Two NOTCH1 O-fucose sites have opposing functions in mouse retinal angiogenesis

Area of Science:

• Developmental Biology
• Molecular Biology
• Glycobiology

Background:

• Fringe glycosylation of NOTCH1's extracellular domain at O-fucose residues in Epidermal Growth Factor-like Repeats (EGFs) 6 and 8 influences NOTCH1 activation by JAG1 and DLL1.
• These glycosylation sites are critical for modulating Notch signaling pathway activity.

Purpose of the Study:

• To investigate the in vivo significance of NOTCH1 O-fucosylation at EGF6 and EGF8 during mammalian development.
• To generate and analyze mouse models with specific NOTCH1 mutations affecting O-fucosylation at these sites.

Main Methods:

• Generation of two C57BL/6J mouse lines with NOTCH1 point mutations (T232V at EGF6, T311V at EGF8) to eliminate O-fucosylation and Fringe activity.
• Assessment of retinal angiogenesis, including vessel density and branching, in the generated mouse models.
• Analysis of gene expression related to the Notch pathway (Notch1, Jag1, Dll4, Lfng, Mfng, Rfng).

Main Results:

• The EGF6 O-fucose mutant (6f/6f) exhibited reduced retinal vessel density and branching, indicating a Notch1 hypermorph phenotype.
• The EGF8 O-fucose mutant (8f/8f) mice were viable and fertile, displaying increased retinal vessel density, consistent with a Notch1 hypomorph phenotype.
• These findings contrast with initial predictions regarding the EGF8 mutant's viability.

Conclusions:

• NOTCH1 O-fucose residues are essential for proper pathway function during mammalian development.
• Specific O-glycan sites on NOTCH1 contain critical signaling information that influences developmental processes like retinal angiogenesis.
• The study highlights the differential impact of O-fucosylation at EGF6 versus EGF8 on Notch signaling outcomes.