Abstract
Purpose :
Angiogenesis of the retina is mediated in part by cell-cell signaling through the NOTCH1 receptor and its ligands Jagged1 (JAG1) and Delta-Like ligand 4 (DLL4). O-Fucosylation of Ser/Thr residues in the Epidermal Growth Factor-like (EGF) repeats by Protein O-fucosyltransferase 1 (POFUT1) is necessary for receptor activation by both ligands, and addition of b1,3-GlcNAc to O-fucose by Fringe glycosyltransferases reduces NOTCH1 activation during interaction with JAG1 while enhancing NOTCH1 activation during interaction with DLL4. We generated a mouse model to study the regulatory contributions by Fringe glycosyltransferases during angiogenic development of the mouse retina.
Methods :
Using CRISPR/Cas9 to knock in a three base pair change from ACC to GTG, we made a T/V substitution at Thr232 of the NOTCH1 protein in a C57BL/6J mouse. This mutation excludes the O-fucose site at EGF6 as a substrate for POFUT1 and Fringe, and previous in vitro studies show that this mutation rescues the inhibitory effect of JAG1 signaling through Fringe-modified NOTCH1 with no changes to the enhancing effect of DLL4 signaling through Fringe-modified NOTCH1. Retinas from P5-P6 and adult mice were stained with Isolectin B4 to visualize the endothelial vasculature, and a Mask Sholl Analysis was performed to quantify the vascular density of the retina.
Results :
Preliminary data from P5-P6 mutant retinas show decreased vascular density compared to wildtype as well as a growth delay from the inner retina to the outer retina. This phenotype extends into the adult stage with reduced endothelial staining and a less severe growth delay.
Conclusions :
A loss of vascular density is consistent with published mouse models showing hyperactivation of NOTCH1 through knockdown of Jag1 or with the opposite gain of vascular density from treatment with the λ-secretase inhibitor DAPT, which is an established inhibitor of Notch signaling. These data suggest that we uncoupled Fringe-mediated regulation of NOTCH1-JAG1 signaling from Fringe-mediated enhancement of NOTCH1-DLL4 during angiogenic development of the mouse retina. We are currently immunostaining and measuring transcript levels of components in the NOTCH1 pathway to confirm the genetic mechanism by which we observe this phenotype.
This work was supported by NIH grants GM061126 and T32GM107004.
This is a 2021 ARVO Annual Meeting abstract.