C. elegans GLP-1/Notch activates transcription in a probability gradient across the germline stem cell pool.

C. elegans Notch signaling maintains a pool of germline stem cells within their single-celled mesenchymal niche. Here we investigate the Notch transcriptional response in germline stem cells using single-molecule fluorescence in situ hybridization coupled with automated, high-throughput quantitation. This approach allows us to distinguish Notch-dependent nascent transcripts in the nucleus from mature mRNAs in the cytoplasm. We find that Notch-dependent active transcription sites occur in a probabilistic fashion and, unexpectedly, do so in a steep gradient across the stem cell pool. Yet these graded nuclear sites create a nearly uniform field of mRNAs that extends beyond the region of transcriptional activation. Therefore, active transcription sites provide a precise view of where the Notch-dependent transcriptional complex is productively engaged. Our findings offer a new window into the Notch transcriptional response and demonstrate the importance of assaying nascent transcripts at active transcription sites as a readout for canonical signaling.