Notch GOF was achieved through overexpression of a constitutively active
Notch 3 construct in retinal pericyte monocultures, an approach that is used widely in the field to uncover transcriptional targets of Notch activity bypassing the requirement for ligand-mediated activation.
48 Notch 3 was selected for this analysis because its expression is restricted to mural cells in adult tissues, and
Notch 3 mutations have been linked to mural cell pathology in animal models and humans with CADASIL.
21 –23 The Notch GOF analysis in monoculture identified 56 genes downstream of Notch activity in pericytes using the same cutoff defined for LOF (
Supplementary Table S3).
Hes1, a canonical downstream target of Notch activity, was the most upregulated gene (4.05).
HeyL,
Hey1, and
Hey2, which were downregulated in Notch LOF, conversely were upregulated in Notch GOF, further validating the approach (2.46, 2.57, 1.89, respectively).
Jagged 2, a ligand that showed significantly lower levels of expression in BRPs compared to BRECs, was the most downregulated gene upon
Notch 3 activation in BRPs (0.22).
Notch 2,
ADAM10, and
LFng were downregulated by Notch GOF, although the differences did not reached the pre-established cutoff (0.63,
P < 0.001; 0.87,
P = 0.03; 0.52,
P < 0.001, respectively). Of the 26 apoptosis genes examined, 20 were misregulated by Notch GOF in BRPs (
Supplementary Table S2). Of these, only
TNFRSF6 was downregulated (0.42), whereas all others were upregulated, including
TNFRSF10A (1.9) and
HMOX1 (2.9, also increased by Notch LOF).