Defining how dominant-negative RBPJ disease alleles impact Notch outcomes

. The Notch pathway is a highly conserved cell signaling pathway that plays essential roles in embryonic development and adult tissue homeostasis. Mutations in the Notch pathway underlie Adams-Oliver Syndrome (AOS), a rare disease defined by scalp aplasia cutis congenita (missing skin and skull tissue) and limb, heart, vascular, and neurological defects. Many AOS patients inherit dominant mutations within the NOTCH1 receptor, the DLL4 ligand, or the RBPJ transcription factor, all of which reside within the Notch signaling pathway. In contrast, mutations in the NOTCH2 receptor and JAG1 ligand are associated with Alagille Syndrome, a disease characterized by liver, eye, kidney, heart, skeleton, and vasculature defects. The differential expression of NOTCH1/DLL4 and NOTCH2/JAG1 receptor/ligand pairs correlates well with the organ specific defects observed in AOS versus Alagille. However, it is unclear how variants in RBPJ, which is the sole transcription factor that mediates target gene activation in the Notch pathway, causes AOS but not Alagille-like phenotypes. The goal of this grant is to use novel conditional mouse models to dissect how AOS associated variants in RBPJ alter Notch signal strength and cellular decisions. Our data supports the hypothesis that AOS- RBPJ variants do not function as loss-of-function alleles, but instead are pathological due to a sequestration mechanism of the Notch signal. Consistent with human patient data, we found that mice with an Rbpj AOS allele develop AOS-like phenotypes and have increased lethality in association with Notch1 haploinsufficiency, but not Notch2 haploinsufficiency. We propose to leverage these animal models to define the molecular defects and underlying tissue-specific pathogenesis of AOS through two aims: (1) Aim1 proposes to use a conditional AOS ‘initiating’ mouse model to study the consequences of AOS alleles on animal growth, homeostasis, wound healing, vascular development, and vascular integrity. (2) Aim2 proposes to determine how Rbpj variants cause AOS but not Alagille phenotypes by testing the non-mutually exclusive hypotheses that differences in Notch1 versus Notch2 signal composition, and/or cellular differences in co-repressor levels underlies the preferential sensitivity of N1-dependent cell types to Rbpj AOS alleles. Since Notch signaling is highly conserved to specify cell fates within virtually all organs and tissues, these studies will reveal insight into AOS pathogenesis and have a broader impact on our understanding of how the widely used Notch pathway can impact the development of specific tissues. Project Number: 1R21HD121139-01 | Fiscal Year: 2026 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: BRIAN GEBELEIN | Institution: CINCINNATI CHILDRENS HOSP MED CTR, CINCINNATI, OH | Award Amount: $445,500 | Activity Code: R21 | Study Section: Development - 2 Study Section[DEV2] View on NIH RePORTER: https://reporter.nih.gov/project-details/11312014