two Notch ligands. An activating Notch ligand on the surface of a cell interacts with a Notch receptor on the surface of an adjacent cell initiating trans-activation and driving the expression of a self-repressing core oscillator in the signal-receiving cell. Expression of an additional ligand that does not appreciably activate the receptor in trans but is competent to interact with the receptor provides additional regulation that alters the level of Notch activation.In the mammalian lineage, interactions between the activating Notch ligand Deltalike1 (DLL1), Notch receptor NOTCH1, and the cis-only acting Notch ligand Deltalike3 (DLL3) that mediate Notch activation are additionally regulated through glycosylation by a glycosyltransferase Lunatic Fringe (LFNG). The involvement of DLL3 in regulating DLL1-NOTCH1 interaction, the role of LFNG in mediating these iiiinteractions, and how DLL3 and LFNG function to alter the ticking of the segmentation clock are not well understood. To begin to understand these mechanisms, I utilized a mammalian cell system to determine how Notch signaling is regulated in this complex environment. I determined that in the absence of DLL3, cells co-expressing DLL1 and NOTCH1 do not appreciably send signal to receiving cells. Induction of DLL3 expression leads to increased loss of NOTCH1 protein through a Notch activation-dependent pathway. The loss of NOTCH1 receptor is coincident with an increased signal sending capacity in these cells and is also coincident with increased presentation of DLL1 at the cell surface. Addition of LFNG into the DLL3-DLL1-NOTCH1 expressing cells abrogates the increased signal sending capacity. Thus, in this work we propose a novel role for DLL3 as a decoy ligand that relieves DLL1-NOTCH1 inhibition, promotes DLL1 surface presentation, and favors signal-sending cell character in a glycosylation- and LFNG-dependent manner. This mechanism establishes roles for DLL3 and LFNG in regulating oscillatory Notch activation that times the vertebrate segmentation clock.This work additionally proposes that this role of DLL3 as a decoy ligand has evolved separately in both the mammalian and avian lineages. We identified an ortholog of DLL3 in chickens (cDLL3) that has lost similar and additional N-terminal domains to the mammalian DLL3; compared to more canonically structured DLL3 orthologs like zebrafish deltaC. Similar to the mammalian DLL3, cDLL3 accumulates in the Golgi, cannot trans-activate the receptor but can cis-inhibit the receptor. Analysis of cDLL3’s post-translational modification determined that it is fucosylated, providing two sites that ivare possible substrates for LFNG glycosylation. Overall, this work proposes that DLL3 has evolved in the mammalian and avian lineages to modulate DLL1-NOTCH1 cisinhibition and aid in establishing levels of Notch activation that time the expression and activity of core oscillators of the vertebrate segmentation clock. DLL3’s activity is further modified by LFNG glycosylation and together DLL1, DLL3, NOTCH1, and LFNG, contribute to the complex signaling system that times the periodic patterning of skeletal precursors during vertebrate development. |
