Microscopy image data containing fluorescently labeled gene loci, recruited RNA polymerase II, and elongating RNA polymerase II. This data set is for the gene iscub and is obtained from fixed zebrafish embryos, collected at the developmental stages oblong, sphere, dome, 30% epiboly, and 50% epiboly (indicated in the file names). Data were recorded using an instant-SIM microscope (iSIM, VisiTech UK) with a 100X TIRF oil immersion objective (Nikon, NA 1.49, CFI SR HP Apo TIRF 100XAC Oil). Two independent experiments were performed (IF1, IF2 in the file name), for each experiment, two samples were prepared per stage and experiment (001, 002 in the file name). The image data are in the ND2 format (Nikon proprietary) and can be imported using the BioFormats importer (Open Microscopy Environment).