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Abstract
The Notch signaling pathway is an evolutionarily conserved mechanism for cell-to-cell communication in metazoan development. Significant pathway regulation occurs through glycosylation of the Notch receptor extracellular domain by the Fringe family of glycosyltransferases at numerous O-fucosylated Epidermal Growth Factor-like (EGF) Repeats. In this study we investigated the in vivo contributions of two Fringe-modified O-fucose sites in NOTCH1 by using CRISPR/Cas9 knock-in technology to generate two lines of C57BL/6J mice carrying point mutations at O-fucose sites in NOTCH1 previously shown to alter signaling in vitro. Because T/V point mutation of EGF8 O-fucose results in reduced Notch receptor activation when stimulated by both Delta-like and Jagged ligands in cellular assays, we predicted that mice carrying two mutated gene copies would have Notch1-null phenotypes and embryonic lethality. Another Fringe-elongated O-fucose site in EGF6 was of great interest for its ability to inhibit Notch activation by Jagged ligand, despite its location outside the NOTCH1 ligandbinding region, without enhancing activation by Delta-like ligand. Moreover, T/V mutation of EGF6 O-fucose in vitro causes no change to Notch activation when unmodified, suggesting that its role is solely regulatory. We chose to monitor changes in the well-documented developmental process of retinal angiogenesis due to its dependence on the coordinated expression of Notch1, Jag1, Dll4, and the three mammalian Fringe genes Lfng, Mfng, and Rfng. Unexpectedly, the EGF8 mice were viable and fertile, indicating that the point mutation did not sufficiently lower pathway activation to cause embryonic lethality, although the O-fucose residue directly participates in ligand-binding events. However, vessels in the EGF8 retinas were densely clustered, consistent with the predicted Notch1 loss-of-function phenotype. Retinas from the EGF6 mice presented with the opposite phenotype, a decrease in vessel density associated with our predicted Notch1 gain-of-function outcome. Overall, these in vivo results recapitulate the in vitro data and confirm the significance of individual NOTCH1 O-glycans in pathway function during mammalian development.
