Genetic mutations in GLP-1/Notch pathway reveal distinct mechanisms of Notch signaling in germline stem cell regulation.

The Notch signaling pathway is crucial for germline stem cell (GSC) regulation in Caenorhabditis elegans, yet the molecular and biological consequences of GLP-1/Notch mutations remain poorly understood. This study systematically analyzes commonly used and pathological glp-1 loss- (lf) and gain-of-function (gf) mutations to investigate their effects on Notch activity at nascent transcript (ATS), mRNA, and germline levels. Using complementary direct readouts of Notch activation, including sygl-1 activation sites, mRNA levels, and germline functional assays of the Notch-responsive 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. Among the commonly used glp-1 alleles we examined, NICD mutations reduced Notch transcriptional activation at cellular and germline levels while having little impact at the chromosomal (ATS) level, whereas partial lf 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.