John, N. S.; Urman, M. A.; Mehmood, M. G.; Lee, C.

The Notch signaling pathway is crucial for germline stem cell (GSC) regulation in C. elegans, yet the molecular and biological consequences of GLP-1/Notch mutations remain poorly understood. This study systematically analyzes commonly used and clinically relevant glp-1 loss- and gain-of-function mutations to investigate their effects on Notch activity at chromosomal, cellular, and tissue levels. Using complementary direct readouts of Notch activation, including sygl-1 activation sites, mRNA levels, and germline functional assays of the GSC pool and progenitor zone (PZ), we demonstrate that the severity of glp-1 mutations is dependent on their position within the GLP-1 protein. Our results reveal that NICD mutations reduce Notch transcriptional activation at tissue and cellular levels while having little impact at the chromosomal level, whereas NECD mutations have minimal effects across all biological levels. Furthermore, a series of regression analyses of sygl-1 activation, mRNA production, and PZ size reveal strong correlations, qualifying these readouts as predictive markers for germline function. These findings provide a comprehensive framework for understanding glp-1 mutation effects and offer new insights into the regulation of Notch signaling in stem cell biology.