The One Click Wonder: a retrained automated segmentation pipeline that enables quantitative and modular analysis of C. elegans 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 elegans, generalist segmentation models often perform poorly due to rapid changes in nuclear size, shape, and density. To overcome this obstacle, we developed One Click Wonder (OCW), 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 Biological Annotation and Association Mapper (BAAM), which integrates segmentation with spot detection, to enable single-cell quantitation. Applied to the pioneer factor pha-4/FoxA, 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 C. elegans embryos.