The data comprises bright-field time-lapse images of zebrafish embryos acquired in multiple batches within multi-well plates using an Acquifer Imaging Machine. Embryos from incrosses of TE zebrafish strains were used for imaging. Small-molecule signaling pathway modulator LDN-193189 was added to wells A3-A6 with final concentrations of 4 µM, to wells B1-B4 with final concentrations of 3.5 µM, and to wells B5-B6 with final concentrations of 12 µM. Small-molecule signaling pathway modulator SB-505124 was added to wells C1-C6 with final concentrations of 5 µM and to wells D1-D6 with final concentrations of 6 µM. Wells A1 and A2 did not receive small-molecule signaling pathway modulator treatment. Imaging started with embryos between 2-2.5 hpf and ended with embryos between 26-26.5 hpf. Images of embryos were acquired at 720 acquisition time points with a time interval of 120 s between each acquisition time point. Individual embryo segments were identified and extracted using a trained neural network for object detection. Within this experiment folder, data are organized by microscope position and embryo number.