Abstract Single molecule RNA fluorescence in situ hybridization (smFISH) has become the standard tool for high spatial resolution analysis of gene expression in the context of tissue organization. This article describes protocols to perform smFISH on whole-mount mouse embryonic organs, where tissue organization can be compared to RNA expression by co-immunostaining of known protein markers. An enzymatic labeling strategy is also introduced to produce low-cost smFISH probes. Important considerations and practical guidelines for imaging smFISH samples using fluorescence confocal microscopy are described. Finally, a suite of custom-written ImageJ macros is included with detailed instructions to enable semi-automated smFISH image analysis of both 2D and 3D images. © 2018 by John Wiley & Sons, Inc.

Throughout metazoans, germ cells undergo incomplete cytokinesis to form syncytia connected by intercellular bridges. Gamete formation ultimately requires bridge closure, yet how bridges are reactivated to close is not known. The most conserved bridge component is centralspindlin, a complex of the Rho family GTPase-activating protein (GAP) CYK-4/MgcRacGAP and the microtubule motor ZEN-4/kinesin-6. Here, we show that oocyte production by the syncytial \textitCaenorhabditis elegans germline requires CYK-4 but not ZEN-4, which contrasts with cytokinesis, where both are essential. Longitudinal imaging after conditional inactivation revealed that CYK-4 activity is important for oocyte cellularization, but not for the cytokinesis-like events that generate syncytial compartments. CYK-4’s lipid-binding C1 domain and the GTPase-binding interface of its GAP domain were both required to target CYK-4 to intercellular bridges and to cellularize oocytes. These results suggest that the conserved C1-GAP region of CYK-4 constitutes a targeting module required for closure of intercellular bridges in germline syncytia.