DISTINCT NOTCH1 AND NOTCH2 SIGNALING PATHWAYS REGULATE NEURAL STEM CELL ACTIVATION AND MAINTENANCE IN MOUSE ADULT NEUROGENESIS

Tight regulation of neural stem cell (NSC) maintenance and differentiation is essential for central nervous system development and adult neurogenesis. In the adult brain, neurogenesis remain active in two specialized niches: the ventricular-subventricular zone (V-SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). Notch signaling is a key regulator in both niches, with multiple receptors—including NOTCH1, NOTCH2, and NOTCH3—expressed in adult NSCs. Previous work from our group showed that NOTCH1 and NOTCH2 are co-expressed in NSC populations, where they exert distinct and opposing functions on adult neurogenesis. NOTCH1 primarily acts on activated NSCs, and its deletion leads to their differentiation and depletion, whereas loss of NOTCH2 activates dormant NSCs, expands the active NSC pool, and increases neurogenesis. Importantly, deletion of RBPJk, the canonical downstream effector of Notch signaling, phenocopies the combined loss of NOTCH1 and NOTCH2, raising the question of how receptor-specific outcomes are achieved through a shared signaling effector. To address this, we generated CRISPR/Cas9-engineered transgenic mouse lines expressing endogenous 3xFLAG-tagged NOTCH1 or NOTCH2 proteins. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq) and immunoprecipitation-mass spectrometry (IP-MS), we mapped the transcriptional targets and protein interactors of NOTCH1, NOTCH2, and RBPJk in adult V-SVZ NSCs. Our findings reveal distinct molecular mechanisms underlying NOTCH1- and NOTCH2-mediated regulation of NSC states, providing new insight into receptor-specific Notch signaling and its role in balancing stem cell quiescence and activation during neurogenesis.