The One Click Wonder: a retrained automated segmentation pipeline that enables quantitative and modular analysis of <i>C. elegans</i> embryos

High-throughput approaches have transformed the study of gene regulation by enabling quantitative, genome-scale analyses in both genomics and imaging. However, applying these methods to intact organisms remains challenging, particularly for high-throughput, 3D imaging. In Caenorhabditis <i>elegans</i> , generalist segmentation models often perform poorly due to rapid changes in nuclear size, shape, and density. To overcome this obstacle, we developed <b>One Click Wonder (OCW)</b> , an automated pipeline that pairs a retrained Cellpose model with stage-specific parameter selection to deliver accurate, high-throughput segmentation of embryos. We further introduce the <b>Biological Annotation and Association Mapper (BAAM)</b> , which integrates segmentation with spot detection, to enable single-cell quantitation. Applied to the pioneer factor <i>pha-4/FoxA</i> , this pipeline revealed distinct cell populations with an eight-fold range in transcriptional burst frequency. These findings demonstrate that OCW and BAAM provide a modular, scalable pipeline for quantitative, single-cell analysis of gene expression in <i>C. elegans</i> embryos.